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Cellular factors involved in mouse hepatitis virus replication

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Cellular factors involved in mouse hepatitis virus replication

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Content Cellular Factors Involved in Mouse Hepatitis Virus Replication by Keum Sook Choi A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfdlment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR MICROBIOLOGY AND IMMUNOLOGY) December 2004 Copyright 2004 Keum Sook Choi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3184217 Copyright 2004 by Choi, Keum Sook All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3184217 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements This dissertation could not have been accomplished without the help of many individuals. Dr. Pei-yong Huang, Dr. Vicky Sung and Dr. Hideki Aizaki trained me about basic techniques related with my research and provided me with experimental materials for my studies. All other members of Lai lab also gave me invaluable advice and training. Dr. Akihiro Mizutani (University of Tokyo, Japan) also generously provided constructs and antibody for the study of SYNCRIP proteins. I thank the members of my dissertation committees, Dr. James Ou and Dr. Ite Laird-Offringa, who gave me a lot of advice and encouragement throughout the course of my research. Finally, I would like to thank my advisor, Dr. Michael Lai, who provided the opportunity to pursue my Ph.D. degree. His support and firm guidance were critical to my intellectual development and scientific thinking. He influenced me strongly on the continuation of research in my life and even beyond scientific endeavor. This influence is a gift that I will carry throughout my future career. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents Acknowledgements ii List of Tables v List of Figures vi Abstract viii Chapter 1: The Molecular Biology of Mouse Hepatitis Virus 1.1 Introduction 1 1.2 Classification 3 1.3 Virion structure 3 1.4 Genome organization 6 1.5 Structural proteins 6 1.6 Nonstructural proteins 13 1.7 Virus replication cycles 15 1.8 Cis- and trans-acting signals 21 1.9 The goal of the research 25 1.10 References 28 Chapter 2: Polypyrimidine-tract binding protein affects transcription but not translation of MHV RNA 2.1 Abtract 34 2.2 Introduction 35 2.3 Materials and methods 39 2.4 Results 44 2.5 Discussion 62 2.6 References 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3: SYNCRIP, a novel MHV RNA binding protein, is involved in MHV RNA synthesis 3.1 Abtract 73 3.2 Introduction 74 3.3 Materials and methods 77 3.4 Results 82 3.5 Discussion 102 3.6 References 107 Chapter 4: Coronivirus involves lipid rafts virus entry and cell-cell fusion, but not virus release 4.1 Abtract 111 4.2 Introduction 112 4.3 Materials and methods 115 4.4 Results 120 4.5 Discussion 135 4.6 References 140 Chapter 5: Conclusions and Future directions 5.1 Roles of host factors involved in MHV replication 144 5.2 Implication of lipid rafts in MHV replication 148 5.3 References 152 Alphabetized bibliography 153 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Tables Tab. 1.1 Classification of coronaviras Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Chapter 1 Fig. 1.1 Morphology of coronavirus Fig. 1.2 Genomic organizationlntroduction Fig. 1.3 S, M and E proteins Fig. 1.4 Putative motifs in gene 1 of coronavirus Fig. 1.5 Life cycle of coronavirus Fig. 1.6 Replication, transcription, translation and their cis-acting signals Chapter 2 5 7 9 14 16 22 Fig. 2.1 PTB binds to MHV RNA in PTB- overexpressing cell lines. 46 Fig. 2.2 Interaction between PTB and N in vitro and PTB-overexpressing cell lines. 48 Fig. 2.3 Characterization of cell lines overexpressing wild-type and truncated PTB 50,51 Fig. 2.4 Inhibition of syncytia formation and virus production in the stable cell lines 53 Fig. 2.5 Inhibition of viral RNA and protein syntheis in the stable cell lines 55 Fig. 2.6 Transcriptional effects of PTB 57 Fig. 2.7 Translational effects of PTB 59,61 Chapter 3 Fig. 3.1 Specific association of cellular proteins with 5’-UTR and c5’-UTR, and RNA affinity purification of MHV-RNA binding proteins from DBT cells 83, 85 Fig. 3.2 Schematic diagram of SYNCRIP and confirmation of p70 as SYNCRIP protein 87 Fig. 3.3 In vitro binding of the recombinant SYNCRIP and analysis of its binding sites on 5’-UTR 89, 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3.4 In vivo binding of SYNCRIP in MHV- 92 infected 293A cells Fig. 3.5 Syncytia formation of 293A cells 94 overexpressing F-SYN and N-SYN Fig. 3.6 RNAi-mediated reduction of SYCRIP 96 in DBT cells Fig. 3.7 Retardation of MHV replication by 97, 98 reduction of endogenous SYNCRIP Fig. 3.8 The effect of SYNCRIP on the 100,101 translation of DI RNA Chapter 4 Fig. 4.1 Cholesterol depletion by M(3CD and its 121, 123 effects on MHV replication in DBT cells Fig. 4.2 The effects of M(3CD on the viral RNA uptake 124 Fig. 4.3 Effects of cholesterol depletion on virus 126 binding and association of MHV receptor with lipid rafts Fig. 4.4 Redistribution of MHV during virus entry 128 Fig. 4.5 Lack of incorporation of lipid rafts into 130 MHV virion Fig. 4.6 Raft-association of viral S proteins on 131, 132 plasma and Golgi membranes Fig. 4.7 Inhibition of cell-cell fusion by M(3CD 134 Fig. 4.8 Determination of raft-association domains 136 of S proteins Chapter 5 Fig. 5.1 Roles of host factors in MHV replication 145 Fig. 5.2 S protein on plasma and Golgi membranes 149 Fig. 5.3 Redistribution process during virus entry 151 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract Cellular Factors Involved in Mouse Hepatitis Virus Replication Host factors are involved in various steps of virus replication, including virus entry, replication, and virus release. In my research, I have attempted to identify host factors and characterize their roles in MHV replication. Polypyrimidine-tract binding protein (PTB) which was previously identified as an MHV-RNA binding protein, was directly involved in MHV RNA synthesis, based on the study of dominant- negative mutants of PTB. Dominant-negative form of PTB significantly inhibited MHV RNA synthesis when expressed stably in cells, indicating that PTB modulate MHV RNA synthesis. However, PTB did not affect viral translation. In addition to PTB, synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP) was identified as a novel MHV RNA-binding protein using RNA affinity purification method. Knock-down of SYNCRIP in cells greatly delayed MHV RNA synthesis, but not translation, thus indicating that SYNCRIP is directly involved in MHV RNA synthesis like PTB. Not only viral RNA synthesis, we also found that host factors are required for virus entry and cell-cell fusion. The study of involvement of lipid rafts in MHV replication showed that virus entry and cell-cell fusion requires yet unidentified factors that interact with viral spike proteins, which were associated with lipid rafts. Interestingly, there was a redistribution process of which virus shifts from non-rafts viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to rafts during virus entry. Host factors likely mediate the shifts of MHV on the membranes. Therefore, overall of my research emphasize that various host factors play an important role to modulate MHV replication cycles. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 1 The Molecular Biology of Mouse Hepatitis Virus 1.1 Introduction Coronavirus is considered as ‘big’ virus, compared with other RNA viruses. The nature of coronavirus genome, which is non-segmented, single-stranded and positive-sense RNA, is remarkably big, 27 to 32kb. The envelope of virus is composed of highly glycosylated proteins, which gives the virus the appearance of a crown or coronet. The prefix corona was chosen because of the crownlike shape of the surface projections. In 1931, Schalk and Hawn gave the first description of disease caused by coronavirus from respiratory illness of chickens, and then, the responsible agent was recovered by Beaudette and Hudson in 1937 (Beaudette, 1937). Another group of coronaviruses, murine hepatitis virus (Cheever, 1949) and porcine transmissible gastroenteritis virus (Doyle, 1946) was recognized in 1949 and 1946, respectively. In 1965, human coronavirus was discovered from a schoolboy with a cold. Following these discoveries, the organ culture technique was used to discover other strains of 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. coronavirus (Tyrrell, 1965) and thereafter, the entire genus was distinguished on the basis of its distinctive morphology and antigenicity (Tyrrell, 1968). The number and importance o f animal coronaviruses grew rapidly, with the eventual inclusion of viruses causing disease in mice, rats, chickens, turkeys, swine, dogs, cats, rabbits, and cattle. Several coronavirus species caused gastroenteritis, respiratory disease, hepatitis, neurological disease and others in these animals. Besides infections on farm animals, human also were known to suffer from the infection of coronavirus, which is the cause of the common cold. In 1970s and early 1980s, coronavirus virion proteins and nested-set arrangements of mRNA were identified and the discontinuous nature of coronavirus transcription was demonstrated. As with the first published sequence of a coronavirus gene in 1983, the whole of the genomes of four coronaviruses were cloned into pieces and the system of defective-RNA (DI) was developed for studying coronavirus RNA replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors. Although the infection of coronavirus has been devastating in farm animals and vaccination against it has been routinely performed, the lack of firm association of coronaviruses with any serious human disease had hampered the public’ s interest in this virus family. However, the recent outbreak of SARS coronavirus makes the studies on coronavirus important both scientifically and medically, which provided a blueprint for understanding the SARS virus. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2 Classification Coronaviruses are members of the family Coronaviridae, which contains coronavirus and torovirus (Lai and Cavanagh, 1997). Three serologically distinct groups of coronaviruses have been identified (Table 1-1). Most coronaviruses naturally infect only one species or several closely related species. Virus replication in vivo can be disseminated, causing systemic infections, or restricted to a few cell types, causing localized infections. 1.3 Virion Structure Coronavirus virions are round, moderately pleomorphic, medium-sized particles measuring 100 to 150 nm in diameter and covered with a distinctive fringe of widely spaced, club-shaped surface projections (Fig 1-1) (McIntosh, 1974). The projections are about 20 nm in length. Virion particles are enveloped with typical bilayer external membrane and inside virion, viral genome is covered by nucleocapsid proteins. The genome is a single-stranded, positive-sense RNA, which is capped and polyadenylated. The RNA genome is associated with the nucleocapsid phosphoprotien, N to form a long, flexible, helical nucleocapsid. In thin sections, the nucleocapsid appears as tubular strands 9 to 11 nm in diameter. The envelopes of coronaviruses contain 2 to 4 different types of viral glycoproteins, membrane protein (M), spike protein (S), small glycoprotein (E) and hemagglutinin-esterase protein (HE), which is most found in the envelopes of group II coronaviruses. 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Antigenic Group Virus Host Disease HCV-229E Human R TGEV Pig R/E CCV Dog E FECV Cat E FIPV Cat R/E/H/N HCV-OC43 Human R I MHV Mouse R/E/H/N I SDAV Rat N HEV Pig E BCV Cow E RbEVC Rabbit R/E TCV Turkey R/E IBV Chicken R/H Table 1-1 Classification of Coronaviruses Coronavrius is classified based on antigenic group. Group I includes HCV-229E (human coronavirus 229E), TGEV (transmissible gastroenteritis coronivirus), CCV (canine coronavirus), FECV (feline enteritis coronavirus), and RbCV (rabbit coronavirus). Group II includes HCV-OC43 (human coronavirus OC43), MHV (mouse hepatitis virus), SDAV (sialodacryoadenitis virus), HEV (swine hemagglutinating encephalomyelitis virus), BCV (bovine coronavirus), and TCV (turkey coronavirus). Group 3 includes IBV (infectious bronchitis virus).R: respiratory, E: enteric, H: hepatitis, N: neurological Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 1-1 Morphology of Coronavirus Virion particles are enveloped with structural proteins; S (spike protein), M (integral membrane protein) and E (small glycoprotein). Inside virion, viral genome is covered by N (nucleocapsid protein). 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4 Genome organization Coronavirus genomic RNAs are capped and polyadenylated, which can function as mRNAs and are infectious (Lai and Cavanagh, 1997). The genomic RNA contains 7-10 functional genes, 4 or 5 of which encode structural proteins (Fig 1-2). At the 5’ end is the 60- to 80-nucleotide-long leader RNA that is followed by about 200 to 500 untranslated nucleotides (5’-UTR). At the other end of the genome is a 3’-UTR of 200-500 nucleotides followed by a poly (A) tail. The first half of the length of the genome from the 5’ end, approximately 20kb, consists of two overlapping open reading frames (ORF la and ORF lb) that encode the viral RNA-dependent RNA polymerase, protease, and other yet unidentified proteins. The rest of genome encodes the structural proteins, in the order of S-E-M-N and other nonstructural/structural proteins, of which functions are unknown. Each ORF has a stretch of consensus sequence, UCUAAAC, or a related sequence, at sites immediately upstream of most of the genes. This sequence represents signals for transcription of subgenomic mRNAs. 1.5 Structural Proteins 1.5.1 Spike protein (S) S protein is the outermost component of the virion, and is responsible for the attachment of the virus to cells (Kubo, Yamada, and Taguchi, 1994) and for the 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Protease and RNA polymerase HE A A s ▲ E M ▲ A N A 31 kb Fig. 1-2 Genomic Organization of Coronavirus MHV genome contains 7 ORFs. ORF1 encodes protease and RNA polymerase. The downstream ORF encode structural proteins in the order of HE (hemagglutinin-esterase), S (spike protein), E (envelope protein), M (membrane protein) and N (nucleocapsid protein) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fusion of the virus-cell and cell-cell (Taguchi, 1993). S protein is a heavily glycosylated 180kDa protein, which has many potential N-linked glycosylation sites and contains four structural domain (Fig 1-3): a short carboxyterminal cytoplasmic domain, a transmembrane domain, and two large external domains called SI and S2 (Lai and Cavanagh, 1997). S protein is cleaved into two 90kDa subunits, SI and S2 (Weismiller et al., 1990); the extent of its cleavage varies greatly among the species. Cytoplasmic domain is rich in cysteine residues, which may mediate virus assembly and interaction with other viral and cellular proteins. Transmembrane domain is attached to S2 subunits. N-terminus SI subunit contains the receptor-binding domain that is mapped to first 330 amino acids, and C-terminus S2 subunit has the membrane-anchoring domain and is responsible for fusion events. S2 subunit has two regions of heptad repeats with a coiled-coil structure, which helps form oligomers of S proteins. There is considerable diversity in both lengths and nucleotides of SI subunits, as compared with S2 subunits (Banner, Keck, and Lai, 1990). This diversity in SI may result from mutation and recombination between coronaviruses, giving the altered antigenicity and virulence of virus (Gallagher, Parker, and Buchmeier, 1990). The tertiary structure of S appears to be a trimer of S proteins held together by noncovalent bonds (Delmas and Laude, 1990) (Xu et al., 2004). S protein has two important biological activities for the virus. Fist, S proteins induce membrane fusion, which is required for viral entry into cells or for cytopathic effect on target cells. Several reports suggest that S2 ectodomain contains the major 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. S protein M protein E protein S1 domain -4 S2 domain SP HR1 HR2 TM CL O TM CP TM CP S protein F u n c tio n S tru c tu re Receptor a L M binding ■ M H S1 (globular domain) Oligomerization! domain - Leucine Ezipper S2 Oligomerization domain LLeucine thinner Plasm a mem brane (stalk domain) M em brane anchor j] Hydrophobic domain Cytoplasmic tail; I Cysteine-rich domain cytosol membrane lumen M protein N-terminal cytosol membrai Golgi targeting lumen C-terminal E protein Fig. 1-3 Schematic diagram of S, M and E proteins, and their proposed topology. Upper diagrams show the schematic organization of S, M and E proteins. Bottom panels show the expected topology of structural proteins. TM: transmembrane domain HR: heptad repeats CP: cytoplasmic tail Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. determinants for the membrane fusion (Luytjes et al., 1989) (Gallagher, Escarmis, and Buchmeier, 1991); however, S2 does not contain hydrophobic domains typical of fusion proteins like HA of influenza virus, suggesting that several disparate regions may contribute to the fusion activity. It is not clear that cleavage activity is essential for the fusogenic activity of S proteins, since cleavage-defective mutant of S in MHV was able to fuse, although with a lower efficiency (Taguchi, 1993). Moreover, it has not been established whether virus-cell fusion and cell-cell fusion follow the same pathway or not. Secondly, S protein mediates virus binding to the receptors on the target cells. The most N-terminus of SI subunit is responsible for the receptor binding (Suzuki and Taguchi, 1996), while S2 subunit is not involved in the binding. 1.5.2 Integral Membrane Glycoprotein (M) The M protein is the most abundant protein in virions and comprises 225-230 amino acids that have a small number of glycosylation sites. It spans the membrane bilayer three times, displaying a short amino-terminal domain on the virion exterior surface and a large carboxy-terminal tail in the virion interior (Fig 1-3) (Locker et al., 1992) (Escors et al., 2001). M protein has been suggested to play a role in virus assembly. When expressed alone, M accumulates in the Golgi complex in homomultimeric complexes, which is the site o f virus assembly (Tooze, Tooze, and Warren, 1984). The retention site of M protein is slightly different from that for viral particle budding (Klumperman et al., 1994). However, in combination with E protein, 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M is retained in the budding compartment and is incorporated into virus-like particle (VLP), which are exported from the cells (Maeda, 1999). In case of TGEV, M protein has an additional biological activity: induction of a- interferon, suggesting that M may be involved in viral pathogenesis (Laude et al., 1992). 1.5.3 Small envelope protein (E) The E protein is a minor, yet critical, structural component in the virus assembly, as demonstrated in the study of virus-like particle (VLP) (Maeda, 1999). The E protein varies from 84 to 109 amino acids, corresponding to molecular weights of 9100 to 12,400 Da. The E protein contains three common features (Fig 1-3); hydrophobic region in the N-terminus, conserved proline residues in the middle and abundant charged residues in the C-terminus, which localize to Golgi membrane (Corse and Machamer, 2002). The E protein is anchored in the membrane by the sequence of N-terminus, with the C-terminus being exposed outside the cells (Maeda et al., 2001). The exact function of E protein is not known in virus assembly. 1.5.4 Hemagglutinin-esterase Glycoprotein (HE) The HE protein is an approximately 65kDa glycoprotein and is detected in virions of HEV, MHV, HCV-OC43, BCV and TCV, but not other coronaviruses. It has been suggested that mature protein exist in the virion as a dimer, anchored by the C terminus, forming a fringe of short spikes (Dea and Tijssen, 1988). In case of 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MHV, the existence of HE proteins depends on strains; HE is expressed in neurotropic JHM strain, but it exists as a psedogene in hepatropic A59 (Luytjes et al., 1988) (Yokomori and Lai, 1991). The HE protein has the two biological activities, as implied in the name. First, HE alone can mediate hemagglutination and hemadorption (Pfleiderer et al., 1991), similar to S protein, with a weaker activity (Schultze et al., 1991). Second, HE has esterase acitivity, specifically neuraminate-O-acetylesterase. It hydrolyzes the 9-0- acetylated sialic acid on erythrocytes, thereby reversing hemagglutination induced by the HE or S proteins. With this function, HE is considered a receptor-destroying enzyme (Yokomori et al., 1989). The functional significane of HE for coronaviruses is not known. Among coronaviruses, only BCV requires HE for infectivity (Deregt et al., 1989); However, the presence of HE may affect the pathogenenicity of some coronaviruses, as evidenced by the facts that passive administration of HE-specific antibody in mice altered MHV pathogenicity and that MHVs with an HE have different neuropathogenicity form those without HE (Yokomori et al., 1995). Alternatively, HE may be involved in virus binding to target cells with S protein. 1.5.5 Nucleocapsid protein (N) The N protein is a 50- to 60-kDa phosphoprotein that forms a helical nucleocapsid (RNP) together with the genomic RNA. The N protein is highly basic and has a high serine content, poteintial targets for phosphorylation. 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The regulatory roles of N protein in MHV replication have been suggested based on several reports; The N protein is able to bind to leader sequence of RNA in case of MHV (Baric et al., 1988) and 3’-UTR in case of IBV (Zhou et al., 1996). In an in vitro RNA replication system, the addition of MHV N-specific antibodies inhibited viral RNA syntheis, indicating that N proteins is a component of replication complex (Compton et al., 1987). N protein is able to bind to the membrane (Anderson and Wong, 1993), where viral RNA syntheis occurs in the membrane fraction of the infected cells. Although N proteins interact with the cytoplasmic domain of M protein, requiring the existence of viral RNA for the interaction (Narayanan et al., 2000), N protein itself is not incorporated into VLP, implying that N protein is not directly involved in virus budding. 1.6 Non-Structural proteins 1.6.1 The polymerase Gene 1 encodes the several proteins involved in RNA replication, including polymerase. The predicted size from this gene is 700-800kDa protein, but, it has not been detected in coronavirus-infected cells, presumably due to co-translational polyprotein processing. Gene 1 encodes two ORFs, ORF la and lb, which is in -1 reading frame with respect of ORF la. ORF lb is translated following ribosomal frameshifting. 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0RF1a 0RF1b PLP1 X PLP2 MD3CLPMD POL MB HEL ADRP GFL ExoN 2’-0-MT XendoU CPD Fig. 1-4 Putative motifs in gene 1 of coronavirus PLP : papain-like protease MD : membrane domain CLP : picornavirus-3C- like protease GFL : growth factor-like POL : RNA-dependent RNA polymerase MB : metal-binding motif HEL : helicase ADRP : adenosine diphosphate-ribose 1’’-phosphate ExoN : 3’-5’ exonuclease XendoU : poly(U)-specific endoribonuclease 2’-0-MT : S-adenosylmethionine- dependent ribose 2'-0-methyltransferase CPD : cyclic phosphodiesterase Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A number of functional domains within gene 1 have been predicted by motif analyses (Lee et al., 1991). Three motifs have been identified in ORF la (Fig 1-4): papain-like cysteine proteases (PLP), chymotrypsin/picomaviral 3C-like protease (3CLP) and cystein-rich growth factor-related protein (GFL). The functional domains associated with RNA synthesis are located within the more conserved ORF lb. These include domains for an RNA-dependent RNA polymerase, a nucleoside triphosphate (NTP)-binding/helicase domain, and a zinc-fmger nucleic acid-binding domain. The recent studies on SARS virus revealed that there are putative domains for RNA processing enzymes, suggesting that ORF1 contains other activities and products besides proteases and polymerases (Snijder et al., 2003). 1.7 Virus replication cycle (Fig 1-5) 1.7.1 Attachment and Penetration The first step in the replication cycle is the binding of the virus to the target cells. Coronavirus that express the HE glycoprotein may bind to 9-O-acetylated neuraminic acid moieties on membrane macromolecules via HE or the S gylcoproteins (Schultze et al., 1990). Then, S protein binds to its specific receptor protein on the cell surface. The first identified viral receptor was the MHV receptors (Dveksler et al., 1991). MHV receptor is CEACAM, which is the murine homolog of a member of the carcinoembryogenic antigen (CEA) family and belongs to the biliary glycoprotein (bgp) subfamily. It has an immunoglobulin-like structure, 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Genomic RNA (+) NegativeStrand ( - ) .3' Template v ' mRNAs (+ )< Genomic , . RNA (+) r RNA# Proteins 3 2 — *-N S 2 A / 2-1— smooth-walled vesicles * - A / 6 Leader None nucleocapsid >$®S) Fig. 1-5 Life cycle of Coronavirus MHV enter the cells through the interaction with MHV receptor. After binding and penetrating the cells, viral genome is released into cytoplasm. Genomic RNA is translated to encode viral proteins and then, negative stranded viral RNA is generated by viral RNA-dependent RNA polymerase. Subsequently, several subgenomic mRNAs are produced from negative strand genomic RNA. Viral proteins and genomic RNAs assemble on Golgi. Virus bud out of Golgi. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. consisting of four immnoglobulin-like loops in the N-terminus that interacts with viral S protein. The sequence of the C terminus of the receptor is not essential. Subsequently, receptors for TGEV and HCV-229E have been identified: aminopeptidase (APN) of the porcine and human species (Delmas et al., 1992) (Yeager et al., 1992). APN is a member of the membrane-bound metallopeptidase family and is widely distributed on diverse cell types. In case of BCV, sialic acid- containing glycoproteins are probably a component required for virus infection, since the removal of sialic acids inhibits BCV infection and resialylation restores virus infectivity (Schultze and Herrler, 1992). However, it should be determined whether it functions as a primary receptor. After binding of S to its receptor, the next step is virus entry into cells, which involves fusion of the viral envelope with either the plasma membrane or endosomal membranes. In case of MHV, virus enters cells by both acidic-dependent (endocytosis) and -nondependent pathways, depending on cell types and virus strains. The mechanism of virus uncoating, that is, the release of virion RNA from the nucleocapsid after the internalization, remains unclear. However, it is suggested that ubiquitin-proteasome pathway is involved in releasing MHV-RNA from endosome to cytoplasme during MHV entry (Yu and Lai, 2004, in submission) 1.7.1 Primary translation Following virus uncoating, RNA-dependent RNA polymerase (RdRP) is predicted to be synthesized from the incoming viral genomic RNA, as with the other 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. positive-stranded RNA virus. However, these primary gene products have not yet been detected. 1.7.2 Transcription of viral RNA The plus-strand viral genomic RNA is transcribed into minus-strand RNA, which is used as the template for the synthesis of plus-strand viral subgenomic mRNAs (6-8 in numbers) and genomic RNAs. Genomic and subgenomic mRNA have a nested-set structure, and all of them contain sequences starting at the 3’-terminus and extending to various distances toward the 5’-end. Except for the smallest mRNA, all of the mRNAs are structurally polycistronic, but, functionally monocistronic. A leader sequence of approximately 60-90 nucleotides exist in the 5’-ends, which is derived form the 5’-end of the genomic RNA. The leader sequences of all the mRNAs are identical for a given strain of virus. The leader sequence of MHV ranges in length from 72 to 82 nucleotides, which contains 2-to 4 copies of a UCUAA repeat. At the mRNA start sites on the viral genomic RNA, there is a short stretch of sequence that is nearly homologous to the 3’-end of the leader RNA, which is called as intergenic (IG) sequence. IG sequence is used as a signal for subgenomic mRNA transcription. The core sequence of Igs in MHV is UCUAAAC. The homologous nucleotides (UCUAA) at the 3’-end of the leader and IG sites serve as fusion sites for the leader and mRNAs. 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Two major models of mRNA syntheis, which are not mutually exclusive, have been suggested to explain the discontinuous fusion between leader and IG sequence (Lai and Cavanagh, 1997). One model is the leader-primed transcription, in which the leader RNA would bind to intergenic sequences downstream on the minus-strand template. This model is based on the report that infected cells contained only genome-length, negative-strand templates (Lai, Patton, and Stohlman, 1982). An alternative model is the discontinuous transcription during minus-strand RNA synthesis. During minus-strand RNA synthesis from the genomic template, the polymerase would pause at one of the intergenic sequences and then jump to the 3’- end o f the leader seuqnece near the 5’-end o f the genomic RNA template, generating subgenomic minus-strand RNAs. Data available at this time do not unequivocally rule out either model. 1.7.3 Replication of Viral genomic RNA Unlike the discontinuous synthesis of mRNA, the production of full-length, plus- strand genomic coronaviurs RNA requires uninterrupted synthesis using the full- length, minus-strand template. Therefore, the mechanism of RNA replication and subgenomic mRNA transcription differ in some respects. Based on the study o f DI RNA system, it has been revealed that several elements are required for the replication of genomic RNA: approximately 400 nucleotides at both 5’- and 3’-ends of the genomic RNA and free leader RNA (Lin and Lai, 1993). However, the precise mechanism of replication remains to be determined. 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.7.4 Translation of viral proteins Viral proteins are encoded from the subgenomic RNAs, from which only the 5’- most ORF in the mRNAs is translatable and the downstream ORFs are usually functionally silent. However, proteins from genel are generated from the ribosomal frame shifting mechanism and posttranslational processing and modifications. Gene lb is translated into one polyprotein by -1 ribosomal frameshifting and both gene la and lb polyproteins are processed into multiple proteins by viral and host proteases. Alternatively, some ORF is translated by internal ribosomal entry site (IRES), which allows ribosome to bypass upstream ORFs (Thiel and Siddell, 1994). As a cis-regulatory region, 5’-UTR including leader sequence is important in modulationg translation (Tahara et al., 1998). 1.7.5 Virus assembly and release The assembly of virus particles starts with the formation of RNP, which includes the interaction between genomic RNA and N proteins. The encapsidation of RNA is followed by binding of nucleocapsids to intracellular membranes that contain M protein to form a virion. The major determinant factor for the site of virus assembly appears to be the site of localization of the M protein, which is in the Golgi complex (Klumperman et al., 1994). The M protein exists as monomers in the ER, but it oligomerizes to form various sized complexes during transport through the Golgi and trans-Golgi network. It is likely that the M molecules in the virus particles are in complexed form. S 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. glycoprotein is not required for the formation or release of virions, since only M and E protein are required for the production of VLPs (Vennema et al., 1996) (Maeda, 1999). The large and small spikes formed by S and HE, respectively, are incorporated into virions at the time of budding. After budding, virions accumulate in large, smooth-walled vesicles that apparently fuse with the plasma membrane to release virus. 1.8 Cis- and Trans-acting signals for MHV replication and transcription 1.8.1 Cis-acting signals (Fig 1-6) 1.8.1.1 IG sequence The IG sequence, which is also known as transcription-regulating sequence (TRS), is the promoter element for transcription. It also serves as the mRNA start site and the site o f fusion between leader RNA and body sequence of mRNAs. A seven-nucleotide core sequence, UCUAAAC, is sufficient to initiate mRNA synthesis (Makino, Joo, and Makino, 1991). These seven nucleotides represent the minimum promoter; deletion of a nucleotide results in complete ablation of mRNA transcription. The effects of the sequence near the promoter on transcription are contradictory: in certain situations, the nature of the neighboring sequence did not affect transcription, but under other circumstances, it did. Therefore, the strength of 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IG 3 4 1 n t (A )n 3 ’ + 5 ’- + 5 " 3 ’' l e a d e r I (-) R N A s y n t h e s i s -O IG "CD 1 5 5 n t - n 3’ 4 7 0 n t 1 3 5 n t ^ R e p l i c a t i o n 4 3 6 n t .............................................................................. .... ..... + 5 ’ 1 r T r a n s c r i p t i o n 3 ’ 5 ’ 5 ’ 3 ’ 5 ’ 3 ’ 3 0 5 n t " m R N A C a p - d e p e n d e n t T r a n s l a t i o n V ir a l p r o t e i n s Fig. 1-6 Replication, transcription and translation of coronavirus, and their cis-acting signals cis-acting signal for negative-strand synthesis cis-acting signal for replication cis-acting signal for transcription 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the promoter appears to depend on the context of the overall RNA sequence and structure. 1.8.1.2 5’-UTR 5’-UTR contains the two cis-acting regions: leader sequence and nine-nucleotide sequence, UUUAUAAAC. The leader sequence at the 5’-end of the viral genomic RNA becomes the leader sequence o f subgenomic mRNAs; thus, it fills a structural role for mRNA synthesis. Deletion of this cis-acting leader abolished transcription and the sequence of this leader RNA affects the efficiency of transcription from certain IG sequences on the DI RNA (Zhang, Liao, and Lai, 1994). Nine-nucleotide sequence located immediately downstream from the UCUAA repeats at the 3 ’ end of the leader RNA in the viral genome and play an important role in RNA synthesis. This sequence serves as an mRNA start signal, allowing transcription of genome- length mRNA (Zhang and Lai, 1996). In addition, this sequence appears to regulate the mechanism by which the leader fused to the subgenomic mRNAs (Zhang, Liao, and Lai, 1994). 1.8.1.2 3’-UTR Partial deletion of the 3’-UTR completely abolished transcription from the upstream IG in the DI RNA (Lin et al., 1996). This stretch of 3’-UTR is involved in positive-strand RNA synthesis, since the length of this required sequence (305 nt) is significantly longer than that required for negative-strand RNA synthesis (55 nt). 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The requirement of 3’-end in postive-strand RNA synthesis suggest that 3’-end may interact with the 5’-end and possibly with IG sequence during transcription. 1.8.2 Trans-acting factors 1.8.2.1 Leader sequence Besides the function of cis-acting signal, leader sequence can function in trans; the leader RNA of the subgenomic mRNAs is not derived exclusively from the leader RNA o f the same RNA (Zhang, Liao, and Lai, 1994). 1.8.2.2 Viral and cellular proteins Temperature-sensitive mutants of MHV that are defective in RNA synthesis at the nonpermissive temperature have been divided into five complementation groups, all of which are mapped within gene 1 region (Baric et al., 1990). However, the precise nature of the gene 1 products involved in RNA synthesis has yet to be determined. In addition, N protein is also involved in RNA synthesis by binding to 5’-UTR (Baric et al., 1988). In addition to viral proteins, cellular factors may also be involved in RNA synthesis. Several cellular proteins have been shown to bind to the regulatory elements o f MHV RNA, including the 5’ and 3’ ends of the genomic RNA and the 3’ end of the negative-strand RNA and IG sites. So far, four proteins have been known to function as trans-acting factors; polypyrimidine tract-binding protein (PTB) (Li et al., 1999), heterogeneous ribonucleoproteins A l (hnRNP A l) (Shi et al., 2000), 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mitochondrial aconitase (Nanda and Leibowitz, 2001), and poly (A)-binding protein (PABP) (Spagnolo and Hogue, 2000). PTB and hnRNP Al belong to the hnRNP family, which are involved in alternative splicing pathway in the cells. While PTB binds to 5’-UTR and c3’-UTR (complementary to 3’-UTR), hnRNP A l binds to c5’-UTR, IG sequence and 3’-UTR. The studies of dominant-negative mutant of both forms showed that PTB and hnRNP A l are involved in MHV RNA synthesis directly. Moreover, the binding sites of PTB and hnRNP A l was complementary, thus suggesting that two proteins may mediate the crosstalk of 5’-3’ ends (Huang and Lai, 2001). Mitochondria aconitase and PABP have been shown to bind to 3’-UTR.The increased expression of mitochondria aconitase enhanced the virus production and viral protein expression in the early infection. However, the significance of the involvement o f mitochondrial proteins or mitochondria in MHV replication has yet to be determined. The binding of PABP to poly (A) tail also has shown to be important in MHV replication (Spagnolo and Hogue, 2000), since serial deletion of poly (A) tail affected the MHV RNA synthesis. 1.9 The Goal of the research The overall objective o f my research is to understand how MHV replication cycles are regulated by cellular components. Most steps of virus infections require various interactions between viral components and host factors. Host factors 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. participate in virus entry, gene expression, virion assembly, and release. Accordingly, it is important to identify the host factors and address the function in MHV replication. UV-crosslinking method has been used to identify the MHV RNA-binding proteins from host cells. In the previous studies, PTB was one of host factors UV- crosslinked to MHV RNA and several in vitro studies indeed showed that PTB was the MHV RNA binding proteins. However, the in vivo significance of PTB in MHV replication has not yet been addressed. HnRNP A l was another MHV RNA binding proteins, which has been shown to be directly involved in MHV RNA synthesis based on the study o f dominant-negative mutants of hnRNP A l. Moreover, in addition to PTB and hnRNP A l, UV-crosslinking experiments showed that there were several unknown proteins that crosslinked to MHV RNA. The identities of those proteins have not yet been determined. Besides the direct involvement of host factors in MHV replication, virus replication can be modulated by the change of cellular compartments. Since MHV entry, replication and assembly require a specific membrane compartment in the cells, the changes of membrane composition and morphology may also affect the various steps of MHV replication. Therefore, specific aims of my research include the following: 1) In vivo function of PTB in MHV replication. PTB was identified as a 58 kDa MHV RNA-binding protein and PTB was able to bind to 5’-UTR, especially UCUAA repeats. Deletion of the binding site in 5’-UTR inhibited the 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transcription of a DI reporter RNA. This suggests that PTB may be involved in MHV RNA synthesis. However, it has not been studied whether PTB is directly involved in MHV RNA synthesis and how PTB functions in the regulation of MHV replication. 2) Identification and characterization of other host factors involved in MHV replication. A number of efforts have been made to identify MHV RNA binding proteins. So far, four proteins have been shown to function as regulatory factors of MHV RNA synthesis. However, several proteins still remain unidentified. Therefore, it is important to identify other unknown factors and address their functions in MHV replication. 3) Involvement of lipid raft in MHV replication cycles. Various membranous compartments are important in various steps of MHV infection; virus entry, replication and assembly. For the virus to enter the cells, virus needs to be fused with plasma membrane or endosomal membrane. Moreover, it has been reported that virus replication require the intracellular membranous structure, and assembly and budding occur on Golgi membrane. Recently, the studies of membrane structure showed that lipid raft localize on the membrane, functioning as platforms of various cellular events as well as virus replication cycles. Therefore, it is important to address whether MHV replication involves lipid rafts and, if so, what the mechanism is. 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.10 References Anderson, R., and Wong, F. (1993). Membrane and phospholipid binding by murine coronaviral nucleocapsid N protein. Virology 194(1), 224-32. Banner, L. R., Keck, J. G., and Lai, M. M. (1990). A clustering of RNA recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus. Virology 175(2), 548-55. Baric, R. S., Fu, K., Schaad, M. C., and Stohlman, S. A. (1990). Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups. Virology 177(2), 646-56. Baric, R. S., Nelson, G. W., Fleming, J. O., Deans, R. J., Keck, J. G., Casteel, N., and Stohlman, S. A. (1988). Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription. Journal o f Virology 62(11), 4280-7. Beaudette, F. R., Hudson C. B. (1937). Cultivation of the virus of infectious bronchitis. J. Am. Vet. Med. Assoc 1937(90), 51-60. Cheever, F. S., Daniels J. B., Pappenheimer A. M., Bailey O. T. (1949). A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelis. Isolation and biological properties of the virus. J. Exp. Med. 90, 121-194. Compton, S. R., Rogers, D. B., Holmes, K. V., Fertsch, D., Remenick, J., and McGowan, J. J. (1987). In vitro replication of mouse hepatitis virus strain A59. Journal o f Virology 61(6), 1814-20. Corse, E., and Machamer, C. E. (2002). The cytoplasmic tail of infectious bronchitis virus E protein directs Golgi targeting. Journal o f Virology 76(3), 1273-84. Dea, S., and Tijssen, P. (1988). Identification of the structural proteins of turkey enteric coronavirus. Archives o f Virology 99(3-4), 173-86. Delmas, B., Gelfi, J., L'Haridon, R., Vogel, L. K., Sjostrom, H., Noren, O., and Laude, H. (1992). Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357(6377), 417-20. Delmas, B., and Laude, H. (1990). Assembly of coronavirus spike protein into trimers and its role in epitope expression. Journal o f Virology 64(11), 5367-75. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Deregt, D., Gifford, G. A., Ijaz, M. K., Watts, T. C., Gilchrist, J. E., Haines, D. M., and Babiuk, L. A. (1989). Monoclonal antibodies to bovine coronavirus glycoproteins E2 and E3: demonstration of in vivo virus-neutralizing activity. Journal o f General Virology 70(Pt 4), 993-8. Doyle, L., Hutchings LM. (1946). A transmissible gastroenteritis in pigs. J. Am. Vet. Assoc 108, 257-259. Dveksler, G. S., Pensiero, M. N., Cardellichio, C. B., Williams, R. K., Jiang, G. S., Holmes, K. V., and Dieffenbach, C. W. (1991). Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV. J Virol 65(12), 6881-91. Escors, D., Camafeita, E., Ortego, J., Laude, H., and Enjuanes, L. (2001). Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core. Journal o f Virology 75(24), 12228-40. Gallagher, T. M., Escarmis, C., and Buchmeier, M. J. (1991). Alteration of the pH dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein. Journal o f Virology 65(4), 1916-28. Gallagher, T. M., Parker, S. E., and Buchmeier, M. J. (1990). Neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino- terminal half of the spike glycoprotein. Journal o f Virology 64(2), 731-41. Huang, P., and Lai, M. M. (2001). Heterogeneous nuclear ribonucleoprotein al binds to the 3'-untranslated region and mediates potential 5'-3'-end cross talks of mouse hepatitis virus RNA. Journal o f Virology 75(11), 5009-17. Klumperman, J., Locker, J. K., Meijer, A., Horzinek, M. C., Geuze, H. J., and Rottier, P. J. (1994). Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. Journal o f Virology 68(10), 6523-34. Kubo, H., Yamada, Y. K., and Taguchi, F. (1994). Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein. Journal o f Virology 68(9), 5403-10. Lai, M. M., and Cavanagh, D. (1997). The molecular biology of coronaviruses. Advances in Virus Research 48, 1-100. Lai, M. M., Patton, C. D., and Stohlman, S. A. (1982). Replication of mouse hepatitis virus: negative-stranded RNA and replicative form RNA are of genome length. Journal o f Virology 44(2), 487-92. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Laude, H., Gelfi, J., Lavenant, L., and Charley, B. (1992). Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus. Journal o f Virology 66(2), 743-9. Lee, H. J., Shieh, C. K., Gorbalenya, A. E., Koonin, E. V., La Monica, N., Tuler, J., Bagdzhadzhyan, A., and Lai, M. M. (1991). The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology 180(2), 567-82. Li, H. P., Huang, P., Park, S., and Lai, M. M. (1999). Polypyrimidine tract-binding protein binds to the leader RNA of mouse hepatitis virus and serves as a regulator of viral transcription. Journal o f Virology 73(1), 772-7. Lin, Y. J., and Lai, M. M. (1993). Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement o f an internal and discontiguous sequence for replication. Journal o f Virology 67(10), 6110-8. Lin, Y. J., Zhang, X., Wu, R. C., and Lai, M. M. (1996). The 3' untranslated region of coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. Journal o f Virology 70(10), 7236-40. Locker, J. K., Rose, J. K., Horzinek, M. C., and Rottier, P. J. (1992). Membrane assembly of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation. Journal o f Biological Chemistry 267(30), 21911-8. Luytjes, W., Bredenbeek, P. J., Noten, A. F., Horzinek, M. C., and Spaan, W. J. (1988). Sequence of mouse hepatitis virus A59 mRNA 2: indications for RNA recombination between coronaviruses and influenza C virus. Virology 166(2), 415- 22 . Luytjes, W., Geerts, D., Posthumus, W., Meloen, R., and Spaan, W. (1989). Amino acid sequence of a conserved neutralizing epitope of murine coronaviruses. Journal o f Virology 63(3), 1408-12. Maeda, J., Repass, J. F., Maeda, A., and Makino, S. (2001). Membrane topology of coronavirus E protein. Virology 281(2), 163-9. Maeda, J. M., A. Makino, S. (1999). Release of coronavirus E protein in membrane vesicles from virus-infected cells and E protein-expressing cells. Virology 263(2), 265-72. Makino, S., Joo, M., and Makino, J. K. (1991). A system for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. Journal o f Virology 65(11), 6031-41. 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. McIntosh, K. (1974). Coronaviruses: a comparative review. Curr. Top. Microbiol. Immunol 63, 85-129. Nanda, S. K., and Leibowitz, J. L. (2001). Mitochondrial aconitase binds to the 3' untranslated region of the mouse hepatitis virus genome. Journal o f Virology 75(7), 3352-62. Narayanan, K., Maeda, A., Maeda, J., and Makino, S. (2000). Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells. Journal o f Virology 74(17), 8127-34. Pfleiderer, M., Routledge, E., Herrler, G., and Siddell, S. G. (1991). High level transient expression of the murine coronavirus haemagglutinin-esterase. Journal o f General Virology 72(Pt 6), 1309-15. Schultze, B., Gross, H. J., Brossmer, R., and Herrler, G. (1991). The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant. Journal o f Virology 65(11), 6232-7. Schultze, B., Gross, H. J., Brossmer, R., Klenk, H. D., and Herrler, G. (1990). Hemagglutinating encephalomyelitis virus attaches to N-acetyl-9-O- acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza C virus. Virus Res 16(2), 185-94. Schultze, B., and Herrler, G. (1992). Bovine coronavirus uses N-acetyl-9-O- acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells. Journal o f General Virology 73(Pt 4), 901-6. Shi, S. T., Huang, P., Li, H. P., and Lai, M. M. (2000). Heterogeneous nuclear ribonucleoprotein A l regulates RNA synthesis of a cytoplasmic virus. EMBO Journal 19(17), 4701-11. Snijder, E. J., Bredenbeek, P. J., Dobbe, J. C., Thiel, V., Ziebuhr, J., Poon, L. L., Guan, Y., Rozanov, M., Spaan, W. J., and Gorbalenya, A. E. (2003). Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J M ol Biol 331(5), 991-1004. Spagnolo, J. F., and Hogue, B. G. (2000). Host protein interactions with the 3' end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication. Journal o f Virology 74(11), 5053-65. Suzuki, H., and Taguchi, F. (1996). Analysis of the receptor-binding site of murine coronavirus spike protein. Journal o f Virology 70(4), 2632-6. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Taguchi, F. (1993). Fusion formation by the uncleaved spike protein of murine coronavirus JHMV variant cl-2. Journal o f Virology 67(3), 1195-202. Tahara, S. M., Dietlin, T. A., Nelson, G. W., Stohlman, S. A., and Manno, D. J. (1998). Mouse hepatitis virus nucleocapsid protein as a translational effector of viral mRNAs. Advances in Experimental Medicine & Biology 440, 313-8. Thiel, V., and Siddell, S. G. (1994). Internal ribosome entry in the coding region of murine hepatitis virus mRNA 5. Journal o f General Virology 75(Pt 11), 3041-6. Tooze, J., Tooze, S., and Warren, G. (1984). Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions. European Journal o f Cell Biology 33(2), 281-93. Tyrrell, D., Almeida JD., Berry DM. (1968). Coronavirus. Nature, 220-650. Tyrrell, D., Bynoe ML. (1965). Cultivation of a novel type of common-cold virus in organ culture. Br. Med. J. 1, 1467-1470. Vennema, H., Godeke, G. J., Rossen, J. W., Voorhout, W. F., Horzinek, M. C., Opstelten, D. J., and Rottier, P. J. (1996). Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. Embo J 15(8), 2020-8. Weismiller, D. G., Sturman, L. S., Buchmeier, M. J., Fleming, J. O., and Holmes, K. V. (1990). Monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions. Journal o f Virology 64(6), 3051-5. Xu, Y., Liu, Y., Lou, Z., Qin, L., Li, X., Bai, Z., Pang, H., Tien, P., Gao, G. F., and Rao, Z. (2004). Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core. Journal o f Biological Chemistry 279(29), 30514-22. Yeager, C. L., Ashmun, R. A., Williams, R. K., Cardellichio, C. B., Shapiro, L. H., Look, A. T., and Holmes, K. V. (1992). Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357(6377), 420-2. Yokomori, K., Asanaka, M., Stohlman, S. A., Makino, S., Shubin, R. A., Gilmore, W., Weiner, L. P., Wang, F. I., and Lai, M. M. (1995). Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression.[see comment]. Journal o f Neurovirology 1(5-6), 330-9. 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Yokomori, K., La Monica, N., Makino, S., Shieh, C. K., and Lai, M. M. (1989). Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus. Virology 173(2), 683-91. Yokomori, K., and Lai, M. M. (1991). Mouse hepatitis virus S RNA sequence reveals that nonstructural proteins ns4 and ns5a are not essential for murine coronavirus replication. Journal o f Virology 65(10), 5605-8. Zhang, X., and Lai, M. M. (1996). A 5'-proximal RNA sequence of murine coronavirus as a potential initiation site for genomic-length mRNA transcription. Journal o f Virology 70(2), 705-11. Zhang, X., Liao, C. L., and Lai, M. M. (1994). Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis. Journal o f Virology 68(8), 4738-46. Zhou, M., Williams, A. K., Chung, S. I., Wang, L., and Collisson, E. W. (1996). The infectious bronchitis virus nucleocapsid protein binds RNA sequences in the 3' terminus of the genome. Virology 217(1), 191-9. 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2 Polypyrimidme-tract-binding protein affects transcription but not translation of mouse hepatitis virus RNA 2.1 Abstract Polypyrimidine-tract-binding protein (PTB) has been shown to bind specifically to the 5’end of mouse hepatitis virus (MHV) RNA. To further characterize the function of PTB in MHV replication, I generated dominant-negative mutant cell lines that express a wild-type PTB or a truncated form of PTB, which includes only the N- terminal half of the molecule, retaining the protein-dimerization domain. The truncated form of PTB was localized in the cytoplasm, while the wild-type PTB was present mainly in the nucleus. The truncated form can interact with the full-length PTB in vitro. I observed that both the full-length and truncated PTB, when over expressed, functioned in a dominant-negative manner in MHV replication. However, the truncated mutant protein exhibited more severe effects on syncytia formation, virus production, and synthesis of viral RNA and viral proteins. To clarify the precise function of PTB in MHV replication, I separated viral transcription from viral 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. translation by transfecting different types of MHV defective-interfering (DI) RNA that contain various reporter genes into these stable cell lines. Transcription of the DI RNA was greatly inhibited in these mutant cell lines infected with MHV, indicating that PTB modulates MHV transcription. In contrast, translation of the DI RNA was not affected by either PTB depletion in in vitro translation in rabbit reticulocyte lysates or in in vivo translation experiments in cells. Given that PTB interacts with viral N protein, which is one of the components of the MHV replication complex, PTB may exert its function on virus replication/transcription by association with other viral and cellular molecules of the replication complex. 2.2 Introduction Polypyrimidine-tract-binding protein (PTB), a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, is a 57kDa protein that binds to pyrimidine-rich RNA sequences, which are present mostly in the introns and untranslated regions of cellular and viral RNA (Ghetti et al., 1992). As an RNA- binding protein, PTB has fmy RNA-recognition motifs (RRM). RRM 1 and 2, which are in the N-terminal half of PTB, are required for PTB oligomerization and other protein-protein interactions, whereas RRM 3 and 4, which are in the C- terminal half of PTB, are necessary for the RNA-binding activity (Oh et al., 1998). Though PTB is a nuclear protein in a static state, it can shuttle between the nucleus and cytoplasm. It has been shown that the N-terminal 60 amino acids are required 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for the nuclear localization of PTB (Li and Yen, 2002; Perez et al., 1997). Although the mechanism for nuclear export has not been completely characterized, the recent experiments have mapped the nuclear export signal (NES) to the N-terminal 25 amino acids of PTB (Li and Yen, 2002), which is not separable from its nuclear localization signal (NLS). Since the RRM 1-containing NLS localizes exclusively in the nucleus, and the addition of RRM 2 domain to RRM 1 enhances the nucleocytoplasmic shuttling (Kamath et al., 2001), it is possible that RRM2 facilitates the nuclear export of PTB, though it is not the primary determinant for the nucleocytoplasmic shuttling. However, RRM 3 and 4 may also contribute to the nuclear localization of PTB, since the wild-type PTB localizes mostly in the nucleus. It is possible that by binding to nuclear RNA, the export of PTB is retarded; alternatively, the presence of RRM 3 and 4 may induce a conformational change of the protein to favor its nuclear localization. PTB is involved in multiple steps of pre-mRNA processing, including tissue- specific splicing (Wagner and Garcia-Bianco, 2001), mRNA localization in Xenopus oocytes (Cote et al., 1999; Schnapp, 1999) and regulation of polyadenylation (Moreira et al., 1998). In addition, PTB has been shown to mediate the export of hepatitis B virus (HBV) unspliced mRNA from the nucleus (Zang et al., 2001). Cytoplasmic PTB has been shown to have an effect on viral and cellular IRES-dependent translation of RNA (Belsham and Sonenberg, 1996). For example, it has been shown that encephalomyocarditis virus (EMCV) and foot-and-mouth disease virus (FMDV) IRES-dependent translation is abolished by the depletion o f 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PTB from the rabbit reticulocyte lysate (RRL) or HeLa cell lysate (Hellen et al., 1994; Hellen et al., 1993; Kaminski et al., 1995). In contrast, PTB can inhibit IRES- dependent Bip mRNA translation (Kim et al., 2000). Thus, PTB may enhance or inhibit translation, depending on the type of IRES. Although the mechanism by which PTB have an effect on translation is not yet clear, it is probable that PTB may affect the IRES structure or may interact with some general or specific translation factors. MHV belongs to the Coronaviridae family and contains a single-stranded, 31 kb, positive-sense RNA (Lai and Cavanagh, 1997). MHV RNA replication and transcription are carried out in the cytoplasm, and mediated by its own RNA- dependent RNA polymerase and probably other viral and cellular proteins. Although the exact mechanism by which viral transcription, replication and translation of viral RNA are regulated is not known, a number of evidence suggest that several cis-and trans-acting RNA elements, including the untranslated regions of viral RNA, and cellular proteins are involved in these processes. In case of MHV, these cis-acting elements include the intergenic sequence (Makino et al., 1991), leader sequence (Zang et al., 2001) and 3’-UTR (Lin et al., 1996) of viral RNA. While the leader RNA can function in RNA synthesis both in cis and trans (Zhang et al., 1994), several trans-acting factors, such as viral and cellular proteins, can bind to this region (Furuya and Lai, 1993) Numerous cellular proteins have been known to function as frara.v-regulatory factors for viral replication, transcription and translation (Lai, 1998). In poliovirus, 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. La autoantigen (Meerovitch et al., 1989) binds to IRES and enhances viral RNA translation in in vitro translation systems. Another cellular protein, poly(C)-binding protein (PCBP) also binds to a 5’-cloverleaf structure, inducing the circularization of poliovirus RNA in concert with polyA-binding protein (PABP) and 3 CD viral protein. This circular RNP complex has been shown to be required for negative- strand RNA synthesis, thus affecting viral replication (Herold and Andino, 2000; Herold and Andino, 2001). In addition, Sam68 interacts with RNA-dependent RNA polymerase 3D (McBride et al., 1996), and nucleolin binds to 5’-UTR and 3’-UTR of the poliovirus RNA (Izumi et al., 2001; Waggoner and Samow, 1998), though the exact role of Sam68 and nucleolin in viral replication is not yet clear. For MHV, several cellular proteins have been identified to bind to the untranslated regions of viral RNA by UV crosslinking methods (Furuya and Lai, 1993). These include PTB (Li et al., 1999), hnRNP A l (Li et al., 1997), mitochondrial aconitase (Nanda and Leibowitz, 2001), polyA-binding protein (PABP) (Spagnolo and Hogue, 2000) and several other unidentified proteins. PTB binds to the UCUAA repeats sequence within the leader and the sequence complementary to the 3’-UTR (c3’-UTR) at two different sites (Huang and Lai, 1999; Li et al., 1999). The PTB binding induced a conformational change in RNA structure (Huang and Lai, 1999). Site-directed mutagenesis of the PTB-binding site in either 5’-leader or c3’-UTR inhibited the replication and transcription of MHV genomic and DI RNA, suggesting that PTB may play a role in regulating viral RNA synthesis. In contrast, hnRNP A l binds to the leader and intergenic sequence (IG) of 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. negative strand and 3’ UTR region of positive strand (Furuya and Lai, 1993; Li et al., 1997). Similar to PTB, site-directed mutagenesis of IG sequence, which causes reduced binding of hnRNP A l, also inhibited the transcription of MHV DI RNA (Li et al., 1997; Zhang and Lai, 1995). The effect of hnRNP A l on MHV RNA transcription was further confirmed in cell lines expressing a dominant-negative mutant of hnRNP A l (Shi et al., 2000). Although MHV can replicate in cell lines deficient in hnRNP A l (Shen and Masters, 2001), recent studies showed that other hnRNP A 1-related proteins substitute for the functions of hnRNP A l in these cell lines (Shi et al, unpublished). While the previous studies imply that PTB may play a role in viral RNA synthesis, there is no direct evidence that PTB is involved in viral replication in virus-infected cells. In order to address this issue, I generated stable cell lines that express the wild-type PTB and a truncated form of PTB with deletion of the C- terminus, which function in a dominant-negative manner for viral RNA synthesis. The over-expression of this mutant PTB caused severe inhibition of both viral RNA and viral protein synthesis. I further established that the primary effect of the mutant PTB was the inhibition of viral RNA synthesis, but not the direct translation of viral RNA. These studies established that PTB may participate in viral RNA synthesis probably by interacting with the viral replication complex. 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3 Material and Methods Cells and Virus DBT cell (Hirano et al., 1974), a mouse brain astrocytoma cell line, was cultured in Eagle’s minimal essential medium (MEM) supplemented with 7% newborn calf serum (NCS), 10% tryptose phosphate broth, and 1% streptomycin- penicillin. MHV-JHM was amplified in DBT cells and maintained in MEM with 1% NCS. Establishment of stable cell lines DBT cells were transfected with pcDNA3.1-FLAG-F-PTB, pcDNA3.1-FLAG- N-PTB or pcDNA3.1 empty vector using DOTAP reagents (Boehringer Mannheim, Indianapolis, IN). After 4 hmy, the transfected cells were incubated in the presence of 500ug/ml G418 (Omega Scientific, Tarzana, CA) for 10 days. Individual surviving cells were selected and cultured for two weeks before screening for the expression of proteins. Antibodies The mouse hybridoma cell, BB7, which produces anti-PTB monoclonal antibody, was purchased from ATCC (Manassas, VA). The hybridoma cells were cultured for 3 days and antibodies were collected from the supernatant and purified by T-gel purification kit (Pierce, Rockford, IL). The monoclonal antibodies against 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VSV-G protein and actin, and the polyclonal antibody against FLAG were purchased from Sigma (St. Louis, MO). Immunoprecipitation and RPA PTB-overexpressing stable cell lines were cultured in 10-cm plates. At 80% confluency, cells were infected with MHV-JHM, at an M.O.I. of 10. At 8 hour p.i., cells were rinsed with PBS once and lysed with Buffer K (20mM Tris-HCl pH7.5, lOOmM NaCl, 0.2mM EDTA pH8.0), followed by incubation on ice for 30 minutes. After incubation, samples were further passed through the G21 needle five times and centrifuged at 4000 rpm for 5 minutes. The supernatant was incubated with the antibody for 2 hours at 4°C and then, protein A sepharose beads (Zymed Laboratories Inc, San Francisco, CA) were added into the supernatants. Following a 2-hour incubation, the beads were washed 5 times with buffer K and then used for RNA extraction by incubating with RNA elution buffer (0.3M Na acetate pH 5.2, 0.2% SDS, ImM EDTA pH 8.0, proteinase K lOug/ml) for 10 minutes at 65°C. The eluted RNA was further extracted with phenol/chloroform (Ambion, Austin, TX) and then precipitated with ethanol. To prepare RPA probe, 5’-UTR region (nt 77-327 from the 5’-end) plus 38 extra nucleotides containing the T7 promoter was amplified from the purified MHV-JHM viral RNA by RT-PCR; the PCR product was used directly for in vitro transcription. In vitro transcription and RPA was done using RPA III ™ kit from Ambion (Austin, TX). 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Immunofluorescence staining Cells were fixed with 4% formaldehyde for 15 minutes at room temperature and permeabilized with ice-cold acetone for 5 minutes at -20°C. Then, cells were washed three times with PBS and incubated with the primary antibody, diluted in 5% BSA/PBS buffer, for 1 hour at room temperature. After washing with PBS three times, cells were incubated with FITC-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA) for 1 hour and mounted. GST pull-down assay GST-PTB was immobilized onto the glutathione sepharose beads (Pierce, Rockford, IL). In vitro translated, S3 5 -labeled proteins were added and incubated for 2 hour at 4°C in GST-binding buffer (40mM HEPES pH 7.5, lOOmM KC1, 20mM 2-mercaptoethanol) containing 0.3% NP 40. After binding, beads were washed with the same buffer containing five times and the bound proteins were eluted, run on 10% SDS-PAGE and detected by autoradiography. [3 H]-uridine labeling of viral RNA RNA labeling was performed as described previously (Shi et al., 2000). In brief, cells were infected with MHV-JHM at an M.O.I. of 2 and cells were treated with 5 ug/ml Actinomycin D for one hmy prior to [3 H]-uridine labeling. At various time points (1,9, 11, 13, 15, and 24 hmy), [3 H]-uridine (lOOuCi/ml) (NEN, Boston, MA) was added into the medium and incubated for one hour. Cytoplasmic extract was 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. prepared and spotted on 3-mm paper and washed with 5% trichloroacetic acid (TCA). The paper was counted using a Beckman Scintillation counter. CAT assay of DI RNA Transcription assay of 25 CAT RNA was performed as described previously (Liao and Lai, 1994). Briefly, plasmid p25CAT was linearized by X bal and transcribed by T7 RNA polymerase. DBT cells were infected with JHM and at 1 hour p.i., transfected with 5ug of 25CAT RNA using DOTAP. After an 8-hour incubation, cells were collected and freeze/thawed three times. After centrifugation at 12,000 rpm for 10 minutes to remove cell debris, the supernatant was used for CAT assay as described previously (Lin et al., 1996). Immunodepletion and in vitro translation To coat protein A-sepharose beads with antibodies, beads were first rinsed with PBS containing BSA (O.lmg/ml) three times. Fifty microliters of the beads (50% slurry) were then incubated with lOul of mouse monoclonal antibody against PTB (BB7) for 2 hour at 4°C, and unbound antibody was washed away from the beads. The antibody-coated beads were added into 200ul rabbit reticulocyte lysate (RRL) (Promega, Madison, WI) and incubated for 2 hour at 4°C. Beads were removed from RRL and the depletion procedure was repeated three times. After depletion, 3ul RRL was used for immunoblotting and 20ul RRL was used for in vitro translation. 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For in vitro translation, the in vitro transcribed, capped DI RNA was generated using mMessage machine kit from Ambion (Austin, TX). In vitro translation was performed according to the manufacturer’s instruction (Promega, Madison, WI). In vitro translated proteins were run on 10% SDS-PAGE and signals were detected by autoradiography. Construction of MHV-UTR-LUC, RNA transfection and luciferase assay pGL3-basic vector (Promega, Madison, WI) was used as a luciferase reporter. MHV 5’-UTR (184nt) plus 54 nucleotides from the N-terminus of ORFla and 3’- UTR (400nt), including poly (A) tail, were amplified from the purified MHV-JHM RNA by RT-PCR and cloned into pGL3-basic vector. The construct was linearized with E coR V prior to in vitro transcription. In vitro transcribed, LUC DI RNA (5ug) was transfected into the cells using DMRIE-C reagent (Invitrogen, Carlsbad, CA). At 8 hour post-transfection, cells were harvested and used for luciferase assay, using luciferase assay system from Promega (Madison, WI). For translation study in MHV-infected cells, cells were infected with MHV- JHM and at 1 hour p.i., cells were transfected with 5ug of LUC DI RNA and luciferase activity was assayed as above at 8 hour post-transfection. 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.4 Results PTB binds to the 5’-untranslated region of MHV RNA in virus-infected cells. PTB was identified as a UV-crosslinked protein to MHV RNA using uninfected cell extracts in in vitro crosslinking reactions (Furuya and Lai, 1993). Several subsequent in vitro binding studies have shown that PTB binds to the 5’-leader sequence and the sequence complementary to the 3’-UTR of MHV RNA (Huang and Lai, 1999; Li et al., 1999). To establish that PTB indeed binds to viral RNA in the virus-infected cells, I immunoprecipitated PTB from the MHV-infected cells; the viral RNA was then detected by RNase protection assay using a 5’-UTR-specific probe. I used a DBT stable cell line that overexpresses a FLAG-tagged PTB (see below) to increase the amount of PTB for easier detection (Fig 2-1). The control experiments showed that MHV RNA was detected in the virus-infected cell lysates by this probe, while no protected band was detected in the uninfected cells (Fig 2-1, lanes 2 and 3). Immunoprecipitation of PTB with either anti-PTB antibody or anti- FLAG antibody also pulled down the viral RNA (Fig 2-1, lanes 5 and 6). Anti-VSV- G protein or anti-actin antibodies yielded only background signals similar to that using the bead only (lanes 4, 8 and 9). Anti-N antibody also precipitated the viral RNA, consistent with the finding that N protein binds to the 5’-UTR of MHV RNA (Stohlman et al., 1988) (Fig 2-1, lane 7). These results indicate that PTB does bind to MHV RNA in the virus-infected cells. 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 270nt 250nt Fig. 2-1. PTB binds to MHV RNA in the PTB-overexpressing cell line. Total RNA from uninfected and MHV-JHM-infected DBT cells were directly used for RPA using a negative strand 5’-UTR probe (270nt) (lanes 2 and 3). Cytoplasmic extract from MHV-infected DBT cells was immunoprecipitated with beads only (lane 4) or specific antibodies (lane 5-9) before RPA. The predicted size of the protected band is 250 nt long. Lane 1 indicates 1% unhybridized probe without RNase digestion. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PTB interacts with the viral N protein in vitro and in vivo Next, I asked whether PTB interacts with viral proteins in the virus-infected cells. Since N protein binds to 5’-UTR, and PTB also does so, I hypothesized that there may be an interaction between PTB and N proteins. I first tested in vitro interaction between PTB and N by GST pull-down assay (Fig 2-2A). In vitro translated, 35S-methionine-labeled N protein was pulled down by GST-PTB, but not by GST (Fig 2-2A, lanes 2 and 3). Under the same conditions, N protein was also pulled down by GST-hnRNP A l, confirming the previous report (Wang and Zhang, 1999) (Fig 2-2A, lane 4). I further examined whether this interaction occurred in the virus-infected cells. PTB-overexpressing DBT cells were infected with MHV; at 8 hour p.i., cytoplasmic extracts were immunoprecipitated with anti-PTB antibody, followed by immunoblotting with anti-N antibody to detect the N protein (Fig 2-2B). In accord with the in vitro GST pull-down assay, N was specifically coimmunoprecipitated with PTB, proving that PTB interacts with N protein in the virus-infected cells (Fig 2-2B, lane 5). Under the same conditions, neither anti-VSV-G protein antibody nor bead alone brought down the N protein (Fig 2-2B, lanes 3 and 6). Interaction between endogenous PTB and N protein was also confirmed in PTB- overexpressing cell line (data not shown). These results suggest that PTB may form a ribonucleoprotein complex with viral RNA, N protein and other viral and cellular proteins. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 a c I — w C D Q Q I- 0- I h w C D C / > C D ■4- N p r o te in B a IP c ------------------------ aVSVg aPTB | -------------- .a ■4- N p r o te in Fig. 2-2. Interaction between PTB and N in vitro and in PTB- overexpressing cell line A. In vitro binding between PTB and N. GST (lane 2), GST-PTB (lane 3) and GST-hnRNP A1 (lane 4) were incubated with 3 5 S-labeled, in vitro translated N protein. After washing, samples were separated by 10% SDS-PAGE, and signals were detected by autoradiography. Lane 1 indicates 10% of input N protein. B. In vivo binding between PTB and N. Cytoplasmic extract from MHV-JHM-infected DBT cells was immunoprecipitated with beads (lane 6), anti-PTB antibody (lanes 4 and 5), and anti-VSV-G protein antibody (lane 2 and 3). The immunoprecipitated proteins were separated by 10% SDS-PAGE, and detected by immunoblotting with polyclonal MHV anitbody (1:3000 dilution). Lane 1 indicates 10% input of the total lysates. 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Analysis of stable cell lines that express wild-type and truncated PTB To assess the function of PTB in MHV replication, I generated several stable cell lines expressing FLAG-tagged wild-type PTB (F-PTB) and C-terminus- truncated PTB (N-PTB) in murine DBT cell line. The truncated form of PTB includes only the N-terminal two RRM motifs (RRM 1 and RRM 2), which is responsible for the oligomerization of PTB and contains the signal for nucleocytoplasmic shuttling, but lacks the C-terminal half, which is required for the RNA-binding activity of PTB (Oh et al., 1998) (Fig 2-3A). Both wild-type PTB (57kDa) and the truncated form of PTB (29kDa) were well expressed in the stable cell lines, as shown by immunoblotting with anti-FLAG antibody (Fig 2-3B). The expression level of N-PTB was slightly higher than F-PTB, probably due to the difficulty in extracting proteins from the nucleus, where most of F-PTB resides. Over-expression of wild-type PTB and N-PTB did not affect the cell growth kinetics (Fig 2-3C). Overexpressed N-PTB localizes predominantly in cytoplasm and can bind to F- PTB in vitro Next, I determined the subcellular localization of the overexpressed wild-type PTB and truncated PTB. Immunofluorescence staining showed that F-PTB was localized in the nucleus, similar to the endogenous PTB, while N-PTB was predominantly in the cytoplasm, though a small fraction was observed in the nucleus (Fig 2-3D). Thus, in the cytoplasm, the amount of the overexpressed N-PTB 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 0 60 152 238 305 429 496 546 614 621 F-PTB NLS RRM1 RRM2 RRM3 RRM4 k - PTB dimerization -J RNA binding ► 0 60________________ 152 238______________ 305 NLS RRM1 RRM2 B DBT-VEC DBT-F-PTB DBT-N-PTB 68- 4 3 - 2 9 - c hour Fig. 2-3-1. Characterization of cell lines overexpressing wild- type PTB and truncated PTB A. Structure of the wild-type PTB and truncated PTB. RRM: RNA recognition motif. B. Protein expression of wild-type PTB and truncated PTB in the stable cell lines. Lysates were used for immunoblotting with anti-FLAG antibody from vector control cell (lanes 1 and 2), F-PTB (lanes 3 and 4) and N-PTB (lanes 5 and 6)-overexpressing cell lines. C. Growth kinetics of vector (o), F-PTB (□) and N-PTB (▲). Same number of cells (105) were seeded onto 10-cm plates and counted every 24 hours. 50 M F-PTB < N-PTB i > i : » > 7.00E+0& 6.00E+06 -e -V E C -a-F -P T B N-PTB 5.00E+06 4.00E+061 3.00E+0& 2.00E+0& 0.00E+0I 0 /1 A Q 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E G S T 1 0 % i n p u t G S T -P T B ffl h- Q . C Q I- 0. C Q I- Q . C Q I- Q . I Z C Q I- Q . I LL C Q I- Q . 4 5 6 1 2 3 Fig. 2-3-2. Characterization of cell lines overexpressing wild- type PTB and truncated PTB D. Localization of F-PTB and N-PTB in stable cell lines. Cells were stained with anti-FLAG antibody. E. In vitro binding between F-PTB and N-PTB. S3 5 _ labeled, in vitro translated F-PTB or N-PTB were incubated with GST (lanes 1 and 2) or GST-PTB (lanes 5 and 6) and separated by 12% SDS-PAGE. Lanes 3 and 4 indicate 10% input of the in vitro translated F- PTB and N-PTB. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. likely far exceeds the amount of the endogenous PTB, rendering it likely that N- PTB could affect the cytoplasmic functions of PTB. I hypothesized that the truncated PTB may form a dimer with the endogenous PTB, resulting in disruption of the normal function of cytoplasmic PTB. To examine this possibility, I carried out an in vitro binding experiment to test the interaction of N-PTB with F-PTB. As seen in Fig 2-3E, GST-PTB binds to both wild-type F-PTB and N-PTB, though the binding ability of N-PTB was slightly weaker than F-PTB. The low protein binding ability of N-PTB is consistent with the report that, even though RRM2 domain contributes the most to PTB dimerization, other RRM domains help the stabilization of the dimer (Perez et al., 1997). Nonetheless, because of the high expression level of N-PTB in the cytoplasm, it is expected to sequester the endogenous, cytoplasmic PTB by forming a dimer with F-PTB and influence the normal functions of PTB in the cytoplasm. Syncytia formation and virus production are retarded in PTB-overexpressing stable cell lines I next examined the possible effects of F-PTB and N-PTB on virus replication. To begin with, I investigated the morphological changes induced by MHV infection in these stable cell lines (Fig 2-4A). Compared with the vector control cells, the N- PTB stable cell line showed significantly delayed syncytia formation at every time points p.i., especially at the earlier time points, indicating that N-PTB functions in a dominant-negative manner in viral replication. Surprisingly, overexpression of F- 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A DBT-VEC DBT-F-PTB DBT-N-PTB B 6 o T - 0) o VEC F-PTB N-PTB Q . 0 20 25 5 10 15 Hours post infection Fig. 2-4. Inhibition of syncytia formation and virus production in the stable cell lines. A. Vector, F-PTB-and N-PTB-overexpressing cell lines were infected with MHV-JHM, and syncytia formation was observed at various time points (7, 15 and 24 hour p.i.). B. Virus titers were measured at various time points (7, 9, 14 and 25 hour) after infection. 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PTB also caused delayed syncytia formation, although to a lesser level than N-PTB. The dominant-negative effect of F-PTB is similar to the “squelching effect” commonly observed with the overexpression of transcription factors (Katja and Hurt, 2001); thus, overexpression of PTB may impair the virus replication in a dominant-negative manner by titrating out some factors involved in virus replication. Similar to cellular morphology, virus production also was inhibited by nearly 100 fold in N-PTB stable cell line at 7-15 hours p.i. and lesser inhibition in F-PTB stable cell line at same time points (Fig 2-4B). At 25 hr p.i., the virus titer from the PTB-overexpressing cell lines became higher than the vector control cells, probably due to the fact that the control cells have already completely lysed; thus, the virus particles could have been inactivated during the long incubation period. The synthesis of viral RNA and proteins are inhibited in the F-PTB and N-PTB stable cell lines Next, I examined the effect of F-PTB and N-PTB on viral RNA and protein synthesis. For viral RNA synthesis, cells were labeled with [3 H]-uridine for one hmy in the presence of antinomycin D (Fig 2-5A). In vector control cells, viral RNA synthesis started to increase from 6 hours p.i., and peaked at 10 hours p.i.. In contrast, in both F-PTB- and N-PTB-expressing stable cell lines, maximum incorporation of [ H]-uridine occurred at 12 hour p.i., and the amounts of incorporated uridine were at least 2.5-fold less than that in vector control cells. This 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3000 & 2500 .E | 2000 | 1 1500 4- a 1000 X u 8 500 c o ~ V e c t o r =— P T B -F u ll PTB -N 10 15 h o u r 30 B 15h 2 4 h DBT-VEC DBT-Full PTB DBT-N-PTB co in C M £ 0 0 T l- ^ N protein Fig.2-5. Inhibition of viral RNA and protein synthesis in the stable cell lines. A. Viral RNA synthesis was determined by [3H]-uridine incorporation from the various cell lines. At various times p.i., cells were pretreated with 5 ug/ml Actinomycin D, and labeled with [3H]-uridine for 1 hour. TCA-precipitable counts were determined. B. Northern blotting analysis of viral RNAs at various time points (7, 15 and 24 hour) p.i.. 32P-labeled antisense mRNA 7 was used as a probe. Numbers indicate various MHV RNA species. C. Synthesis of viral N protein from various cell lines. Immunoblotting was done using a monoclonal anti-N antibody. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. delayed and reduced uridine incorporation in the F-PTB and N-PTB stable cell lines indicated that overexpression of PTB may affect directly viral RNA synthesis. To further determine whether all viral RNA species were affected by the overexpression of F-PTB and N-PTB, northern blotting experiments were performed. The results showed that all of the MHV RNAs were reduced in the F- PTB and N-PTB stable cell lines at 7 hours p.i. as compared with the vector control cells (Fig 2-5B). At 15 hours p.i., the amounts of all viral RNA species increased in the F-PTB and N-PTB stable cell lines, but were still less than that in the control cells. At 24 hours p.i., only a trace amount of viral RNAs was detected in vector control cells due to the loss of the dead cells. In combination with [H3 ]-uridine incorporation experiment, this result showed that viral RNA synthesis is inhibited in both F-PTB and N-PTB stable cell lines. I further examined the effect of PTB on viral protein synthesis. I examined the expression of N protein by immunoblotting at various time points (Fig 2-5C). N proteins were detected from 7 hours p.i., and peaked at 14 hours p.i. in the vector control cells. However, in F-PTB and N-PTB stable cell lines, the detection of N proteins began later (visible at 9 hours p.i. in F-PTB stable cell line and at 14 hours p.i. in N-PTB stable cell line) and peaked at 24 hours p.i.. From these experiments, I concluded that PTB affects both viral RNA and protein synthesis either directly or indirectly. PTB directly regulates viral RNA transcription, but not viral translation 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B > 120 25CAT DI RNA IG CAT * 25CAT mRNA * CAT B 100 N-PTB VEC F-PTB Fig. 2-6. Transcriptional effect of PTB A. Organization of 25CAT DI RNA. L: leader sequence, IG: MHV intergenic sequence, CAT: chloramphenicol acetyl transferase. B. CAT activity in the vector control, F-PTB and N-PTB-overexpressing cell lines. MHV DI 25CAT RNA was transfected into MHV-JHM-infected stable cell lines, and CAT activity was determined at 8 hours post-transfection. Error bars represent average standard deviations. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The finding that the overexpression of PTB retarded both viral RNA and protein synthesis could be due to the possibilities that PTB is directly involved in both viral transcription and translation or that PTB affects transcription, which, in turn, affects translation, or vice versa. To distinguish these possibilities, I designed experiments to examine transcription/replication and translation separately. First, I assessed the transcriptional effect of PTB using a DI reporter RNA, 25CAT RNA (Liao et al., 1995), which can not be translated unless a subgenomic mRNA from the inserted intergenic sequence is made in the presence of helper virus; thus, the expression of CAT mirrors the transcription of CAT RNA (Fig 2-6A). PTB-overexpressing stable cell lines were infected with MHV and transfected with 25 CAT RNA (Fig 2-6B). At 8 hours p.i., CAT activity was 4 fold lower in N-PTB stable cell line than in control cells. In F-PTB stable cell line, CAT activity was also inhibited, though to a lesser extent. Next, I examined whether PTB directly affected viral translation. For this purpose, I performed in vitro immunodepletion of PTB from the rabbit reticulocyte lysate (RRL) (Ali and Siddiqui, 1995; Hellen et al., 1993); the immunodepleted lysates were used for translation of a natural DI RNA DE25 (Makino et al., 1988) (Fig 2-7A, top diagram), in which part of ORFla is fused to ORF7 encoding the N protein. Different from 25CAT RNA, the DE 25 RNA can be translated directly from the DI RNA without mRNA transcription. RRL was incubated with beads coated with anti-PTB antibody. Immunoblotting showed that endogenous PTB was completely depleted from the lysate (Fig 2-7A, bottom, left). In vitro translation of 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MHV-5’UTR 0RF1a DE25 RNA MHV-3’UTR N ^ Translation 0RF1a-N fusion protein In vitro translation WB O ' O' Nuclease-treated a a > ■ a _i c _j a 0! ° 0 £ ® o ' ? o r 9 N uclease-u ntreated O' o' a a > T 3 cc ? _ l a- a : ® o' 9 ORF1a- N protein < PTB Fig. 2-7-1. Translational effect of PTB A. In vitro translational effect of PTB. Diagram shows the structure of DE25 RNA. After depleting endogenous PTB from RRL, lysates were used for immunoblotting (bottom panel) and in vitro translation of DE25 DI RNA (top panel). Right one is from nuclease-treated lysates and left panel is from nuclease-untreated lysates. 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25CAT DI RNA in this lysate showed that PTB depletion did not affect translation. Since this RRL had been pre-treated with nuclease to remove most of endogenous cellular RNA, thus allowing exclusive translation of the exogenous RNA, it is possible that PTB is not required when translation occurs in the absence of competing cellular mRNA. To examine this possibility, I tested another RRL without prior nuclease pretreatment so that the viral RNA has to compete with cellular mRNA. Under this condition, PTB depletion also did not have any effect on the DI RNA translation (Fig 2-7A, bottom, right). I further tested the possible translational effect of PTB in the stable cell lines. For this purpose, I constructed another reporter DI RNA, which contains the authentic MHV 5’-and 3’-UTR and expresses luciferase as a reporter (Fig 2-7B, upper diagram). The luciferase ORF is fused with ORFla, so that the translation of luciferase mimics closely that of the natural MHV RNA. This RNA was transfected into the F-PTB and N-PTB stable cell lines; at 8 hours post-transfection, luciferase activity was assayed using the transfected cell line lysates (Fig 2-7B, bottom). No difference in luciferase activity was observed between the vector control cells and the stable cell lines expressing F-PTB or N-PTB. In addition, I expressed F-PTB and N-PTB transiently in 293A cells, followed by the transfection of MHV-UTR- LUC RNA; again, no difference in luciferase activity was observed (data not shown). Based on the in vitro and in vivo results, I concluded that PTB does not affect MHV translation directly. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B MHV UTR - LUC MHV-5’UTR T 7 ^ Luciferase MHV-3’UTR E * 3 5 ‘E ;> u < 0 0 ) (A ( 0 O 3 E * 3 5 ’E 3 > 3 O re a > (/> re o 3 ORF1a 18a.a. 80000 70000 60000 50000 40000 30000 20000 10000 0 80000 70000 60000 50000 40000 30000 20000 10000 0 I Translation LUCIFERASE ■ f V E C F - P T B s t a b l e c e l l l i n e N - P T B V E C F - P T B M H V i n f e c t e d s t a b l e c e l l l i n e N - P T B Fig. 2-7-2. Translational effect of PTB B. In vivo translational effect in the stable cell lines. Diagram shows the structure of MHV-UTR-LUC construct. In vitro transcribed LUC DI RNA were transfected into the various cell lines, and the lysates were subject to luciferase activity assay at 8 hour post-transfection. C. In vivo translation in MHV-infected stable cell lines. Cells were infected with MHV-JHM and, at 1.5 hours p.i., transfected with LUC DI RNA. Luciferase activity was assayed at 8 hour post transfection. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To examine whether PTB affects MHV RNA translation in the context of MHV infection, I further performed the translation study in the MHV-infected PTB stable cell lines. F-PTB- and N-PTB-overexpressing cell lines were infected with MHV and then transfected with MHV-UTR-LUC RNA. Even in the presence of MHV infection, I did not observe any difference in luciferase activity (Fig 2-7C), indicating that PTB has no effect on MHV translation. Combining the transcription and translation experiments, I conclude that the effects of PTB overexpression on MHV RNA synthesis were not due to the indirect effect of inhibition of viral protein synthesis. Therefore, PTB plays a direct role in MHV RNA synthesis. 2.5 Discussion My previous studies have shown that PTB binds to the 5’-UTR of both positive and negative strand of MHV RNA and that PTB binding requires the UCUAA repeats sequence of the 5’-UTR of the positive strand RNA, and its binding to the negative-strand RNA caused conformational change of the RNA (Huang and Lai, 1999; Li et al., 1999). Furthermore, RNA mutations that interfered with PTB binding reduced the transcription of the DI RNA, suggesting that PTB may regulate MHV RNA transcription and replication. In this study, I used dominant-negative mutants of PTB to demonstrate the direct involvement of PTB in viral RNA synthesis. I showed that the expression of a dominant-negative mutant of PTB 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inhibited viral RNA synthesis, but did not affect viral RNA translation. Thus, the primary effect of PTB appears to be on viral RNA synthesis. Combined with the finding that PTB interacts with viral N protein, which also binds to 5’-UTR (Stohlman et al., 1988) and may modulate viral RNA synthesis (Compton et al., 1987), my findings suggest that PTB is complexed with the viral transcription/replication machinery. This may represent another new cytoplasmic function of PTB in the cells. Despite the binding of PTB to 5’-UTR of MHV genomic RNA, my study showed that PTB is not directly implicated in MHV translation. The 5’-UTR has been shown to regulate the translation of MHV RNA; specifically the N protein binding to leader sequence of 5’-UTR enhanced the translation of MHV, indicating that 5’-UTR is necessary for efficient MHV translation (Tahara et al., 1998). However, since genomic and subgenomic mRNAs of MHV are capped, polyadenylated and translated by a cap-dependent translation pathway, it may not need noncanonical factors for the efficient translation. Nevertheless, I could not rule out the possibility that PTB may regulate MHV translation under certain conditions. In influenza A virus, 5’-UTR of viral RNA is not necessary for the efficient translation (Cassetti et al., 2001). In this study, I established stable cell lines overexpressing F-PTB or N-PTB, and showed that the truncated form of PTB functions in a dominant-negative manner in MHV RNA synthesis. There are several possible mechanisms for the dominant- negative effects of the truncated form of PTB. First, since this truncated form has 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. only RRM1 and 2, which are responsible for dimerization and protein-protein interaction, N-PTB may interact with the wild-type PTB, resulting in the dysfunction of the endogenous PTB. My in vitro binding experiment between F- PTB and N-PTB supports this possibility (Fig 2-3E). Secondly, N-PTB may sequester other RNA-binding proteins, such as hnRNP A l and other un-identified proteins, which may be involved in MHV transcription/replication. The third possibility is that the overexpressed N-PTB may bind to viral RNA, replacing the binding of cytoplasmic, endogenous PTB, since N-PTB retains a weak RNA- binding activity (Oh et al., 1998). The fourth possibility is that N-PTB may interact with N proteins, thus blocking the interaction between the endogenous PTB and N proteins. In any case, the amount of the overexpressed N-PTB in the cytoplasm likely far exceeds the amount of the cytoplasmic, endogenous of PTB, enough to disturb the normal function of PTB in the cytoplasm. On the other hand, it is puzzling why the wild-type PTB also functions in a dominant-negative manner, despite the fact that the overexpressed PTB binds to viral RNA fairly well (Fig 2-1). It is possible that the recombinant PTB, which has a FLAG tag, may be less efficient in its biological activity than the endogenous PTB. Furthermore, overexpression of PTB may titrate out one or more replication components, thus resulting in the inhibition of replication/transcription, in a mechanism akin to the “squelching effects” frequently observed for the overexpressed transcription factors. It is intriguing to note that overexpression of a truncated form of hnRNP A l also inhibited MHV RNA synthesis, but overexpression of the full-length hnRNP A l 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. accelerated MHV RNA synthesis (Shi et al., 2000). This difference may reflect the different abundance and protein-interacting properties of these two hnRNPs. Multiple hnRNP A 1-related proteins exist, most of which can interact with MHV RNA in a similar mechanism to hnRNP A l (Shi et al., unpublished data). Thus, hnRNP Al may be more difficult to titrate out. The mechanism by which PTB shuttles between the nucleus and cytoplasm is still not clear. Mapping studies have shown that both NLS and NES are confined to the N-terminus of PTB, and overlap in sequence. However, it is unlikely that the import/export of PTB is determined by the primary sequence alone. Considering that N-PTB has both NLS and NES and predominantly localizes in the cytoplasm, I speculate that RRM 3 and 4 domains may affect the nuclear localization by either changing the overall protein conformation or RNA-binding properties to favor nuclear localization. Given that a truncated mutant of PTB consisting of only RRM3 and RRM4 also localizes in the cytoplasm (unpublished data), it is likely that the localization of PTB in the cells is determined by multiple factors besides the NLS/NES signals, such as neighboring sequence, RNA-binding properties, and interaction with import/export machinery. Though it is not clear how much PTB exists in the cytoplasm in the static state, it is possible that in certain situations, such as viral infection or transcription inhibition, PTB may relocalize from the nucleus to cytoplasm to carry out its cytoplasmic functions. Recently, it has been shown that actinomycin D treatment or poliovirus infections induced the movement of PTB from the nucleus to cytoplasm 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Back et al., 2002). I have also studied PTB relocalization during MHV infection, but I could not demonstrate unequivocally the relocalization of PTB by immunofluorescence staining or immunoblotting methods. One possible explanation is that the existing cytoplasmic PTB is sufficient for the function of PTB on MHV transcription/replication; alternatively, my immunostaining and immunoblotting procedure may not be sensitive enough to detect the subtle movements of PTB. So far, there have been at least four cellular proteins bound to the 5’-and 3’- UTR o f MHV RNA and participate in MHV RNA synthesis. These are hnRNP A l, PTB, mitochondrial aconitase (Nanda and Leibowitz, 2001) and polyA-binding protein (Spagnolo and Hogue, 2000). Although a recent study using an hnRNP A l- deficient cell line claimed that hnRNP A l is not necessary for MHV replication (Shen and Masters, 2001), my recent study showed that, in this particular cell line, other hnRNP Al-related proteins substitute for hnRNP A l (Shi et al., unpublished data). In the current study, I further demonstrated the modulating role of PTB in MHV RNA synthesis. I propose several possible mechanisms for the involvement of PTB in MHV RNA transcription and replication, which are not mutually exclusive. First, PTB may facilitate the recruitment of the transcription/replication factors at the replication site, through the protein-protein-interacting domain. Second, PTB may change the conformation of viral RNA into one that is more favorable for replication and transcription, probably functioning as an RNA chaperon to unfold the highly structured untranslated region. This proposal also is supported by the 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. previous report that PTB binds to 5’-UTR of MHV negative-strand RNA, and alters its conformation (Huang and Lai, 1999). Finally, PTB may mediate the 5’-3’ crosstalk by interacting with hnRNP A l proteins (Huang and Lai, 2001) or its related proteins, causing the circularization of viral genome, which may help virus replication. This genomic circularization and its importance in viral RNA replication have been reported for poliovirus (Herold and Andino, 2001). More experiments should be done to prove that this circularization is also important for the MHV replication. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.6 References Ali, N., and Siddiqui, A. (1995). Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation. J. Virol. 69, 6367-6375. Back, S. H., Kim, Y. K., Kim, W. J., Cho, S., Oh, H. R., Kim, J. E., and Jang, S. K. (2002). Translation of polioviral mRNA is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3C(pro). J. Virol. 76, 2529-2542. Belsham, G. J., and Sonenberg, N. (1996). RNA-protein interactions in regulation of picomavirus RNA translation. Microbiol. Rev. 60, 499-511. Cassetti, M. C., Noah, D. L., Montelione, G. T., and Krug, R. M. (2001). Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5 'untranslated region. Virology 289, 180-185. Compton, S. R., Rogers, D. B., Holmes, K. V., Fertsch, D., Remenick, J., and Mcgowan, J. J. (1987). In vitro replication of mouse hepatitis virus strain A59. J. Virol. 61, 1814-1820. Cote, C. A., Gautreau, D., Denegre, J. M., Kress, T. L., Terry, N. A., and Mowry, K. L. (1999). A Xenopus protein related to hnRNP I has a role in cytoplasmic RNA localization. Mol. Cell 4, 431-437. Furuya, T., and Lai, M. M. C. (1993). Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA. J. Virol. 67, 7215-7222. Ghetti, A., Pinol-Roma, S., Michael, W. M., Morandi, C., and Dreyfuss, G. (1992). hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs. Nucleic Acids Res. 20, 3671-3678. Hellen, C. U., Pestova, T. V., Litterst, M., and Wimmer, E. (1994). The cellular polypeptide p57 (pyrimidine tract-binding protein) binds to multiple sites in the poliovirus 5' nontranslated region. J. Virol. 68, 941-950. Hellen, C. U., Witherell, G. W., Schmid, M., Shin, S. H., Pestova, T. V., Gil, A., and Wimmer, E. (1993). A cytoplasmic 57-kDa protein that is required for translation of picomavirus RNA by internal ribosomal entry is identical to the 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nuclear pyrimidine tract-binding protein. Proc. Natl. Acad. Sci. U S A 90, 7642- 7646. Herold, J., and Andino, R. (2000). Poliovirus requires a precise 5' end for efficient positive-strand RNA synthesis. J. Virol. 74, 6394-6400. Herold, J., and Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Mol. Cell 7, 581-591. Hirano, N., Fujiwara, K., Hino, S., and Matumoto, M. (1974). Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture. Arch. Gesamte Virusforsch. 44, 298-302. Huang, P., and Lai, M. M. C. (1999). Polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3' untranslated region, thereby altering RNA conformation. J. Virol. 73, 9110-9116. Huang, P., and Lai, M. M. C. (2001). Heterogeneous nuclear ribonucleoprotein al binds to the 3'-untranslated region and mediates potential 5'-3'-end cross talks of mouse hepatitis virus RNA. J. Virol. 75, 5009-5017. Izumi, R. E., Valdez, B., Banerjee, R., Srivastava, M., and Dasgupta, A. (2001). Nucleolin stimulates viral internal ribosome entry site-mediated translation. Virus Res. 76, 17-29. Kamath, R. V., Leary, D. J., and Huang, S. (2001). Nucleocytoplasmic shuttling of Polypyrimidine Tract-binding protein is Uncoupled from RNA export. Mol. Biol. Cell 12, 3808-3820. Kaminski, A., Hunt, S. L., Patton, J. G., and Jackson, R. J. (1995). Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA. RNA 1, 924-938. Katja, S., and Hurt, E. (2001). Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yralp. Nature 413, 648 - 652. Kim, Y. K., Hahm, B., and Jang, S. K. (2000). Polypyrimidine tract-binding protein inhibits translation of bip mRNA. J. Mol. Biol. 304, 119-133. Lai, M. M. C. (1998). Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology 244, 1- 12. 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lai, M. M. C., and Cavanagh, D. (1997). The molecular biology of coronaviruses. Adv. Virus Res. 48, 1-100. Li, B., and Yen, T. (2002). Characterization of the Nuclear Export Signal of Polypyrimidine Tract-binding Protein. J. Biol. Chem. 277, 10306-10314. Li, H. P., Huang, P., Park, S., and Lai, M. M. C. (1999). Polypyrimidine tract- binding protein binds to the leader RNA of mouse hepatitis virus and serves as a regulator of viral transcription. J. Virol. 73, 772-777. Li, H. P., Zhang, X., Duncan, R., Comai, L., and Lai, M. M. C. (1997). Heterogeneous nuclear ribonucleoprotein A l binds to the transcription-regulatory region of mouse hepatitis virus RNA. Proc. Natl. Acad. Aci. U S A 94, 9544-9549. Liao, C. L., Zhang, X., and Lai, M. M. C. (1995). Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase. Virology 208, 319-327. Lin, Y. J., Zhang, X., Wu, R. C., and Lai, M. M. C. (1996). The 3’ untranslated region of coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. J. Virol. 70, 7236-7240. Makino, S., Joo, M., and Makino, J. K. (1991). A system for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. J. Virol. 65, 6031-6041. Makino, S., Shieh, C. K., Soe, L. H., Baker, S. C., and Lai, M. M. C. (1988). Primary structure and translation of a defective interfering RNA of murine coronavirus. Virology 166, 550-560. Mcbride, A. E., Schlegel, A., and Kirkegaard, K. (1996). Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells. Proc. Natl. Acad. Sci. U S A 93, 2296-2301. Meerovitch, K., Pelletier, J., and Sonenberg, N. (1989). A cellular protein that binds to the 5'-noncoding region of poliovirus RNA: implications for internal translation initiation. Genes & Dev. 3, 1026-1034. Moreira, A., Takagaki, Y., Brackenridge, S., Wollerton, M., Manley, J. L., and Proudfoot, N. J. (1998). The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3' end formation by two distinct mechanisms. Genes c£ Dev. 12, 2522-2534. 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Nanda, S. K., and Leibowitz, J. L. (2001). Mitochondrial aconitase binds to the 3' untranslated region of the mouse hepatitis virus genome. J. Virol. 75, 3352-3362. Oh, Y. L., Hahm, B., Kim, Y. K., Lee, H. K., Lee, J. W., Song, O., Tsukiyama- Kohara, K., Kohara, M., Nomoto, A., and Jang, S. K. (1998). Determination of functional domains in polypyrimidine-tract-binding protein. Biochem. J. 331, 169- 175. Perez, I., Mcafee, J. G., and Patton, J. G. (1997). Multiple RRMs contribute to RNA binding specificity and affinity for polypyrimidine tract binding protein. Biochemistry 36, 11881-11890. Schnapp, B. J. (1999). A glimpse of the machinery. Current Biology 9, R725-727. Shen, X., and Masters, P. S. (2001). Evaluation of the role of heterogeneous nuclear ribonucleoprotein A l as a host factor in murine coronavirus discontinuous transcription and genome replication. Proc. Natl. Acad. Aci. U S A 98, 2717-2722. Shi, S. T., Huang, P., Li, H. P., and Lai, M. M. C. (2000). Heterogeneous nuclear ribonucleoprotein A l regulates RNA synthesis of a cytoplasmic virus. EMBO J. 19, 4701-4711. Spagnolo, J. F., and Hogue, B. G. (2000). Host protein interactions with the 3' end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication. J. Virol. 74, 5053-5065. Stohlman, S. A., Baric, R. S., Nelson, G. N., Soe, L. H., Welter, L. M., and Deans, R. J. (1988). Specific interaction between coronavirus leader RNA and nucleocapsid protein. J. Virol. 62, 4288-4295. Tahara, S. M., Dietlin, T. A., Nelson, G. W., Stohlman, S. A., and Manno, D. J. (1998). Mouse hepatitis virus nucleocapsid protein as a translational effector of viral mRNAs. Adv. Exp. Med. Biol. 440, 313-318. Waggoner, S., and Samow, P. (1998). Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells. J. Virol. 72, 6699-6709. Wagner, E. J., and Garcia-Bianco, M. A. (2001). Polypyrimidine tract binding protein antagonizes exon definition. Mol. Cell. Biol. 21, 3281-3288. Wang, Y., and Zhang, X. (1999). The nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein Al in vitro and in vivo. Virology 265, 96-109. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Zang, W. Q., Li, B., Huang, P. Y., Lai, M. M. C., and Yen, T. S. (2001). Role of polypyrimidine tract binding protein in the function of the hepatitis B virus posttranscriptional regulatory element. J. Virol. 75, 10779-10786. Zhang, X., and Lai, M. M. C. (1995). Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed. J. Virol. 69, 1637-1644. Zhang, X., Liao, C. L., and Lai, M. M. C. (1994). Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis. J. Virol. 68, 4738-4746. 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3 SYCNRIP, a novel MHV RNA binding protein, is involved in MHV RNA synthesis 3.1 Abstract Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5’- and 3’-untranslated region (UTR) of the viral RNA. Here, I identified another cellular protein, p70, which has been shown by UV-crosslinking to bind both the positive- and negative-strand UTR of MHV RNA specifically. I purified p70 by using a one-step RNA affinity purification procedure with biotin-labeled 5’-UTR. MALDI-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of hnRNP family and localizes largely in the cytoplasm. The p70 was crosslinked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to 5’-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of 5’-UTR, requiring the UCUAA repeats sequence. To investigate the functional significance of SYNCRIP in MHV replication, I expressed a full-length or a C-terminus-truncated form of SYNCRIP in mammalian cells 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expressing the MHV receptor. The overexpression of either form o f SYNCRIP inhibited syncytia formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP by using a specific siRNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein, potentially involved in viral RNA synthesis. 3.2 Introduction MHV belongs to the Coronaviridae family and contains a single-stranded, 31-kb, positive-sense RNA (Lai and Cavanagh, 1997). Viral genome is composed of a series of open reading frames (ORF1-7), flanked by untranslated regions (UTR) at the 5’- and 3’-ends. MHV RNA replication and transcription take place in the cytoplasm, and are mediated by its own RNA-dependent RNA polymerase and other viral and cellular proteins. Six to seven subgenomic mRNAs share 5’- and 3’-ends with the genomic RNA and are translated through a cap-dependent mechanism. Regulation of transcription, replication and translation of viral RNA involves several cis- and trans-acting RNA elements, and viral and cellular proteins. In the case of MHV, the cA-acting RNA elements include the intergenic sequence (Makino, Joo, and Makino, 1991), leader sequence (Zhang and Lai, 1995) and 3’-UTR (Lin et al., 1996) of viral RNA. The leader RNA can function in viral RNA synthesis both in cis 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and in trans in the virus-infected cells (Zhang and Lai, 1995). Several trans-acting factors, including viral and cellular proteins, have been shown to bind to this region (Furuya and Lai, 1993). Besides the cis- and trans-acting RNA elements, cellular proteins have been increasingly recognized to play important roles in virus replication, transcription and translation, as well as virus entry, assembly and release (Lai, 1998). For example, poliovirus translation and replication are coordinated by the interaction of host factors with viral factors at the 5’- and/or 3’-ends of viral RNA. These factors include host poly (C)-binding protein (PCBP), poly (A)-binding protein (PABP) and viral polymerase precursor 3CD. PCBP and PABP bind to 5’- and 3’-ends, respectively, thus promoting translation early in the infection. As 3 CD accumulates later in infection and binds to the cloverleaf structure in the 5’-end of viral RNA, PCBP, PABP and 3 CD interact with each other to induce the circularization of poliovirus RNA. This circular RNP complex has been shown to be required for positive-strand RNA synthesis, thus affecting viral replication (Andino et al., 1999; Herold and Andino, 2001), 36). In addition, it has been reported that La autoantigen (Meerovitch et al., 1993), PCBP, PTB and Unr (Boussadia et al., 2003) are involved in poliovirus IRES-dependent translation. For MHV, several cellular proteins have been identified to bind to the untranslated regions of viral RNA by UV-crosslinking methods (Furuya and Lai, 1993). These include polypyrimidine tract-binding protein (PTB) (Li et al., 1999), heterogeneous nuclear ribonucleoproteins A l (hnRNP A l) (Li et al., 1997), 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mitochondrial aconitase (Nanda and Leibowitz, 2001), poly (A)-binding protein (PABP) (Spagnolo and Hogue, 2000) and several other unidentified proteins. PTB binds to the UCUAA repeats sequence within the 5’-UTR and the sequence complementary to the 3’-UTR (c3’-UTR) (Huang and Lai, 1999; Li et al., 1999). The PTB binding induced a conformational change in RNA structure (Huang and Lai, 1999). Site-directed mutagenesis of the PTB-binding site in either 5’- or c3’-UTR inhibited the replication and transcription of MHV genomic and defective-interfering (DI) RNA, suggesting that PTB may play a role in regulating viral RNA synthesis. Moreover, the study of a dominant-negative mutant of PTB showed that PTB affected MHV RNA transcription and replication, but not translation (Choi, Huang, and Lai, 2002). In contrast, hnRNP A l binds to the leader and intergenic sequence (IG) of negative strand and 3’-UTR region o f positive strand (Li et al., 1997). Mutations of IG sequence that caused reduced binding of hnRNP A l also inhibited the transcription of MHV DI RNA to the corresponding extents (Li et al., 1997; Zhang and Lai, 1995). The effect of hnRNP A l on MHV RNA transcription was further confirmed in cell lines expressing a dominant-negative mutant of hnRNP A l (Shi et al., 2000). Although MHV can replicate in cell lines deficient in hnRNP A l (Shen and Masters, 2001), a recent study showed that multiple type A/B hnRNPs substituted for the functions of hnRNP A l in these cell lines (Shi, Yu, and Lai, 2003). In this study, I have attempted to identify and characterize additional MHV RNA-binding proteins that interact with either 5’-UTR or c5’-UTR of MHV. By 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RNA affinity purification, I were able to identify a 70-kDa, novel MHV RNA- binding protein, synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP), which belongs to hnRNP family. My results showed that SYNCRIP binds to either MHV 5’-UTR or c5’-UTR in vitro, and the overexpressed SYNCRIP was able to bind to viral RNA in the MHV-infected cells. In an in vivo study, a truncated form of SYNCRIP with the deletion of its C-terminus functioned as a dominant-negative mutant of viral replication, and delayed syncytia formation associated with virus infection. Furthermore, the downregulation of SYNCRIP by a specific siRNA retarded syncytia formation, viral protein synthesis and viral RNA replication. Since SYNCRIP does not have any effect on MHV RNA translation, I suggest that SYNCRIP is directly involved in MHV RNA synthesis. 3.3 Materials and Methods Cells, Virus and antibodies DBT cell, a mouse astrocytoma cell line (Hirano et al., 1974), was cultured in Eagle’s minimal essential medium (MEM) supplemented with 7% newborn calf serum (NCS), 10% tryptose phosphate broth, and streptomycin-penicillin. The 293A cell line was cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MHV-JHM was amplified in DBT cells and maintained in MEM containing 1% NCS. 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The polyclonal anti-SYNCRIP antibody was a gift from Dr. Katsuhiko Mikoshiba at RIKEN, JAPAN (Mizutani et al., 2000). Anti-PTB antibody (BB7) and anti-Al antibody were purchased from ATCC (Manassas, VA) and Aves Labs, Inc (Tigard, OR), respectively. The monoclonal anti-HA antibody was purchased from the micro core facility at USC. Anti-actin antibody was obtained from Sigma (St. Louis, MO). The mouse monoclonal antibody against the N protein of MHV has been described previously (Fleming et al., 1983). UV-crosslinking assay The UV-crosslinking assay was performed as previously described (Furuya and Lai, 1993). Briefly, 20ug of DBT cytoplasmic extract were preincubated for 10 min at 30°C with 20ug of tRNA and 40U RNasin. Next, in vzfro-transcribed and 3 2 P- labeled (106 cpm) MHV RNA were added and incubated for 10 more minutes. Samples were placed on ice and exposed to UV in a Stratalinker (Stratagene, La Jolla, CA) for lOmin, followed by digestion with 400ug of RNase A per ml for 30 min at 37°C. The protein-RNA complexes were separated by SDS-PAGE and visualized by autoradiography. RNA affinity purification For biotinylation of RNA, either 5’-UTR or c5’-UTR of MHV was in vitro transcribed from pNXl-182 (Furuya and Lai, 1993) by T7 or T3 RNA polymerase with Biotin-UTP (Roche, Indianapolis, IN) using Maxiscript kit from Ambion 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Austin, TX). DBT cytoplasmic extract (lOmg) was incubated with 30ug of biotinylated RNA, 400U RNasin, 400ug of yeast tRNA in the binding buffer (5mM KC1, ImM HEPES pH7.6, 0.4mM MgCl2, O.lmM EDTA, 0.04% glycerol, 0.4mM DTT), overnight at 4°C. RNA-protein complexes were pulled down with streptavidin-agarose beads (Sigma, St. Louis, MO) by incubating for 1 hr at 4°C. The beads were washed with binding buffer containing 200mM KC1 five times and then eluted with 2M KC1. The eluates were separated by SDS-PAGE and stained with Coomassie brilliant blue. Individual bands were excised from the gel and analyzed by MALDI-mass spectrometry in the W.M. Keck Facility at Yale University, New Haven, Conn. Plasmid construction The cDNA of SYNCRIP (a gift from Dr. Katsuhiko Mikoshiba at RIKEN) (Mizutani et al., 2000) was amplified and cloned into pET28a (Novagen, Madison, WI) with His-tag, or pcDNA3.1 (Invitrogen, Carlsbad, CA) with HA-tag. The truncated form of SYNCRIP was similarly constructed using a PCR-amplified fragment that represents SYNCRIP aa 1-407. Immunoprecipitation and ribonuclease protection assay (RPA) Assays were performed as previously described (Choi, Huang, and Lai, 2002). Briefly, 293A cells were transfected with vector, pcDNA3.1/F-SYN or pcDNA3.1/N-SYN using Fugene 6 transfection reagent (Roche, Indianapolis, IN). At 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24 hr post-transfection, cells were infected with MHV-JHM at an M.O.I. of 10. At 8 hr post-infection, cells were lysed with buffer K (20mM Tris-HCl pH 7.5, lOOmM NaCl, 0.2mM EDTA pH8.0). The supernatant was incubated with various antibodies for 2 hr at 4°C and then with protein A-sepharose beads (Zymed Laboratories Inc., San Francisco, CA) for an additional 2 hr. After washing with buffer K five times, RNA was extracted with elution buffer (0.3M Na acetate pH5.2, 0.2% SDS, ImM EDTA pH8.0, lOug/ml proteinase K) for lOmin at 65°C, followed by phenol/chloroform extraction and EtOH precipitation. To prepare the RPA probe, the 5’-UTR region (nt 1-251) was amplified into DNA from the purified MHV-JHM viral RNA by RT-PCR using appropriate primers containing 19nt of T7 promoter and 17nt of noncomplementary sequence. The DNA product was directly used for in vitro transcription (Ambion, Austin, TX). RPA was performed accordining to the manufacturer’s guide (Ambion, Austin, TX). RNAi analysis Target sequences of RNAi duplex were chosen using the siRNA target finder software from Ambion (www.ambion.com/techlib/misc/siRNA finder.html) and chemically synthesized from Intergrated DNA Technogies, Inc. (Coralville, IA). Nonspecific siRNA was purchased from Ambion (Austin, TX). DBT cells were grown in the appropriate media without antibiotics. For transfection, cells were plated to 30% confluency in a 24-well plate. On the following day, 3ul of the 20uM stock of siRNA duplex was mixed with 47ul of Opti- 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MEM media (Invitrogen, Carlsbad, CA). In a separate tube, 3ul of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was resuspended with 47ul of Opti-MEM, followed by incubation at room temperature for 7 minutes. The two mixtures were combined and allowed to sit for 20 min at room temperature. After the incubation, lOOul of mixture was directly added to the well containing 500ul of growth media. Cells were grown and harvested at 2, 4, and 6 days post- transfection for further anaylsis. Kinetic analysis of MHV RNA synthesis DBT cells were transfected with RNAi as described above and, at 2 days post transfection, infected with MHV-JHM at an M.O.I of 1. To label newly synthesized viral RNA, cells were treated with 5ug/ml actinomycin D for 1 hr prior to the addition of [3 H]-uridine (lOOuCi/ml) (NEN, Boston, MA). After incubation with [3 H]-uridine for 1 hr, cytoplasmic extracts were prepared, spotted onto 3MM paper, and washed with 10% trichloroacetic acid (TCA). The radioactivity on the paper was counted using a Beckman scintillation counter. Experiments were repeated three times with duplication of samples. Translation study Translation study was performed as previously described (Choi, Huang, and Lai, 2002). In brief, rabbit reticulocyte lysate (RRL) (Promega, Madison, WI) was incubated with radiolabeled and in vitro transcribed MHV DI RNA (either DE25 or MHV-UTR/LUC) with increasing amounts of bacterially purified SYNCRIP protein 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for 90 min at 30°C. The 35S-labeled translation products were separated by SDS- PAGE and visualized by autoradiography. For in vivo translation studies, 2.5ug of in vitro transcribed MHV-UTR/LUC RNA were transfected into either 293A cells that transiently overexpressed SYNCRIP or DBT cells transfected by the SYNCRIP siRNA, using DMRIE-C transfection reagent (Invitrogen, Carlsbad, CA). At 8 hr post-transfection, cells were harvested and used for luciferase assay (Promega, Madison, WI). Experiments were repeated with triplication of samples. 3.4 Results p70 is purified by one-step RNA affinity method and identified as SYNCRIP. Using either 3 2 P-labeled 5’-UTR or c5’-UTR (complementary to 5’-UTR) as a probe, the UV-crosslinking experiment with DBT cell extract showed that several proteins bound to MHV-UTR RNA specifically (Fig 3-1A). These proteins include not only PTB, which binds to 5’-UTR, and hnRNP A l, which binds to c5’-UTR, but also additional proteins of 70, 100 and 130 kDa. Specific binding of the 70-kDa protein to MHV RNA has been suggested in my previous studies (Furuya and Lai, 1993; Huang and Lai, 1999), but it was not clear whether the binding of the 100- and 130-kDa proteins was specific to MHV RNA. To purify unidentified cellular proteins that interact with 5’-UTR, I developed a one-step purification procedure based on the RNA affinity method (Fig 3-1B). Biotinylated 5’-UTR or c5’-UTR 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5’-UTR c5’-UTR 3 : DBT extract p130 p100 p70 PTB A1 B 5’-UTR c5’-UTR B B I DBT extract ‘ S t i f l e I e l t>5<a | 2M KCI elution Elution and SDS-PAGE Fig. 3-1-1. Specific association of cellular proteins with MHV 5’-UTR and c5’-UTR and RNA-affinity purification of MHV RNA-binding proteins from DBT cells. (A) uv-crossiinking of d b t cytoplasmic extracts with 32P-labeled 5’-UTR and c5’-UTR. Lanes 2 and 4: DBT extract, lanes 1 and 3: no extract (B) Diagram o f RNA-affinity purification procedure. B:biotin, S: streptavidin-agarose beads 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RNA was incubated with DBT extract, pulled down with streptavidin-agarose beads, and the bound RNP complexes were eluted by 2M KC1. By this procedure, we were able to detect PTB and hnRNP A l using 5’-UTR and c5’-UTR as a probe, respectively, confirming that this purification procedure worked properly (Fig 3-1C). In addition to PTB and hnRNP A l, 70-, 100-, 130-kDa and several other minor proteins were pulled down with both 5’-UTR and c5’-UTR, specifically (Fig 3-1 A). The 70-kDa protein was identified as Synaptotagmi n-bindi ng Cytoplasmic RNA- Interacting Protein (SYNCRIP) by MALDI-mass spectrometry analysis, while the 100-kDa protein was HSP90, and 130-kDa protein was an unknown protein. Fourteen matched peptides out of ninety-two measured peptides of the 70-kDa protein are shown in Fig 3-ID. The % coverage of the known sequence for the 70- kDa protein was 32%, which is more than the 25% coverage typically considered to be reliable. Since my previous study based on RNA competition experiment showed that the binding of the 70-kDa protein to MHV UTR was specific (Huang and Lai, 1999), I focused on the characterization of SYNCRIP in this study. SYNCRIP is a member of hnRNP family, and its human homolog is hnRNP Q, which was previously named NS1-associated protein (NSAP1) or glycine and tyrosine-rich RNA-binding protein (Gry-rbp) (Harris, Boden, and Astell, 1999; Hresko and Mueckler, 2002). SYNCRIP has been suggested as a cytoplasmic counterpart of hnRNP R (Mizutani et al., 2000). As presented in Fig 3-2A, SYNCRIP has an acidic domain at N-terminus, followed by three sets of RNA-binding domains (RBD) and 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o z Q ' 1- D I v r > a : H D I G d o 5 ? m in o 130 100 73 58 43 35 2 3 1 m I CBB WB 7 8 large scale prep Peptide sequence Measured mass Computed mass Start To LYNNHEIR 1057.554 1057.530 222 229 LMMDPLTGLNR 1259.696 1259.636 193 203 TGYTLDVTTGQR 1310.704 1310.646 132 142 TKEQILEEFSK 1350.731 1350.702 255 265 NLANTVTEEILEK 1472.781 1472.771 344 356 LKDYAFIHFDER 1552.800 1552.767 370 381 EFNEDGALAVLQQFK 1707.859 1707.846 67 81 VTEGLTDVILYHQPDDK 1941.968 1941.967 266 282 GYAFVTFCTKEAAQEAVK 1961.955 1961.955 204 221 LDEIYVAGLVAHSDLDER 2014.015 2014.000 43 60 DLEGENIEIVFAKPPDQK 2041.018 2041.036 395 412 VTEGLTDVILYHQPDDKK 2070.034 2070.062 266 283 YGGPPPDSVYSGQQPSVGTEIFVGK 2565.197 2565.237 144 168 KYGGPPPDSVYSGQQPSVGTEIFVGK 2693.299 2693.332 143 148 Fig. 3-1-2. Specific association of cellular proteins with MHV 5’-UTR and c5’-UTR and RNA-affinity purification of MHV RNA- binding proteins from DBT cells. (C) Purification of MHV-RNA-binding proteins from DBT cells. After purification, the eluates was separated by SDS- PAGE and stained with Coomasie brilliant blue (CBB) (lanes 1,2, and 3). PTB and hnRNP A1 were detected by immunoblotting with specific antibodies (lanes 5 and 6). Individual bands were excised from the gel of large-scale preparation after CBB staining (lanes 7 and 8) and analyzed by mass spectrometry for protein identification. Sizes are shown in kilodaltons. (D) MALDI-mass spectrometry analysis of p70 protein. The 14 matched peptides sequences out of 92 measured peptides and their locations in the protein are shown. 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. another type of RNA-binding motif (RGG box). Furthermore, a tyrosine-rich motif is present in the C-terminus, which putatively mediates protein-protein interactions. To validate that SYNCRIP was indeed the 70-kDa protein crosslinked to the MHV-UTR, I performed immunoprecipitation of DBT extract crosslinked to 3 2 P- labeled 5’-UTR or c5’-UTR, using anti-SYNCRIP antibody. As shown in Fig 3-2B, the 70-kDa protein crosslinked to either 5’-UTR or c5’-UTR was pulled down with anti-SYNCRIP antibody, but not with the control IgG or anti-actin antibody. The upper protein bands of approximately 85-kDa may be the contaminating hnRNP R, which weakly cross-reacted with anti-SYNCRIP antibody (Mizutani et al., 2000). From this experiment, I established that SYNCRIP is indeed the UV-crosslinked 70- kDa protein. SYNCRIP binds to both MHV-5’-UTR and c5’-UTR in vitro. To test whether SYNCRIP is able to bind to MHV-UTR, I performed in vitro binding analysis. His-tagged, recombinant SYNCRIP was expressed in E.coli, and purified by Ni-NTA-agarose column. I generated two different forms of SYNCRIP, the full-length (F-SYN) and the C-terminus-truncated form (N-SYN) (Fig 3-2A). The N-SYN includes the N-terminal RNA-binding motif, but lacks the C-terminus, which is responsible for protein-protein interaction. Both F-SYNl (70 kDa) and N- SYN (54 kDa) were expressed in E.coli and purified to similar purity. The UV- crosslinking experiment showed that both forms of SYNCRIP bound to MHV 5’- UTR, but not nonspecific RNA, although the binding of N-SYN was slightly 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. . Y-rich F-SYN HA or His 1 N-SYN HA or His 1 130 160 407427 487 561 acidic 3XRBD RGG 561 407 B 5 '-UTR c5' -UTR 3 a c IP IP 3 a. c > - </> b o D ) O re z > - (f) a o O ) o re 130 “ 100 " p70 ► 73 _ PTB ^ A1 ► 54 - 43 - 35 - j | ■ ■ :i':: ■ 5 6 7 8 Fig. 3-2. Schematic diagram of SYNCRIP and confirmation of p70 as a SYNCRIP protein. (A) Structural organization of SYNCRIP. Acidic: protein domain rich in acidic amino acids. RBD: RNA-binding domain. RGG box: RGG RNA-binding domain. Y-rich: protein-protein-interacting domain. The diagrams of F-SYN and N-SYN constructs are shown below the diagram. His: 6X His, HA: 8 amino acids of HA tag. (B) Immunoprecipitation of UV- crosslinked p70 with anti-SYNCRIP antibody. After UV-crosslinking of DBT extracts with 32P-labeled 5’-UTR or c5’-UTR RNA and digestion with RNase A, RNA-protein complexes were immunoprecipitated with anti-SYNCRIP antibody (lanes 2 and 6) or nonspecific antibody (lanes 3, 4, 7 and 8). Lanes 1 and 5 represent 10% of the UV-crosslinked lysates used for each analysis. 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stronger than that of F-SYN (Fig 3-3A). Since N-SYN showed a stronger binding activity, I used N-SYN for further in vitro binding studies. To map SYNCRIP- binding site in 5’-UTR, several deletion mutants were used for the UV-crosslinking experiment (Fig 3-3B). N-SYN bound strongly to the 5’-end 184 nt or 112 nucleotides of viral RNA. The truncated mutant, +56, which does not include the UCUAA repeats, retained a weak binding ability. A4R, which is identical to +184 RNA except for a deletion of 4 copies of UCUAA repeats, bound to N-SYN only weakly. Therefore, the UCUAA repeats sequence is crucial for efficient SYNCRIP binding. However, the 4 copies of UCUAA repeats (4R) alone was not sufficient for binding, indicating that the neighboring sequence is also important for SYNCRIP binding, probably because it induces conformational change in 5’-UTR and/or stabilizing the structure of 5’-UTR. In addition, the binding of N-SYN to radiolabeled 5’-UTR was competed away with excess amounts of both unlabeled 5’-UTR and c5’-UTR, but not with 3’-UTR, c3’-UTR or nonspecific RNA (Fig 3-3C), indicating that SYNCRIP binds also to c5’-UTR. This result is consistent with the previous finding that SYNCRIP was pulled down with either 5’-UTR or c5’-UTR by RNA affinity purification (Fig 3- 2B). Overexpressed SYNCRIP binds to MHV-5’-UTR in virus-infected cells. Next, I asked whether SYNCRIP bound to MHV-5’-UTR in virus-infected cells. To address this question, I performed immunoprecipitation of SYNCRIP from the 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5’-UTR >- 0) >- w pcDNA3.1 z z >- >- C O C O 73 54 B Leader +184 , +112 |- +56 |- I I I II1 1 0 60 80 184 ----------------------1 a4R h 4R +184 +112 +56 a4R 4R Fig. 3-3-1. In vitro binding of the recombinant SYNCRIP and analysis of its binding sites on 5’-UTR. (A) Specific binding of recombinant F-SYN and N-SYN to 5’-UTR. Purified recombinant F-SYN and N- SYN (10ng) were crosslinked to 3 2 P-labeled 5’-UTR (lanes 1 and 2) or vector pcDNA3.1 RNA transcript (200nt) (lanes 3 and 4). (B) Binding region of N-SYN in 5’-UTR. Purified N-SYN (10ng) was crosslinked with 30pmol of 32P-labeled probes. R: UCUAA repeats. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c 5' -UTR c5' -UTR 3'-UTR c3'-UTR pcDNA3.1 (-) x1 x5 x20x50x1 x5 x20x50x1 x5 x20x50x1 x5 x20x50 x1 x5 x20x50 RNA _ ________________________________________________________________ Fig. 3-3-2. In vitro binding of the recombinant SYNCRIP and analysis of its binding sites on 5’-UTR. (C) Competition experiments. 32P-labeled 5’-UTR (30pmol) was UV-crosslinked with 10ng of purified N-SYN in the presence of unlabeled probes, 5’-UTR, c5’- UTR, 3’-UTR, c3’-UTR and nonspecific RNA in serial dilutions (x1, x5, x20 and x50). (-) indicates UV-crosslinked N-SYN without added cold RNA. 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lysates of MHV-infected cells using anti-SYNCRIP antibody; viral RNAs were isolated from the immunoprecipitated complex and detected by RNase protection assay (RPA) using a 5’-UTR as the probe (Fig 3-4A). The HA-tagged, full-length SYNCRIP or the truncated form of SYNCRIP (Fig 3-2A) was expressed together with an MHV receptor (MHVR) in 293A cell and then infected with MHV. As shown in lanes 2 and 3 in Fig 3-4A, MHV RNA was detected with this probe only in virus-infected cells, but not in uninfected cells. Immunoprecipitation of SYNCRIP with anti-HA antibody pulled down the viral RNA (Fig 3-4A, lane 5). Anti-GAPDH antibodies yielded only background signals similar to that obtained using the bead only (lanes 4 and 7). Anti-N (MHV nucleocapsid protein) antibody also precipitated the viral RNA (lane 6), consistent with the finding that N protein binds to the 5’- UTR of MHV RNA (Stohlman et ah, 1988). N-SYN had a lower binding ability than F-SYN (compare lanes 5 and 8). Given that N-SYN binds better than F-SYN in vitro (Fig 3-3A), this finding suggests that in vivo binding of SYNCRIP to MHV-UTR may require other factors, since N-SYN lacks the protein-interacting domain. These results together indicate that SYNCRIP binds to MHV RNA in the virus-infected cells. Syncytia formation is delayed when F-SYN or N-SYN is overexpressed. To address the biological significance of SYNCRIP in MHV replication, I overexpressed either F-SYN or N-SYN together with MHV receptor in 293A cells; at 24 hr post-transfection, cells were infected with MHV-JHM, at an M.O.I. of 1. 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 5’-UTR 1 9 n t : 1 8 4 n t = 6 7 n t 3’-UTR 0RF1-7/ 270nt ( Before RNase digestion) 251 nt ( After RNase digestion) B < D n 2 Q . IP 7 3 T O 0 ) m < x 8 X £ ? s i 8 8 270nt 251 nt Fig. 3-4. In vivo binding of overexpressed SYNCRIP in MHV- infected 293A cells. (A) shows the structure of the probe used. The probe consists of 184 nt of 5’-UTR, 67 nt of ORF1a 5’-end and 19nt of noncomplementary sequence. After RNase digestion, the protected band migrated as 251 nt. (B) 293A cells were cotransfected with HA-tagged F-SYN (lanes 4, 5, 6 and 7) or N-SYN (lane 8) and MFIV receptor, and then infected with MFIV-JFIM at an M.O.I. of 10. At 8 hr post-infection, the cell lysates were immunoprecipitated with various antibodies, anti-HA antibody (lanes 5 and 8), anti-N antibody (lane 6) and anti-GAPDFI antibody (lane 7), followed by extraction of RNA from the beads. Viral RNAs were detected by RPA. Lane 1 indicates the 10% unhybridized probe. As a control, total RNA from uninfected and MFIV-JHM-infected 293A cells without prior immunoprecipitation was used directly for RPA (lanes 2 and 3). 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cells were immunostained with anti-N antibody at 12 hr post-infection. As compared with the vector-transfected cells, the appearance of syncytia formation was significantly delayed in N-SYN-transfected cells (Fig 3-5). However, by 24 hr post infection, N-SYN-transfected cells were fully infected and formed syncytia to the same extent as that of the vector-transfected cells (data not shown). I also examined the surface expression of MHV receptor to rule out the possibility that the delay of syncytia formation was due to down-regulation of MHV receptor. There was no significant difference. Surprisingly, overexpression of F-SYN also caused delayed syncytia formation. Thus, both N- and F-SYN had a dominant-negative effect on MHV replication. These dominant-negative effects may be caused by the “squelching effect” commonly observed with the overexpression of transcription factors (Strasser and Hurt, 2001); it was observed previously with the full-length PTB in MHV RNA transcription. These results show that overexpression of SYNCRIP may impair virus replication by titrating out the factors involved in virus replication. Therefore, these results suggest that SYNCRIP may be involved in MHV replication. In vivo knock-down of SYNCRIP delayed MHV replication. To further examine the role of the endogenous SYNCRIP in MHV replication, I attempted to knock down the endogenous SYNCRIP by using RNA interference method (Elbashir et al., 2001; Hannon, 2002). Three different short-interfering RNA (siRNA) were designed against SYNCRIP (Fig 3-6A). Transfection of DBT cells 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VEC F-SYN N-SYN Syncytia formation WB 70kDa P 54kDa Fig. 3-5. Syncytia formation of 293A cells overexpressing F- SYN or N-SYN. 293A cells were transfected as in Fig. 4 and, at 24 hr post transfection, were infected with MHV-JHM at an M.O.I of 1. At 12 h post infection, cells were incubated with an antibody against the viral N protein, followed by incubation with fj-galactosidase-conjugated secondary antibody. Syncytia formation was visualized by X-gal staining. The bottom panel shows the expression of F-SYN or N-SYN by western blotting (WB) with anti-HA antibody. The vector-transfected cell was used as the control. 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with any one of the SYNCRIP-specific siRNAs (Fig 3-6B, #1, #2, and #3), but not with nonspecific siRNA (Fig 3-6B, (-)), resulted in transient reduction of endogenous SYNCRIP. The three SYNCRIP siRNAs showed reduction of the endogenous SYNCRIP to a similar level, i.e. around 25-40% at 2 days post-transfection and around 40-65% at 4 days post-transfection. The level of SYNCRIP returned to almost the normal level at 6 days post-transfection. To examine the effect of reduction of endogenous SYNCRIP on MFIV replication, DBT cells transfected with either SYNCRIP siRNA #1 or nonspecific siRNA were infected with MHV at an M.O.I. of 1 at 2 days post-transfection. First, I investigated the morphological change induced by virus infection. Compared with the nonspecific siRNA-transfected cells, the SYNCRIP siRNA-transfected cells showed significantly delayed syncytia formation, with at least a 4-hour delay (Fig 3- 7A). Next, I examined the viral protein and RNA synthesis by immunoblotting and [3 H]-uridine labeling, respectively. The kinetics of both viral protein and RNA synthesis were delayed in SYNCRIP siRNA-transfected DBT cells (Fig 3-7B and C). The synthesis of viral protein and RNA peaked at 13 hr post-infection in cells transfected with the nonspecific siRNA, but peaked at 20 hr post-infection in SYNCRIP siRNA-transfected cells. From these results, I conclude that SYNCRIP is a positive regulator of MHV protein and RNA synthesis. 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 241 259 t & t U.GA 980 998 1426 1444 1683 3’ SYNCRIP #1 AGACAGUGAUCUCUCUCAUTT siRNA duplex TTUCUGUCACUAGAGAGAGUA #2 GAUCCUGAUCCUGAAGUUATT TT C UAG GAC UAGGAC U U CAAU #3 CUAUCGUGGUGGAUAUGAATT TTGAUAGCACCACCUAUACUU B 2d 4d 6d siRNA (-) #1 #2 #3 (-) #1 #2 #3 (-) #1 #2 #3 a SYNCRIP o c actin % reduction 4 3 24 32 48 40 66 28 11 37 Fig. 3-6. RNAi-mediated reduction of SYNCRIP in DBT cells. (A) Targeted regions of SYNCRIP siRNA and their sequences (#1, #2 and #3). Nonspecific siRNA from Ambion was used as the negative control. (B) Immunoblotting from RNAi-transfected DBT cells. DBT cells were transfected with SYNCRIP-specific siRNA (#1, #2 and #3) or a nonspecific siRNA ((-)) and harvested at 2, 4 and 6 days post-transfection. 20ug of total cell lysates were immunoblotted with anti-SYNCRIP antibody and anti-actin antibody, separately. Percentage of SYNCRIP reduction was quantified using a densitometer. 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A (-)Ctrl-siRNA SYN-siRNA Ohr 10hr ,*.*> 1 7 * ^ „7i' '■ '5* ' :f v,' - * ' • V * y ' i ’y - ' h ' ' f : ; / I * * ? « - * ® . S a . 1 1 1 A . . T ' - ’ V .* » < : j g S M ^ P l 13hr 16hr Fig. 3-7-1. Retardation of MHV replication by reduction of endogenous SYNCRIP. (A) Syncytia formation in SYNCRIP-specific siRNA-transfected DBT cells. At 2 days post-transfection of siRNA, cells were infected with MHV-JHM at an M.O.I of 1 and maintained in virus growth media. Pictures were taken at 0, 10, 13 and 16 hr post-infection. 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B 8 10 13 16 20 24 P.l. (hr) (-)Ctrl-siRNA SYN-siRNA N protein c 0) (-) Ctrl-siRNA SYN-siRNA X CO ■ o 0) 4 -> (0 o Q. o o E 2 4 6 8 10 13 16 20 24 Hrs P.l. Fig. 3-7-2. Retardation of MHV replication by reduction of endogenous SYNCRIP. (B) Kinetics of expression of viral N protein. Cell lysates were taken at different time points and subject to immunoblotting to detect viral N protein. (C) Kinetics of MHV RNA synthesis. [3H]-uridine labeling was done at different time points after virus infection. % incorporation of [3H]- uridine are shown. 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SYNCRIP directly regulates viral RNA synthesis but not viral translation. The finding that the reduction of endogenous SYNCRIP delayed both viral RNA and protein synthesis could be due to the possibilities that SYNCRIP is directly involved in both viral transcription and translation or that SYNCRIP affects transcription, which, in turn, affects translation, or vice versa. To distinguish these possibilities, I designed experiments to separate viral translation from viral RNA transcription using defective-interfering RNA (DI RNA). DE25 is a natural DI RNA, in which part of ORFla is fused to ORF7 encoding the N protein (Fig 3-8A, top diagram) (Makino et al., 1988). This protein can be directly translated from DE25 RNA. I also used reporter DI RNA, MHV-UTR/FUC, which contains the authentic MHV 5’- and 3’-UTR and expresses an ORF-la-luciferase fusion protein (Fig 3-8A, bottom) (Choi, Huang, and Fai, 2002). Therefore, the mechanism of translation of luciferase reflects faithfully that of the natural MHV RNA. The use of both DI RNAs made it possible to examine the direct effect of SYNCRIP on viral translation by excluding the possible transcriptional effect. First, I performed in vitro translation using rabbit reticulocyte lysate (RRF); RRF was incubated with increasing amounts of recombinant SYNCRIP, and then in vitro transcribed DI RNAs were added for translation. The translation o f DE25 and MHV-UTR/FUC were not affected until the amount of the recombinant SYN reached 15nM, when there was nonspecific inhibition. Similar nonspecific inhibition by SYN was observed with the control RNA, EFla. 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DE25 0RF1 a N 5' -UTR 3' -UTR An MHV-UTR/LUC 5 '-UTR 0RF1 a LUC 3 '-UTR An B rSYN (nM) 100 - 73 - 54 DE25 MHV-UTR/LUC EF1a 0 5 7.5 15 30 0 5 7.5 15 30 5 7.5 15 30 !■§ » • Fig. 3-8-1. The effect of SYNCRIP on the translation of DI RNA. (A) Structure of DI RNAs used for translation study. DE25 produces an 80-kDa ORF1a-N fusion protein, while MHV-UTR-LUC produces a 70-kDa ORF1a- luciferase fusion protein. (B) In vitro translation. Rabbit reticulocyte lysates were incubated with 3 5 S-translabeling mixtures, in vitro transcribed DI RNAs and increasing amounts of purified recombinant F-SYN (up to 30nM). EF1a was used for the negative control. 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120000 1 100000 3 80000 60000 5 40000 20000 i J VEC F-SYN N-SYN Overexpression in 293A cells E > » o < 0 ) 0 5 ( 0 6000 5000 4000 3000 2000 1000 0 I —I I ' (-)Ctrl SYN-siRNA siRNA-transfected DBT cells Fig. 3-8. The effect of SYNCRIP on the translation of DI RNA.(C) In vivo translation of the reporter RNA in SYNCRIP-overexpressing 293A cells or SYNCRIP-specific siRNA-transfected DBT cells. The cells were prepared as Fig 4 and Fig 6. In vitro transcribed MHV-UTR/LUC DI RNAs were transfected into the various cells and luciferase activity was assayed at 8 hr post-transfection. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I further tested the possible translational effect of SYNCRIP in vivo. For this study, I overexpressed F-SYN or N-SYN transiently in 293A cells, followed with the transfection of MFTV-UTR/LUC RNA. Both F-SYN and N-SYN were expressed to the similar level in cells (Fig 3-5B). At 8 hours post-transfection of RNA, luciferase activity was assayed (Fig 3-8C, left). No difference in luciferase activity was observed between the vector-transfected cells and F-SYN or N-SYN overexpressed cells. I further performed the similar experiment in cells in which SYNCRIP was knocked down by the specific siRNA. No difference in luciferase activity was observed between cells transfected with the SYN-specific siRNA or the nonspecific siRNA (Fig 3-8C, right). Therefore, based on the in vitro and in vivo translation studies, I conclude that SYNCRIP does not affect the viral translation directly, implying that SYNCRIP most likely plays a direct role in MHV RNA synthesis. 3.5 Discussion Since most viruses carry relatively small numbers of genes in their genome, most steps in virus replication, including virus entry, gene expression, RNA synthesis, assembly, budding and release, require the participation of host factors, which interact with viral RNA and/or viral proteins (1, 17). I have focused on the host factors involved in the regulation of MHV RNA replication, transcription and translation. By a classical UV-crosslinking method, I have been able to define a set 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of specific MHV RNA-binding proteins (Fig 3-1A). So far, I have characterized two MHV RNA-binding proteins, PTB and hnRNP A l, and several other hnRNP A l- related proteins, which bind to the 5’- and 3’-ends of opposite RNA strands. PTB and hnRNP A l may mediate 5’-3’ crosstalks of viral RNA by interacting with each other (Huang and Lai, 2001), causing circularization of viral genome. This genomic circularization and its importance in viral RNA replication have been reported for polio virus (Herold and Andino, 2001), although they have not been unequivocally proven in the case of MHV. Moreover, the studies of dominant-negative mutants of either PTB or hnRNP A l have shown that these two hnRNP proteins are important for the modulation of MHV RNA synthesis (Choi, Huang, and Lai, 2002; Shi et al., 2000). Besides PTB and hnRNP Al-related proteins, several other proteins, including poly (A) binding protein (PABP) (Spagnolo and Hogue, 2000) and mitochondrial aconitase (Nanda and Leibowitz, 2001), have been reported by other laboratories as trans-regulatory factors for MHV replication. SYNCRIP represents yet another factor in MHV RNA synthesis. The one-step RNA affinity purification used in this study was highly specific, since I were able to detect PTB and hnRNP A l using positive- and negative-strand RNA probes, respectively, but not vice versa. SYNCRIP was originally found as a binding partner o f the ubiquitous synaptotagmin iso forms (Mizutani et al., 2000), and its human homolog, NASP1 or gry-rbp, has been identified from a two-hybrid screen as an interacting partner of NS1, the major nonstructural protein o f parvovirus (Harris, Boden, and Astell, 1999). It was renamed as hnRNP Q (Rossoll et al., 2002). Recent 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. database search revealed that there are three isoforms of hnRNP Q, Q1/2/3, which migrate as 55, 60 and 70 kDa respectively; SYNCRIP corresponds to hnRNP Q3 (Mourelatos et al., 2001). It has been suggested that hnRNP Q is involved in mRNA processing and transport (Harris, Boden, and Astell, 1999), translation-coupled mRNA turnover (Grosset et al., 2000) and mRNA splicing (Neubauer et al., 1998). SYNCRIP is 81.2% similar to hnRNP R, but lacks -70 carboxyl-terminal amino acids that contain a nuclear localization motif, and has been suggested as a cytoplasmic counterpart of hnRNP R (Mizutani et al., 2000). Cytoplasmic localization of SYNCRIP is further confirmed by immunoflurorescence staining of SYNCRIP (Mizutani et al., 2000). Moreover, it has been reported that SYNCRIP is part of a cytoplasmic multiprotein complex that binds to the major determinant of instability (mCRD) of the c-fos proto-oncogene mRNA and regulates its stability and translatability (Grosset et al., 2000). The cytoplasmic localization of SYNCRIP and its putative role in the cytoplasm are particularly noteworthy, since MHV replication occurs in the cytoplasm. It is also noteworthy that most of the MHV RNA-binding proteins identified so far are hnRNPs; SYNCRIP is yet another hnRNP. This fact underscores the importance of RNA processing machinery in MHV RNA synthesis. The importance of SYNCRIP in MHV replication was established in my study using the RNAi approach. Reduction of SYNCRIP in DBT cells resulted in the retardation of syncytia formation, viral protein and RNA synthesis. This finding was further supported by the dominant-negative effect of the truncated form of SYNCRIP on MHV replication. These results suggest that SYNCRIP is a positive regulatory 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. factor in MHV replication. On the other hand, it is puzzling why the overexpression of full-length SYNCRIP also inhibited MHV replication in a dominant-negative manner. Similar inhibitory effect on MHV replication was observed when PTB was overexpressed in cells (Choi, Huang, and Lai, 2002). It is possible that the recombinant SYNCRIP, which has a HA-tag, may be less efficient in its biological activity than the endogenous SYNCRIP. Furthermore, overexpression of SYNCRIP may titrate out one or more replication components, thus resulting in the inhibition of replication/transcription, in a mechanism akin to the “squelching effects” frequently observed for the overexpressed transcription factors (Strasser and Hurt, 2001). So far, SYNCRIP, PTB, and hnRNP A l, all of which are hnRNPs, have been demonstrated to bind to 5’-UTR of both strands. It is likely that they form a large protein complex to regulate viral RNA replication. Interestingly, it has been reported that these hnRNPs are detected in the complex of spliceosome (Neubauer et al., 1998), and SYNCRIP/NSAP1 was detected in the same complex including PABP (Grosset et al., 2000), which binds to 3’-end of MHV RNA. Therefore, it is not surprising that they may form an RNP complex with viral proteins in MHV-infected cells to regulate MHV RNA synthesis. The exact molecular mechanism of this complex in MHV RNA replication remains to be clarified. As a positive regulator of MHV replication, SYNCRIP may play a role through several mechanisms, which are not mutually exclusive. First, SYNCRIP may recruit transcription/replication factors to the replication site, through its protein-protein- interacting domain. Second, SYNCRIP may induce the conformational change in the 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. highly structured viral RNA into a structure that is more favorable for replication and transcription, probably functioning as an RNA chaperon. Finally, SYNCRIP may mediate 5’-3’ crosstalks by interacting with other proteins, causing the circularization of viral genome, which may help viral replication. Finally, my study showed that SYNCRIP is not directly implicated in MHV translation. The 5’-UTR has been shown to regulate the translation of MHV RNA; specifically, the binding of N protein to leader sequence of 5’-UTR enhanced the translation of MHV RNA (Tahara et al., 1998). However, since genomic and subgenomic mRNAs of MHV are capped, polyadenylated and translated by a cap- dependent translation mechanism, it may not need noncanonical factors for efficient translation. Nevertheless, I could not rule out the possibility that SYNCRIP may regulate MHV translation under certain conditions. 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. 6 References Andino, R., Boddeker, N., Silvera, D., and Gamamik, A. V. (1999). Intracellular determinants of picomavirus replication. Trends in Microbiol. 7(2), 76-82. Boussadia, O., Niepmann, M., Creancier, L., Prats, A. C., Dautry, F., and Jacquemin- Sablon, H. (2003). Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus. J. Virol. 77(6), 3353-9. Choi, K. S., Huang, P., and Lai, M. M. C. (2002). Polypyrimidine-tract-binding protein affects transcription but not translation of mouse hepatitis virus RNA. Virology 303(1), 58-68. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.[see comment]. Nature 411(6836), 494-8. Fleming, J. O., Stohlman, S. A., Harmon, R. C., Lai, M. M., Frelinger, J. A., and Weiner, L. P. (1983). Antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to JHM (MHV-4) virus. Virology 131(2), 296-307. Furuya, T., and Lai, M. M. C. (1993). Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA. J. Virol. 67(12), 7215-22. Grosset, C., Chen, C. Y., Xu, N., Sonenberg, N., Jacquemin-Sablon, H., and Shyu, A. B. (2000). A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex. Cell 103(1), 29-40. Hannon, G. J. (2002). RNA interference. Nature 418(6894), 244-51. Harris, C. E., Boden, R. A., and Astell, C. R. (1999). A novel heterogeneous nuclear ribonucleoprotein-like protein interacts with NS1 of the minute virus of mice. J. Virol. 73(1), 72-80. Herold, J., and Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Mol. Cell 7(3), 581-91. 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hirano, N., Fujiwara, K., Hino, S., and Matumoto, M. (1974). Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture. Archiv fur die Gesamte Virusforschung 44(3), 298-302. Hresko, R. C., and Mueckler, M. (2002). Identification of pp68 as the Tyrosine- phosphorylated Form of SYNCRIP/NSAP1. A cytoplasmic RNA-binding protein. J. Biol. Chem. 277(28), 25233-8. Huang, P., and Lai, M. M. C. (1999). Polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3' untranslated region, thereby altering RNA conformation. J. Virol. 73(11), 9110-6. Huang, P., and Lai, M. M. C. (2001). Heterogeneous nuclear ribonucleoprotein al binds to the 3'-untranslated region and mediates potential 5'-3'-end cross talks of mouse hepatitis virus RNA. J. Virol. 75(11), 5009-17. Lai, M. M., and Cavanagh, D. (1997). The molecular biology of coronaviruses. Adv. Virus Res. 48, 1-100. Lai, M. M. C. (1998). Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology 244(1), 1- 12. Li, H. P., Huang, P., Park, S., and Lai, M. M. C. (1999). Polypyrimidine tract- binding protein binds to the leader RNA of mouse hepatitis virus and serves as a regulator of viral transcription. J. Virol. 73(1), 772-7. Li, H. P., Zhang, X., Duncan, R., Comai, L., and Lai, M. M. C. (1997). Heterogeneous nuclear ribonucleoprotein A l binds to the transcription-regulatory region of mouse hepatitis virus RNA. Proc. Natl. Acad. Sci. USA 94(18), 9544-9. Lin, Y. J., Zhang, X., Wu, R. C., and Lai, M. M. C. (1996). The 3' untranslated region of coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. J. Virol. 70(10), 7236-40. Makino, S., Joo, M., and Makino, J. K. (1991). A system for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. J. Virol. 65(11), 6031-41. Makino, S., Shieh, C. K., Soe, L. H., Baker, S. C., and Lai, M. M. C. (1988). Primary structure and translation of a defective interfering RNA o f murine coronavirus. Virology 166(2), 550-60. 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Meerovitch, K., Svitkin, Y. V., Lee, H. S., Lejbkowicz, F., Kenan, D. J., Chan, E. K., Agol, V. I., Keene, J. D., and Sonenberg, N. (1993). La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J. Virol. 67(7), 3798-807. Mizutani, A., Fukuda, M., Ibata, K., Shiraishi, Y., and Mikoshiba, K. (2000). SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms. J. Biol. Chem. 275(13), 9823-31. Mourelatos, Z., Abel, L., Yong, J., Kataoka, N., and Dreyfuss, G. (2001). SMN interacts with a novel family of hnRNP and spliceosomal proteins. EMBO J. 20(19), 5443-52. Nanda, S. K., and Leibowitz, J. L. (2001). Mitochondrial aconitase binds to the 3' untranslated region of the mouse hepatitis virus genome. J. Virol. 75(7), 3352-62. Neubauer, G., King, A., Rappsilber, J., Calvio, C., Watson, M., Ajuh, P., Sleeman, J., Lamond, A., and Mann, M. (1998). Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat. Genet. 20(1), 46-50. Rossoll, W., Kroning, A. K., Ohndorf, U. M., Steegbom, C., Jablonka, S., and Sendtner, M. (2002). Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons? Hum. Mol. Genet. 11(1), 93-105. Shen, X., and Masters, P. S. (2001). Evaluation of the role of heterogeneous nuclear ribonucleoprotein A l as a host factor in murine coronavirus discontinuous transcription and genome replication. Proc. Natl. Acad. Sci. USA 98(5), 2717-22. Shi, S. T., Huang, P., Li, H. P., and Lai, M. M. C. (2000). Heterogeneous nuclear ribonucleoprotein A l regulates RNA synthesis of a cytoplasmic virus. EMBO J. 19(17), 4701-11. Shi, S. T., Yu, G. Y., and Lai, M. M. C. (2003). Multiple type A/B heterogeneous nuclear ribonucleoproteins (hnRNPs) can replace hnRNP A l in mouse hepatitis virus RNA synthesis. J. Virol. 77(19), 10584-93. Spagnolo, J. F., and Hogue, B. G. (2000). Host protein interactions with the 3' end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication. J. Virol. 74(11), 5053-65. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stohlman, S. A., Baric, R. S., Nelson, G. N., Soe, L. H., Welter, L. M., and Deans, R. J. (1988). Specific interaction between coronavirus leader RNA and nucleocapsid protein./. Virol. 62(11), 4288-95. Strasser, K., and Hurt, E. (2001). Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yralp. Nature 413(6856), 648-52. Tahara, S. M., Dietlin, T. A., Nelson, G. W., Stohlman, S. A., and Manno, D. J. (1998). Mouse hepatitis virus nucleocapsid protein as a translational effector of viral mRNAs. Adv. Exp. Med. Biol. 440, 313-8. Zhang, X., and Lai, M. M. C. (1995). Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed. J. Virol. 69(3), 1637-44. 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 4 Coronavirus involves lipid rafts for virus entry and cell-cell fusion, but not virus release 4.1 Abstract Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (Thorp and Gallagher, 2004). However, the MHV receptor is not present in lipid rafts: thus, the mechanism of lipid rafts involvement is not clear. In this study, I defined the mechanism and extent of lipid raft involvement in MHV replication. I showed that MHV viral spike protein bound to non-raft membrane at 4°C but shifted to lipid rafts at 37°C, indicating a redistribution of membrane following virus binding, probably involving a lipid-rafts-associated cellular or viral factors. I found that the spike protein from plasma membranes were associated with lipid raffs, whereas those from Golgi membranes where MHV matures were not. Moreover, the buoyant density of the virion was not changed when viruses were generated from the cholesterol-depleted cells, indicating that MHV virion does not incorporate lipid rafts into the virion. These results indicated that virus release does not involve lipid rafts. Finally cell-cell fusion induced by MHV 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was delayed by M(3CD treatment. It is suggested that the viral entry and cell-cell fusion, but not virus release require specific interaction between the spike protein and unidentified cellular factors, which are associated with lipid rafts. 4.2 Introduction Fluid-mosaic model proposed by Singer and Nicholson (Singer and Nicolson, 1972) has long been proposed to explain the organization of membrane. Subsequently, lipid rafts were proposed as the functional lipid microdomains, which consist of cholesterol, sphingolipid and their associated proteins (Simons and Ikonen, 1997). Although their existence is still debatable, the presence of specific microdomains in biological membranes is a largely accepted concept. Based on the studies of model membranes, it is evident that cholesterol and sphingolipid in the membrane can form a highly ordered microdomains, distinct from the disordered liquid phase membranes of surrounding phospholipids (Lee, 2001). This organization confers their resistance to cold-detergent treatment and flotation to light buoyant density (Brown and London, 1998). Both properties are commonly used to identify the lipid rafts. Recent studies have suggested that lipid rafts play a role in a wide range of cellular events, including signal transduction, apoptosis, cell adhesion, and migration, synaptic transmission, and organization of the cytoskeleton, and protein sorting during endocytosis and exocytosis (Brown and London, 1998) (Simons and Toomre, 2000) (Harris and Siu, 2002) (Tsui-Pierchala et al., 2002). In addition to their roles in the cells, lipid rafts function as a docking site of the entry of viruses, 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bacteria and toxins, as well as of virus assembly and budding (Kovbasnjuk, Edidin, and Donowitz, 2001) (Suomalainen, 2002) (Rawat et al., 2003). Both enveloped and nonenveloped viruses use lipid rafts in various ways to enter the cells (Chazal and Gerlier, 2003). In case of non-enveloped viruses, virus entry begins with the attachment of virus to receptors, followed by subsequent internalization o f virus by the invagination of the plasma membrane and intracytoplasmic vesiculation. Lipid rafts are involved in the direct association of viruses with their receptors and internalization o f virus through caveolae. Simian virus 40 (SV40) is internalized into caveolae (Parton and Lindsay, 1999) after its binding to the receptor, major histocompatibility complex 1, which normally is not detected in lipid rafts (Cemy, Stockinger, and Horejsi, 1996). Echovirus typel (EV1) is also internalized into caveolae through the interaction with its receptor, a2(32- integrin, which is in lipid raft (Marjomaki et al., 2002). The entry of enveloped viruses involves the attachment of virus, followed by fusion between virus and cell membrane, which can be either plasma or endosomal membrane. Therefore, lipid rafts may be involved in the life cycle of enveloped viruses in several different ways, including virus entry; association of envelope glycoproteins with lipid rafts inside virion, interaction of virus envelope proteins with lipid rafts on the target membrane, and association of cellular receptors with lipid rafts. Hemaaglutinin (HA) of influenza virus (Scheiffele, Roth, and Simons, 1997), gpl20-gp40 of human immunodeficiency virus-1 (HIV-1) (Pickl, Pimentel-Muinos, and Seed, 2001) and glycoprotein of Ebola virus (Bavari et al., 2002) are associated with lipid rafts. In 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. case o f Semliki Forest virus (SFV), El protein is inserted selectively to cholesterol- rich microdomains in the target membrane (Ahn, Gibbons, and Kielian, 2002). CD4 and CCR5, the receptor and the coreceptor of HIV-1, respectively, are associated with lipid rafts (Del Real et al., 2002) (Kozak, Heard, and Kabat, 2002) (Popik, Alee, and Au, 2002). Involvement of lipid raft in virus assembly and budding has been well studied in influenza virus, Ebola virus and HIV-1. HA and neuraminidase (NA) of influenza virus cluster in lipid rafts and recruit M l matrix protein, which is critical for the virus assembly, to lipid rafts to promote the virus assembly within the lipid rafts (Ali et al., 2000). The matix protein, VP40 of Ebola virus, which is important in virus assembly and budding, localize and oligomerize in lipid rafts (Panchal et al., 2003). Moreover, Pr55gag of HIV-1, as with gpl20/gp40 (Pickl, Pimentel-Muinos, and Seed, 2001), also associates with lipid rafts during virus assembly (Ono and Freed, 2001) (Holm etal., 2003). Although there was no direct evidence that lipid rafts are involved in coronavirus replication, previous studies have implied that cholesterol and cholesterol-related environment may regulate coronavirus replication cycles; supplement of cholesterol on cells resulted in marked enhancement of MHV-induced cell fusion (Daya, Cervin, and Anderson, 1988) and hypercholesterolaemic diet increased the susceptibility to MHV 3 infection (Braunwald et al., 1991). Moreover, in the case of human coronavirus 229E (HCoV-229E), virus entry was inhibited by the depletion of cholesterol, resulting in the disruption of the viral association with CD 13, which is 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the cellular receptor (Nomura et al., 2004). Moreover, knockdown of caveolin-1 affected the entry of HCoV-229E, but not binding, although the significance of caveolin-1 in virus entry has yet to be answered. On the other hand, Thorp and Gallagher showed that cholesterol-rich microdomains were crucial for the virus entry and fusion of MHV, but MHV receptor (MHVR) did not associate with lipid rafts (Thorp and Gallagher, 2004), thus indicating that cholesterol-rich microdomains are implicated in yet another uncharacterized mechanisms rather than through receptor association in virus entry and cell-cell fusion. Here, I report that MHV does not incorporate the lipid rafts into virion and binds to non-rafts membrane, but shifts to lipid rafts membrane for virus entry. Furthermore, the viral spike (S) protein is not associated with lipid rafts on Golgi membrane, which is the site of virus assembly and budding (Klumperman et al., 1994), but is associated with lipid rafts on the plasma membrane, which is involved in cell-cell fusion. These results explain how lipid rafts are involved in MHV virus entry and cell-cell fusion, but not virus release. I also suggest that MHV entry requires a relocalization of the viral spike protein on cellular membrane during virus entry, probably involving cellular factors. 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3 Material and Methods Cells, viruses and antibodies DBT cell (Hirano et al., 1974), a mouse astrocytoma cell line, was cultured in Eagle’s minimal essential medium (MEM) supplemented with 7% newborn calf serum (NCS), 10% tryptose phosphate broth, and streptomycin-penicillin. 293A cell line was cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MHV-A59 was amplified in DBT cells and maintained in MEM containing 1% NCS. Modified vaccinia virus Ankara (MVA) that expresses T7 RNA polymerase was a kind gift from Dr. Bernard Moss and amplified in BHK21 cells. The polyclonal anti-MHV-A59 antibody was produced in rabbits by injection of purified MHV virion. The monoclonal anti-MHVR (CC1) was kindly provided by Dr. Kathryn V. Holmes. Monoclonal anti-transferrin receptor, anti-flotillin and anti- syntaxin 6 were purchased from Zymed Laboratories (San Francisco, CA), BD Biosciences (San Jose, CA), and Stressgen (Victoria, BC Canada), respectively. Plasmids cDNA encoding the viral spike protein was generated by RT-PCR from RNAs isolated from purified MHV virions and then cloned into pcDNA3.1 TOPO (Invitrogen, Carlsbad, CA). Truncated mutants of S protein were made from the cDNA of spike protein with specific sets o f primers. cDNA that expresses MHV 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. receptor, which is murine carcinoembryonic antigen gene family member (CGM1), was generated from mouse liver and cloned into mammalian expression vector, pECE (Yokomori and Lai, 1992). Cholesterol depletion, replenishment and detection of uptake of viral RNA DBT cells were seeded in 12-well plates and, at 90%-confluency, were incubated with lOmM methyl-(3-cyclodextrin (MpCD, Fluka, Milwaukee, WI) in MEM at room temperature for 30 min. In the replenishment experiments, cells were first incubated with M(3CD in MEM for 30 minutes. Afterwards, media was replaced by MEM containing ImM cholesterol (Sigma, Milwaukee, WI) for an hour at room temperature. After the treatment, media was changed into MEM containing 7% NCS and cells were incubated for up to 24 hr. Cholesterol level was assayed from total extracts of M(3CD-treated cells by collecting samples at every three hours by using Amplex Red Cholesterol Assay kit (Molecular Probes, Eugene, OR). Cell viability was assayed by using WST-1 reagent purchased from Roche (Indianapolis, IN). Briefly, Cells were seeded in 96-well plates; lOul of WST-1 was added to the cells, followed by 1-hr incubation at 37°C. The absorbance was measured using an ELISA reader at wavelength of 420-480nm with a reference wavelength >600nm. To detect viral RNA, untreated or MpCD-treated cells were infected with MHV- A59 at an M.O.I of 10 and then, after 1-hr virus adsorption followed by washing three times with PBS, were harvested at 1,2, 3, and 4 hour P.I. Total RNAs were isolated by phenol/chloroform extraction and ethanol precipitation. 5’-UTR of MHV RNA was detected by RT-PCR using specific primers. 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Purification of [35S]-labeled virion DBT cells were infected with MHV-A59 at an M.O.I of 10, and after 1-hour virus adsorption, cells were further incubated in 1% NCS-containing MEM for 5 hours. Cells were rinsed with Met/Cys-free MEM (Sigma, Milwaukee, WI) once and then starved in the 1% dialyzed NCS-containing MEM for 30min, followed by addition of lOOuCi of [35S]-translabeling mixture (ICN, Irvine, CA). Radiolabeled viruses were harvested at 16 hr P.I. and purified by sucrose gradient centrifugation. Supernatant from the cell culture was clarified by centrifugation at 4,500g for 30 min at 4°C, and then overlaid over 30 %- 50% sucrose in NTE (0.5 M NaCl, 10 mM Tris- HC1, 1 mM EDTA, pH 7.4), followed by ultracentrifugation in a Beckman SW28 rotor at 28,000 rpm for 3.5 hr. The interface was collected and pelleted by the ultracentrifugation at 100,000g for an hour. The purified virions were resuspended in either MEM for the binding experiment or NTE for further purification on continuous sucrose gradients (20%-60%) and centrifuged in a Beckman SW 41 Ti rotor at 37,000 rpm for 16hr. Samples were collected into 1ml fractions. Membrane flotation analysis Cells were lysed with hypotonic buffer (lOmM Tris-HCl, pH7.4, ImM EDTA) and passed through a 21G syringe twenty times. The lysates were centrifuged at l,400g for 5 min at 4°C to remove cell debris and nuclei. The postnuclear supernatant (PNS) were subjected to treatment with 1% TX-100 at 4°C or 37°C for 1 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hr. To separate membrane fractions from cytosolic fractions, detergent-treated PNS was subsequently placed over 10, 55, and 63% discontinuous sucrose gradient and centrifuged in a Beckman SW41Ti rotor at 37,000 rpm for 16hr at 4°C. Samples were collected into 1ml fractions and further used for immunoprecipitation or immunoblotting. In vitro binding assay of virion Radiolabeled and purified virion (lxlO 6 cpm) was incubated with DBT cells (lx l0 6 ) at 4°C or 37°C for an hour. After binding, cells were washed with PBS three times, and then, lysed with hypotonic buffer followed by treatment with 1% TX-100 at 4°C or 37°C for 1 hr. To isolate the detergent-resistant membranes, membrane flotation assay was done as described above. Each fraction further was immunoprecipitated with polyclonal anti-A59 antibody to detect viral proteins. Separation of plasma and Golgi membranes Cells (2.5xl07) were infected with MHV-A59 at an M.O.I of 10. At 6 hr P.I, cells were incubated with lOOuCi of [35S]-translabeling mixture (ICN, Irvine, CA). At 10 hr P.I., cells were harvested and resuspended in 0.5ml homogenization buffer (0.25M sucrose, ImM EDTA, lOmM HEPES-NaOH, pH 7.4), followed by passage through a 21G syringe twenty times. The homogenates were centrifuged at 2,000g for 10 min. The pellet was resuspended in the homogenization buffer; the centrifugation was repeated and the two supernatants were combined. The supernatants were 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. centrifuged at 100,000g for 1 hr to remove all cytosolic proteins, and the pellets, which contained membrane fractions, were dissolved in a 0.75ml of 30%(w/v) iodixanol (Axis-Shield PoC AS, Oslo, Norway). Membrane fractions were loaded at the bottom of a step-density gradient of iodixanol (2.5, 10, 17.5, and 25%(w/v) iodixanol; 2.5ml per step), prepared from a stock solution diluted in homogenization buffer. The gradients were centrifuged at 37,000 rpm for 3.5 hr in a Beckman SW41Ti rotor and fractionated into 0.8ml fractions; each fraction was used for immunoprecipitation and immunoblotting using specific antibodies. For the subsequent membrane flotation analysis, fractions from either plasma membranes or Golgi membranes were pooled and concentrated to 0.1ml using the centricon YM50 (Millipore, Bedford, MA) with repeated dilutions with hypotonic buffer. Samples were further subjected to membrane flotation analysis as described above. 4.4 Results Virus entry, but not other steps, of viral replication requires lipid rafts in the target membranes. To address the involvement of lipid rafts in MHV replication cycles, cholesterol was depleted in DBT cells by treating with lOmM M(3CD for 30 min at room temperature. Cholesterol level in the cells was reduced by almost 50% immediately upon treatment and remained low for nearly 6 hr. It returned to the normal level by 10-20 hr post-treatment (Fig 4-1 A). This treatment was not toxic to the cells as 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A ... ^ . No treatment 10mM MpCD £ O ) 3 O L_ 0 ) 4 - > w a > o J C o 12 15 18 21 24 27 6 -0.5 0 3 9 Hrs post treatment 4> 0.8 o c (0 n i— o V ) n flj 0.6 0.4 0.2 untreated H I 10mM MpCD - t - ■ T 0 2 18 Hrs post treatment Fig. 4-1-1. Cholesterol depletion by MpCD and its effects on MHV replication in DBT cells. A. Reduction of cholesterol level in cells. After treatment with 10mM MpCD for 30 min, cells were collected every three hours up to 27 hr after treatment. Cholesterol amount from lysates was assayed using Amplex Red Cholesterol Assay kit. B. Cytotoxicity of M|3CD to the cells. Cell viability was assayed at 0, 2 and 18 hr post-treatment using WST-1 reagent. 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. compared to untreated cells (Fig 4-1B). To examine the effects of cholesterol depletion on virus replication, cells were treated with lOmM MpCD for 30 min at various time points post-infection (Fig 4-1C, upper diagram), namely, untreated, pretreated, at 3 hr post-infection, and at 6 hr post-infection. Virus titers were determined from the supernatants collected from 6.5 to 10 hr post-infection (Fig 4- 1C). While pretreatment of cells with MPCD completely inhibited the virus production, treatments at any time points post-infection did not affect the virus titers even at a higher concentration of MPCD (20mM). This result indicates that cholesterol depletion affected virus entry, but not the later steps, of virus replication. To confirm the inhibitory effect of MpCD on virus entry, the viral RNAs inside the cells at the early time points after virus infection were detected by RT-PCR using 5’- UTR-specific primers. These RNAs represent the viral RNAs inside the cells as a result of virus entry. In untreated cells, viral RNAs were detected beginning at 1 hr post-infection. In contrast, after the MPCD treatment, the viral RNA was not detected until 4 hr post-infection. Uptake of viral RNA was restored to a similar level as that in the untreated cells after the cholesterol was replenished (Fig 4-2). This result suggests that virus entry was blocked by cholesterol depletion specifically. To understand the mechanism of inhibition of virus entry by cholesterol depletion, I first examined whether MPCD affected virus attachment. DBT cells were pretreated with lOmM MPCD for 30 min, and then incubated with the radiolabeled MHV for 1 hr at 4°C. After washing with PBS twice to remove unbound virion, bound radiolabeled MHV was measured by scintillation counting of the cell 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c i i r un-Tx pre-Tx 3hr post-Tx 6 hr post-Tx 3 3 .5 n — r~ 6 6 . 5 7 i i n 10 ~ r m r ....... : l .. a m i : ¥ . . | : m r a f . I l: ! ! ! S aw I M|3CD n MHV m virus titer 0 ) 6.00E+05 5.00E+05 4.00E+05 w 3.00E+05 2.00E+05 1.00E+05 0.00E+00 ] untreated 3 pre-treated 3hr post-treated 6 hr post-treated I 1 I 10mM MPCD I _ l _ ] ........ ..... .. ....I. 20mM M(3CD Fig. 4-1-2. Cholesterol depletion by MpCD and its effects on MHV replication in DBT cells. C. Virus titers released from MpCD- treated cells. Upper diagram shows the time frames of the experiment. Cells were either pretreated before virus infection or treated at 3 or 6 hr P.I., with 10mM or 20mM MpCD for 30 min. Culture supernatant was collected from 6.5 hr to 10 hr P.I., and plaque assay was performed. Samples were duplicated and experiments repeated three times. Arrow bars indicate the standard deviations of three independent experiments. 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O h 1h 2h 3h 4h Untreated 10mM MpCD 10mM MpCD+ 1mM Cholesterol Untreated 10mM MpCD 10mM MpCD+ 1mM Cholesterol MHV- 5’-UTR Act in Fig. 4-2. The effect of MpCD on the viral RNA uptake. Cells were either untreated or treated with 10mM MpCD for 30 min, and then infected with MHV-A59. In the cholesterol replenishment experiment, MpCD was removed after 30 min-treatment and cells were incubated with 1mM cholesterol in MEM for an hour before viral infection. Total RNAs were isolated at 0, 1, 2, 3, and 4 hr post-infection. MHV RNA was detected by RT-PCR using 5’-UTR-specific primers. Actin was used as a loading control. 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. extracts. Similar amount (16-20%) of the radiolabled MHV bound to both untreated and MpCD-treated cells (Fig 4-3A), indicating that virus attachment was not affected by the MpCD treatment. In addition, the MPCD treatment did not affect the surface expression level of MHV receptor (data not shown). Next, I addressed the possibility that MHVR associates with lipid rafts. Lysates from the MHVR-ovexpressing 293A cells were treated with 1% TX-100 for an hour at 4°C, and then membrane flotation analysis was performed. In the absence o f detergent treatment, most of MHVR was detected in membrane fractions; but, after treatment with 1% TX-100 at 4°C, most of MHVR was in the detergent-soluble fractions (data not shown). As controls, flotillin, lipid rafts marker, was in the detergent-resistant membrane, whereas transferrin receptor was found in the detergent-soluble membrane. In addition, I addressed the possibility that virus infection might induce the relocalization of MHVR to lipid rafts. However, most of MHVR still was detected in detergent-soluble fractions upon virus infection. My results were consistent with the results done by Thorp and Gallagher (Thorp and Gallagher, 2004), which also showed that MHVR was not associated in lipid rafts. Together, these results exclude the possibility that MpCD inhibits virus entry by affecting the virus binding to MHVR on lipid rafts. Nevertheless, it is still possible that virus entry may require the interaction of the viral proteins with lipid rafts in the target membrane after virus binding. To address this possibility, the radiolabeled virions were incubated with MHVR-overexpressing 293A cells at 37°C for 1 hr and the lysates were subjected to membrane flotation 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c o ■> > X o D ) C £ £ 1 5 25 20 15 10 5 0 10mM OmM MpCD B Top > X MHVR ' -MHV -+M H V flotillin TfR Bottom ► No detergent 1% TX-100, 4°C Fig. 4-3. Effects of cholesterol depletion on virus binding and association of MHV receptor with lipid rafts. A. Binding of radiolabeled MHV virion. [3 5S]-labeled MHV virion (1x10® cpm) was incubated with either untreated cells or cells treated with 10mM MpCD for an hour at 4°C. Unbound virion was removed by washing with PBS three times. Cells were harvested and resuspended in hypotonic buffer, and the radioactivity was determined in a scintillation counter. Each sample was triplicated and arrow bars indicate the standard deviation. B. Association of MHVR with lipid rafts. MHVR-overexpressing 293A cells were either uninfected or infected with MHV- A59, and membrane flotation analysis was done after treatment with 1% TX- 100 for an hour at 4°C. MHVR was detected by immunoblotting. Flotillin and transferrin receptor were used as positive and negative control, respectively, for lipid rafts-associated proteins. 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. assay, followed by immunoprecipitation with polyclonal anti-A59 antibody. The results showed that after the virus binding at 4°C, most of the viral S and N proteins were associated with the membrane that was disrupted by treatment with 1% TX-100 at 4°C and 37°C, consistent with the previous interpretation that virus binds to MHVR on the non-rafts membrane (Fig 4-4A). However, after virus binding at 37°C, viral S protein was resistant to treatment with 1% TX-100 at 4°C (Fig 4-4B). The membrane association of S protein was disrupted with treatment o f 1% TX-100 at 37°C, consistent with the interpretation that it is associated with lipid rafts. These results indicate that S proteins interact with lipid rafts during virus entry, evidently not through the MHVR. This interaction may come from either direct association of S protein with lipid rafts in the virion or association of S proteins with lipid rafts as a result of interaction with unidentified cellular factors, which are in lipid rafts. These possibilities will be addressed later. These results imply that redistribution of viral and cellular proteins on membranes may occur during virus entry, and that the process involves the shift of viral and/or cellular factors from non-raft to rafts. Lipid rafts are not incorporated into MHV virion. I next explored the possibility that MHV particles contained lipid rafts. To manipulate cholesterol levels in the virion, cells were first treated with M(3CD and MHV were produced from the cholesterol-depleted cells. Buoyant density of the virus was compared with that of the virus produced from the untreated cells (Fig 4- 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o > c ■ 5 c o o 130 f 100 73 54 130 100 73 54 O ) c ' ■ 5 c 5 o o N . C O 130 100 73 B 54 V Top < - Bottom -> 1 2 3 4 5 6 7 8 9 H s No Tx * N 130 100 r 130 100 130 100 V Tx-100,4°C Tx-100,37°C No Tx Tx-100,4°C Tx-100,37°C Fig. 4-4. Redistribution of MHV virion during virus entry. [3 5 S]- labeled MHV virion (1x106 cpm) was incubated with MHVR-overexpressing 293A cells for an hour at ether 4°C (A) or 37°C (B). Lysates were treated with 1% TX-100 for an hour at 4°C or 37°C, and subsequently membrane flotation analysis was done. Viral S and N proteins were detected by immunoprecipitaiton with polyclonal anti-A59 antibody. 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5A). Virus particles generated from M(3CD-treated cells showed the same buoyant density as that from untreated cells, suggesting that lipid rafts are not a significant component of MHV virion. Furthermore, I performed the membrane flotation analysis of the virus particles to examine whether any viral proteins are associated with lipid rafts in the virion. S and N proteins were mostly detected in the membrane fractions without any treatment with detergent and shifted to detergent-soluble fractions by treatment with 1% TX-100 at 4°C for 1 hr, indicating that viral proteins in the virion did not associate with lipid rafts (Fig 4-5B). Next, I examined whether intracellular viral proteins associate with lipid rafts during virus assembly and budding. Since virus assembly and budding of MHV occurs on Golgi membranes (Klumperman et al., 1994), I fractionated Golgi membranes from the total membranes by iodixanol gradients (Fig 4-6A). Syntaxin6 (Bock et al., 1997) and transferrin receptor (Harder et al., 1998) were used as the markers for Golgi and plasma membranes, respectively. As expected, S proteins were detected in both plasma and Golgi membranes. Fractions from plasma membranes or Golgi membranes were pooled separately, and detergent-resistance of viral proteins was examined by membrane flotation analysis. All of S and N proteins from Golgi membranes were detected in detergent-soluble fractions, confirming that virus assembly and budding does not require lipid rafts (Fig 4-6B, right). In contrast, small fractions of S protein from plasma membranes was resistant to treatment with 1% TX-100 at 4°C (Fig 4-6B, left). 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20-60% continuous sucrose gradient < ► 1 2 3 4 5 6 7 8 9 10 11 12 13 14 OmM MpCD 10mM MpCD N B Top No-Tx Bottom Top TX-100, 4°C Bottom < ------------------------------------ ► A------------------------------------ ► 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 130- 100 - 7 3 - 4 3 - Fig. 4-5. Lack of incorporation of lipid rafts into MHV virion. A. Buoyant density of virion produced from cells untreated or treated with 10mM M(3CD. Culture supernatant was collected, purified by two-step sucrose centrifugations. Samples were collected into 1ml fractions and viral N proteins were detected by immunoblotting. B. Lack of association of viral proteins with lipid rafts in the virion. [3 5 S]-labeled MHV virion was treated with 1% TX-100 for an hour at 4°C, and then membrane flotation analysis was done. The viral S and N proteins were detected by immunoprecipitation with polyclonal anti-A59 antibody. 130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 2.5-30% discontinuous loxadinol gradient < ► 1 2 3 4 5 6 7 8 9 10 11 1213 1415161718192021 22 TfR syntaxin6 S protein Plasma membrane Golgi Fig. 4-6-1. Raft-association of the viral S proteins in plasma and Golgi membranes. A. Fractionation of total membranes into plasma and Golgi membranes. Plasma and Golgi membranes were fractionated in 2.5%, 10%, 17.5%, and 25% iodixanol step gradient. Samples were collected into 0.8ml fractions. Transferrin receptor (TfR) and syntaxin6 were used as plasma and Golgi markers, respectively. S proteins were detected by radio- immunoprecipitation. 131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B PM 1 2 3 4 5 6 7 8 9 180' TX-100 100 4°C 180 TX-100 100 37°C 73 54‘ 4 s 4 N Golgi 1 2 3 4 5 6 7 8 9 TX-100 4°C 180 100 4 S 4 N Fig. 4-6-2. Raft-association of the viral S proteins in plasma and Golgi membranes. B. Association of viral proteins from plasma and Golgi membranes with lipid rafts. Fractions from #8-#14 or #21 and #22 of panel A were pooled and analysed by membrane flotation gradients. Viral proteins were detected by immunoprecipitation with polyclonal anti-A59 antibody. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These results were consistent with the previous experiments (Fig 4-1C), which showed that virus titers were not affected when M(3CD was treated after the virus infection. I conclude that MHV assembly and release do not involve lipid rafts, in contrast to many other enveloped viruses (Suomalainen, 2002). Cell-Cell fusion requires lipid rafts MHV infection induces the morphological changes in cells by inducing the cell cell fusion. Therefore, I asked whether cell-cell fusions are affected by cholesterol depletion. DBT cells were treated with lOmM M(3CD either before virus infection or after virus infection (2 and 4 hr P.I.). Syncytia formation was examined at 6 and 8 hr post-infection (Fig 4-7). When cells were pretreated with M(3CD, cell-cell fusion was completely blocked, obviously as a result of the inhibition of virus entry. Surprisingly, cell-cell fusion was much delayed when M(3CD was treated at 2 and 4 hr P.I.. Under this condition, the virus titer released from the cells was not affected (Fig 4-1). This result indicates that cell-cell fusion also requires lipid rafts. Since fusion processes are mediated mainly by viral S protein, I examined whether S proteins are involved in lipid rafts during cell-cell fusion. As shown above (Fig 4-6B), a small fraction of S protein on plasma membrane was associated with detergent-resistant membrane fractions, which shifted to detergent-soluble fractions after treatment with 1% TX-100 at 37°C. Finally, I found that some of S protein, when overexpressed alone in 293A cells, localized in detergent-resistant fractions 133 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MpCD: UT 6hr P.l. 8hr P.l. pre-Tx 2hr P.l. 4hr P.l. A is _ ■ , * i" .V ‘ 41* - $ t J - \ ,v * ■; I* * M " i t ] " >«■ 5'/ - K t~ „ ^ „ „r * / W / »V . . .... - ■ •:* . 0 : y pr* * * s m ^ ' 9 ' * * “ & * * " v - n * * - v ' » * . 5 . * 4 i ! .'"t',. ^ k ■ * , Fig. 4-7. Inhibition of cell-cell fusion by MpCD. Cells were treated with 10mM M(3CD at several different time points during virus infection; pretreatment, treated at 2 and 4 hr P.I.. Syncytia formation was examined at 6 and 8 hr post-infection under the microscope. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Fig 4-8A), thus indicating that S proteins do not require other viral proteins for the association with lipid rafts. Finally, I tried to identify the domains in the S protein required for the association with lipid rafts. Three truncated mutants of S protein were generated by manipulating the transmembrane domain and cytoplasmic tails (Fig 4-8B, left top); S-C9, which contained only 9 amino acids at the C-terminal ends, S-CO, which did not have any cytoplasmic tails, and S-CV, of which transmembrane domain was replaced by the transmembrane domain of vesicular somatitis virus glycoprotein (VSVG). It has been reported that VSVG protein does not associate with lipid rafts (Scheiffele et al., 1999) (Vincent, Gerlier, and Manie, 2000). All three truncated mutants of S protein were expressed in similar amounts in the cells (Fig 4-8B, left bottom). S-C9 and S- C0 of S protein were still able to associate with lipid rafts, whereas S-CV did not (Fig 4-8B, right). Thus, transmembrane domain of S protein is important for its interaction with lipid rafts. 4.5 Discussion Thorp and Gallagher (Thorp and Gallagher, 2004) first reported that virus entry and cell-cell fusion was greatly affected by the depletion of cholesterol. This result was puzzling since MHVR was not associated with lipid rafts. I also showed that MHV did not incorporate lipid rafts into virion. My studies presented here provided explanations for these observations. I found that MHV binds initially to the non-raft 135 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TX-100 37°C Top TX-100 4°C Bottom ► < s I B ™ s ------------------------------------------------------------ s-co ---------------------------------------------- 1 SC9 ------------------------------------- 1 VSVG-TM Vec S S-CO S-C9 S-CV Top TX-100, 4°C Bottom — ► 1 2 3 4 5 6 7 8 9 S-CO S-C9 S-CV Fig.4-8. Determinination of rafts-association domains of S proteins. A. Rafts-association of S protein overexpressed in 293A cells. B. Domains required for lipid rafts association of S protein. Top shows the diagram of truncated mutants of S protein. Expression of truncated mutants by radioimmunoprecipitation. Membrane flotation analysis for the truncated mutants. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. membrane (at 4°C), presumably through MHVR. As virus entry occurs (at 37°C), the S protein became associated with lipid rafts (Fig 4-4). Thus, there is a redistribution of MHV from non-rafts membrane to lipid rafts during virus entry. This redistribution very likely involves cellular factors, which interact with either MHVR or viral S protein after it binds to MHVR. This membrane shift has also been observed in another coronavirus HCoV-229E (Nomura et al., 2004). The internalization of HCoV-229E induced by the incubation of virus at 37°C was decreased by the depletion of cholesterol, while the binding of virus was not affected. Therefore, this redistribution process of shifting to lipid rafts may be the common process in coronavirus. In case of HcoV-229E, redistribution process involves the sequestration o f the viral receptor, CD13, to caveolae. At this time, it has not been demonstrated whether caveolae is also required for MHV entry. It is possible that some host factors, which may interact with S protein, may be relocalized to caveolae during MHV entry. However, this will not be through MHVR, since MHVR did not relocalize to lipid rafts during virus infection. Moreover, it has been shown that forced association of MHVR with lipid rafts by adding the GPI anchor did not enhance the virus infectivity, thus indicating that the association of MHVR with lipid rafts is not crucial for the virus entry. Similar case was observed in Herpes simplex virus (HSV) (Bender et al., 2003); cellular receptors, herpesvirus entry mediator (HVEM) and nectin-1, did not associate with lipid rafts, but one of glycoprotein of HSV, gD, was found associated with lipid rafts, suggesting that unknown cellular cofactors may mediate the recruitment of gD to lipid rafts. 137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I also found that MHV assembly and release did not involve lipid rafts. Consistent with this observation, I found that the viral S protein on the Golgi membrane, where MHV virion is assembled and released, is not associated with lipid rafts, even though the Golgi membrane contains lipid rafts (Simons and Ikonen, 2000). This finding is in contrast to many other enveloped viruses, such as HIV, which contains lipid rafts in the virion and viral proteins that are required for virus assembly and budding are associated with lipid rafts. Significantly, the MHV S protein is associated with lipid rafts in the plasma membrane; correspondingly, the cell-cell fusion induced by MHV is affected by cholesterol depletion. The possible explanation for this different dependency is that S proteins may be recruited to lipid rafts through the interaction with unidentified factors, which is missing in Golgi. These factors obviously are not MHVR or other viral factors; since MHVR did not associate with lipid rafts, and S protein alone could associate with lipid rafts. My observations that both virus entry and cell-cell fusion were inhibited by cholesterol depletion implied that lipid rafts might be involved in both processes following the common mechanism. Both processes are initiated by binding of S protein to MHVR, followed by the fusion between two different membranes (virus cell and cell-cell). In the case of virus entry, the S-MHVR binding triggers the relocalization of the virus to a lipid rafts membrane. In the case of cell-cell fusion, the S protein is localized on lipid rafts on the plasma membrane. Thus, the association of S protein with lipid rafts is crucial for virus entry and cell-cell fusion. 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A number of studies have showed that enveloped virus, such as HIV, contain lipid rafts in the virion, and viral proteins that are required for virus assembly and budding associate with lipid rafts (Holm et al., 2003) (Graham et al., 2003). While all studies focus on the viruses that bud out of plasma membrane, it has not been studied on viruses that bud out of the intracellular membranes. However, cholesterol as a major component of lipid rafts is rich mostly in plasma membranes and endosome, but still is in Golgi membrane via the recycling of cholesterol transport, thus making it likely that lipid rafts also exist on Golgi membrane (Simons and Ikonen, 2000). Therefore, it could not be ruled out that MHV, of which assembly and budding occurs on Golgi membrane, may require lipid rafts for their release. In summary, I conclude that MHV does require lipid rafts in virus entry and cell cell fusion, but it does not in virus release. The involvement of lipid rafts in MHV is mediated by the interaction of S protein with lipid rafts. 139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.7 References Ahn, A., Gibbons, D. L., and Kielian, M. (2002). The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains. J Virol 76(7), 3267-75. Ali, A., Avalos, R. T., Ponimaskin, E., and Nayak, D. P. (2000). Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M l protein. J Virol 74(18), 8709-19. Bavari, S., Bosio, C. M., Wiegand, E., Ruthel, G., Will, A. B., Geisbert, T. W., Hevey, M., Schmaljohn, C., Schmaljohn, A., and Aman, M. J. (2002). Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J Exp M ed 195(5), 593-602. Bender, F. C., Whitbeck, J. C., Ponce de Leon, M., Lou, H., Eisenberg, R. J., and Cohen, G. H. (2003). Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry. J Virol 77(17), 9542-52. Bock, J. B., Klumperman, J., Davanger, S., and Scheller, R. H. (1997). Syntaxin 6 functions in trans-Golgi network vesicle trafficking. Molecular Biology o f the Cell 8(7), 1261-71. Braunwald, J., Nonnenmacher, H., Pereira, C. A., and Kim, A. (1991). Increased susceptibility to mouse hepatitis virus type 3 (MHV3) infection induced by a hypercholesterolaemic diet with increased adsorption of MHV3 to primary hepatocyte cultures. Res Virol 142(1), 5-15. Brown, D. A., and London, E. (1998). Structure and origin of ordered lipid domains in biological membranes. Journal o f Membrane Biology 164(2), 103-14. Cemy, J., Stockinger, H., and Horejsi, V. (1996). Noncovalent associations of T lymphocyte surface proteins. Eur J Immunol 26(10), 2335-43. Chazal, N., and Gerlier, D. (2003). Vims entry, assembly, budding, and membrane rafts. Microbiol M ol Biol Rev 67(2), 226-37, table of contents. Daya, M., Cervin, M., and Anderson, R. (1988). Cholesterol enhances mouse hepatitis virus-mediated cell fusion. Virology 163(2), 276-83. Del Real, G., Jimenez-Baranda, S., Lacalle, R. A., Mira, E., Lucas, P., Gomez- Mouton, C., Carrera, A. C., Martinez, A. C., and Manes, S. (2002). Blocking of HIV- 1 infection by targeting CD4 to nonraft membrane domains. J Exp M ed 196(3), 293- 301. 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Graham, D. R., Chertova, E., Hilbum, J. M., Arthur, L. O., and Hildreth, J. E. (2003). Cholesterol depletion of human immunodeficiency virus type 1 and simian immunodeficiency virus with beta-cyclodextrin inactivates and permeabilizes the virions: evidence for virion-associated lipid rafts. J Virol 77(15), 8237-48. Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998). Lipid domain structure of the plasma membrane revealed by patching of membrane components. Journal o f Cell Biology 141(4), 929-42. Harris, T. J., and Siu, C. H. (2002). Reciprocal raft-receptor interactions and the assembly of adhesion complexes. Bioessays 24(11), 996-1003. Hirano, N., Fujiwara, K., Hino, S., and Matumoto, M. (1974). Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture. Archiv fur die Gesamte Virusforschung 44(3), 298-302. Holm, K., Weclewicz, K., Hewson, R., and Suomalainen, M. (2003). Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100. J Virol 77(8), 4805-17. Klumperman, J., Locker, J. K., Meijer, A., Horzinek, M. C., Geuze, H. J., and Rottier, P. J. (1994). Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. Journal o f Virology 68(10), 6523-34. Kovbasnjuk, O., Edidin, M., and Donowitz, M. (2001). Role of lipid rafts in Shiga toxin 1 interaction with the apical surface of Caco-2 cells. J Cell Sci 114(Pt 22), 4025-31. Kozak, S. L., Heard, J. M., and Kabat, D. (2002). Segregation of CD4 and CXCR4 into distinct lipid microdomains in T lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus. J Virol 76(4), 1802-15. Lee, A. (2001). Membrane structure. Current Biology 11(20), R811-4. Marjomaki, V., Pietiainen, V., Matilainen, H., Upla, P., Ivaska, J., Nissinen, L., Reunanen, H., Huttunen, P., Hyypia, T., and Heino, J. (2002). Internalization of echovirus 1 in caveolae. J Virol 76(4), 1856-65. Nomura, R., Kiyota, A., Suzaki, E., Kataoka, K., Ohe, Y., Miyamoto, K., Senda, T., and Fujimoto, T. (2004). Human coronavirus 229E binds to CD 13 in rafts and enters the cell through caveolae. J Virol 78(16), 8701-8. 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ono, A., and Freed, E. O. (2001). Plasma membrane rafts play a critical role in HIV- 1 assembly and release. Proc Natl Acad Sci U S A 98(24), 13925-30. Panchal, R. G., Ruthel, G., Kenny, T. A., Kallstrom, G. H., Lane, D., Badie, S. S., Li, L., Bavari, S., and Aman, M. J. (2003). In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding. Proc Natl Acad Sci U S A 100(26), 15936-41. Parton, R. G., and Lindsay, M. (1999). Exploitation of major histocompatibility complex class I molecules and caveolae by simian virus 40. Immunol Rev 168, 23-31. Pickl, W. F., Pimentel-Muinos, F. X., and Seed, B. (2001). Lipid rafts and pseudotyping. J Virol 75(15), 7175-83. Popik, W., Alee, T. M., and Au, W. C. (2002). Human immunodeficiency virus type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into CD4(+) T cells. J Virol 76(10), 4709-22. Rawat, S. S., Viard, M., Gallo, S. A., Rein, A., Blumenthal, R., and Puri, A. (2003). Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review). M ol Membr Biol 20(3), 243-54. Scheiffele, P., Rietveld, A., Wilk, T., and Simons, K. (1999). Influenza viruses select ordered lipid domains during budding from the plasma membrane. J Biol Chem 274(4), 2038-44. Scheiffele, P., Roth, M. G., and Simons, K. (1997). Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain. E m boJ 16(18), 5501-8. Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387(6633), 569-72. Simons, K., and Ikonen, E. (2000). How cells handle cholesterol. Science 290(5497), 1721-6. Simons, K., and Toomre, D. (2000). Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1(1), 31-9. Singer, S. J., and Nicolson, G. L. (1972). The fluid mosaic model o f the structure of cell membranes. Science 175(23), 720-31. Suomalainen, M. (2002). Lipid rafts and assembly of enveloped viruses. Traffic 3(10), 705-9. 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thorp, E. B., and Gallagher, T. M. (2004). Requirements for CEACAMs and cholesterol during murine coronavirus cell entry. J Virol 78(6), 2682-92. Tsui-Pierchala, B. A., Encinas, M., Milbrandt, J., and Johnson, E. M., Jr. (2002). Lipid rafts in neuronal signaling and function. Trends Neurosci 25(8), 412-7. Vincent, S., Gerlier, D., and Manie, S. N. (2000). Measles virus assembly within membrane rafts. J Virol 74(21), 9911-5. Yokomori, K., and Lai, M. M. (1992). The receptor for mouse hepatitis virus in the resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection. J Virol 66(12), 6931-8. 143 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 5 Conclusion and Future directions 5.1 Roles of Host factors involved in MHV replication A number of studies have shown that host factors play important roles in virus replication, including virus entry, replication, assembly and budding. Our studies showed that several proteins that belong to hnRNP family are involved in MHV replication, especially modulation MHV RNA synthesis. However, it is still not clear how these host factors function mechanistically to regulate MHV replication. They may function in several ways (Fig 5-1), which are not mutually exclusive. First, they may play a role as an RNA chaperon, thus making the highly structured untranslated regions favorable for replication and transcription. Second, they may promote protein-protein interactions to recruit factors required for replication at the site of replication. Third, they may mediate 5’-3’ end-crosstalk by interacting with other proteins or viral RNA, thus facilitating the replication of virus. Fourth, They may be involved in modulating the switches of the replicationl events during virus replication. Fifth, they may function to protect viral RNA against cellular or viral RNA processing enzymes. Sixth, they may direct the viral RNA to the specific site of replication. Lastly, they may regulate the stability of viral RNA. 144 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5’ RNA chaperon? “= > Recruiting proteins? 5’-3’ crosstalk? Switching of replicational events |1 Protective role against cellular attack Fig 5-1 Roles of host factors in MHV replication Reproduced with permission of the copyright owner. Further reproduction prohibited without permission PTB, as an abundant hnRNP protein, modulate a number of different steps in gene expression. The well-known function of PTB is to regulate alternative splicing, acting as a selective splicing repressor by competing for the binding site with U2AF that functions in a positive way for the regulation of splicing (Valcarcel and Gebauer, 1997). PTB has also been shown to play a role in modulating IRES-dependent translation of both viral and cellular RNA (Valcarcel and Gebauer, 1997). In addition, it has been shown that PTB was involved in 3’-end processing of mRNA by modulating efficiency of polyadenylation (Castelo-Branco et al., 2004). Besides PTB, several reports also have shown that other host factors are involved in regulating viral translation. For example, in case of picomavirus, PTB, La autoantigen, upstream of n-ras protein (unr) and poly (rC)-binding protein 2 (PCBP2) have been implicated in IRES-dependent translation (Jackson, 2002). In addition, a very recent paper showed that NSAP1, human homolog of SYNCRIP, enhanced IRES-dependent translation of HCV (Kim, 2004). Although MHV RNAs are translated by host translational machinery in a cap-dependent way, I still could not exclude the possibility that PTB and SYNCRIP may be involved in viral RNA translation. In contrast to poliovirus and HCV, our studies showed that at least PTB and SYNCRIP were not directly involved in MHV translation, although other unidentified factors may be required for the regulation of MHV translation. These results suggest that PTB and SYNCRIP are involved in some steps of MHV replication, other than the regulation of viral translation. 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Huang and Lai (Huang and Lai, 2001) showed that PTB and hnRNP A l bound to 5’- and 3’-ends, respectively of MHV RNA, and interact with each other, thus mediating the 5’-3’ end crosstalk. It has been suggested that 5’-3’ end crosstalk is important in modulating the replication of virus. In the case of poliovirus, an RNP complex formed around the 5’-cloverleaf RNA structure interacts with the poly (A) binding protein bound to the 3 ’ poly (A) tail, thus linking the ends of the viral RNA and effectively circularizing it (Herold and Andino, 2001). At this time, it is not clear whether SYNCRIP may also play a role in the formation of MHV genomic circularization. It will be interesting to ask whether SYNCRIP interacts with PTB or hnRNP A l and promote 5’-3’ ends crosstalk of MHV RNA. It is still not clear how SYNCRIP functions in the cells, although it has been suggested that it may be involved in the regulation of mRNA stability. A recent report showed that SYNCRIP was detected in RNA-transporting granules that direct RNAs to specific sites for its normal functions (Kanai, Dohmae, and Hirokawa, 2004). Therefore, it is possible that SYNCRIP may also function in guiding MHV RNA to a certain compartment inside cells during replication. Interestingly, SYNCRIP, but not PTB and hnRNP A l, was detected inside MHV virion (data not shown), thus suggesting that SYNCRIP may have an additional role in MHV replication cycle, such as helping the encapsidation of viral RNA. 147 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.2 Implication of lipid rafts in MHV replication Based on my studies, it is clear that lipid rafts are important in regulating virus entry and cell-cell fusion, but not virus release. The involvement of lipid rafts was ediated through the viral S proteins, as supported by the results that S proteins were associated with lipid rafts during virus entry and cell-cell fusion, whereas they were not associated with lipid rafts during virus release or inside the virion. Moreover, these results are consistent with the finding that S proteins on plasma membrane, which is the site for virus-cell and cell-cell fusion, were associated with lipid rafts, but S proteins on Golgi membrane, which is the site for virus assembly and budding, were not (Fig 5-2). It is interesting to ask how S proteins show different dependency on lipid compositions on the plasma and the Golgi membranes. Conceivably, S proteins may interact with a cellular factor that is associated with lipid rafts in plasma membranes, is missing in Golgi membranes. Therefore, identification of cellular factors will be crucial to address the mechanism of lipid rafts involvement in MHV replication. Surprisingly, I found that there is a redistribution process during virus entry from non-rafts to lipid rafts (Fig 5-3). This shift was also observed in human coronavirus, HCoV-229E, thus indicating that this may be a common mechanism in coronavirus life cycle (Nomura et al., 2004). In the case of HCoV-229E, this shift requires the sequesterization of its receptor to caveolae, which is in lipid rafts. However, in the case of MHV, MHVR did not localize in lipid rafts and did not relocalize to lipid 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Viral RNA and protein Non-raft t Spike protein Fig 5-2 Different rafts-dependence of spike protein on Golgi and plasma membrane. Spike protein on Golgi membrane, which is the site of virus assembly and budding, is not associated with lipid rafts, whereas it is associated with lipid rafts on plasma membrane, which is the site of virus entry and fusion. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. rafts during virus infection. Therefore, it is likely that other unidentified factors that are in lipid rafts, but not MHVR, mediate the redistribution process during virus entry. These unidentified factors obviously will interact with S proteins, since S proteins were shifted from non-rafts to lipid rafts during virus entry. Moreover, it will be interesting to address whether caveolae is important during MHV entry, similar to HCoV-229E. Finally, it is intriguing to ask why lipid rafts are required for virus entry and cell cell fuion. Generally, lipid rafts provide the functional microdomains by clustering proteins involved in various cellular events. Therefore, it is suggested that several proteins required for virus-cell and cell-cell fusion will be sequestered in lipid rafts, thus facilitating the fusion events and subsequent signaling events. However, it is still questionable why lipid rafts are not involved in virus release, though Golgi membranes contain the lipid rafts. In summary, my research has shown that host factors are important in various steps in MHV replications, including virus entry, replication/ transcription and fusion. 150 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U b .U U U Non-raft (4°C binding) redistribution process nrff»nnn»n u m a u u u f l u Lipid rafts (37°C binding) unknown factors?? Fig 5-3 Redistribution process during virus entry. MHV binds to the cells by interacting with MHV receptor, which is not associated with lipid rafts (4°C binding). During entry, it relocalizes to lipid rafts, probably by interacting with unidentified cellular factors (37°C binding). 151 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.3 References Castelo-Branco, P., Furger, A., Wollerton, M., Smith, C., Moreira, A., and Proudfoot, N. (2004). Polypyrimidine tract binding protein modulates efficiency of polyadenylation. Mol Cell Biol 24(10), 4174-83. Herold, J., and Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Molecular Cell 7(3), 581-91. Huang, P., and Lai, M. M. (2001). Heterogeneous nuclear ribonucleoprotein al binds to the 3'-untranslated region and mediates potential 5'-3’ -end cross talks of mouse hepatitis virus RNA. Journal o f Virology 75(11), 5009-17. Jackson, R. J. (2002). "Proteins involved in the function of picomavirus internal ribosome entry sites." Molecular biology of picomavirus (B. L. S. a. E. Wimmer, Ed.) ASM press, Washington DC. Kanai, Y., Dohmae, N., and Hirokawa, N. (2004). Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron 43(4), 513-25. Kim, J. H„ Paek, K.Y., Ha, S.H., Cho, S., Choi, K„ Kim, C.S., Ryu, S.H., Jang, S.K (2004). A Cellular RNA-Binding Protein Enhances Internal Ribosomal Entry Site- Dependent Translation through an Interaction Downstream of the Hepatitis C Vims Polyprotein Initiation Codon. Mol Cell Biol 24(18), 7878-7890. Nomura, R., Kiyota, A., Suzaki, E., Kataoka, K., Ohe, Y., Miyamoto, K., Senda, T., and Fujimoto, T. (2004). Human coronavirus 229E binds to CD 13 in rafts and enters the cell through caveolae. J Virol 78(16), 8701-8. Valcarcel, J., and Gebauer, F. (1997). Post-transcriptional regulation: the dawn of PTB. Curr Biol 7(11), R705-8. 152 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Alphabetized Bibliography Ahn, A., Gibbons, D. L., and Kielian, M. (2002). The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains. J Virol 76(7), 3267-75. Ali, A., Avalos, R. T., Ponimaskin, E., and Nayak, D. P. (2000). Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M l protein. J Virol 74(18), 8709-19. Ali, N., and Siddiqui, A. (1995). Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation. J. Virol. 69, 6367-6375. Anderson, R., and Wong, F. (1993). Membrane and phospholipid binding by murine coronaviral nucleocapsid N protein. Virology 194(1), 224-32. Andino, R., Boddeker, N., Silvera, D., and Gamamik, A. V. (1999). Intracellular determinants of picomavirus replication. Trends in Microbiol. 7(2), 76-82. Back, S. H., Kim, Y. K., Kim, W. J., Cho, S., Oh, H. R., Kim, J. E., and Jang, S. K. (2002). Translation of polioviral mRNA is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3C(pro). J. Virol. 76, 2529-2542. Banner, L. R., Keck, J. G., and Lai, M. M. (1990). A clustering of RNA recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavims. Virology 175(2), 548-55. Baric, R. S., Fu, K., Schaad, M. C., and Stohlman, S. A. (1990). Establishing a genetic recombination map for murine coronavims strain A59 complementation groups. Virology 177(2), 646-56. Baric, R. S., Nelson, G. W., Fleming, J. O., Deans, R. J., Keck, J. G., Casteel, N., and Stohlman, S. A. (1988). Interactions between coronavims nucleocapsid protein and viral RNAs: implications for viral transcription. Journal o f Virology 62(11), 4280-7. Bavari, S., Bosio, C. M., Wiegand, E., Ruthel, G., Will, A. B., Geisbert, T. W., Hevey, M., Schmaljohn, C., Schmaljohn, A., and Aman, M. J. (2002). Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg vimses. J Exp M ed 195(5), 593-602. 153 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Beaudette, F. R., Hudson C. B. (1937). Cultivation of the virus of infectious bronchitis. J. Am. Vet. Med. Assoc 1937(90), 51-60. Belsham, G. J., and Sonenberg, N. (1996). RNA-protein interactions in regulation of picomavirus RNA translation. Microbiol. Rev. 60, 499-511. Bender, F. C., Whitbeck, J. C., Ponce de Leon, M., Lou, H., Eisenberg, R. J., and Cohen, G. H. (2003). Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry. J Virol 77(17), 9542-52. Bock, J. B., Klumperman, J., Davanger, S., and Scheller, R. H. (1997). Syntaxin 6 functions in trans-Golgi network vesicle trafficking. Molecular Biology o f the Cell 8(7), 1261-71. Boussadia, O., Niepmann, M., Creancier, L., Prats, A. C., Dautry, F., and Jacquemin- Sablon, H. (2003). Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus. J. Virol. 77(6), 3353-9. Braunwald, J., Nonnenmacher, H., Pereira, C. A., and Kim, A. (1991). Increased susceptibility to mouse hepatitis virus type 3 (MHV3) infection induced by a hypercholesterolaemic diet with increased adsorption of MHV3 to primary hepatocyte cultures. Res Virol 142(1), 5-15. Brown, D. A., and London, E. (1998). Structure and origin of ordered lipid domains in biological membranes. Journal o f Membrane Biology 164(2), 103-14. Cassetti, M. C., Noah, D. L., Montelione, G. T., and Krug, R. M. (2001). Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region. Virology 289, 180-185. Castelo-Branco, P., Furger, A., Wollerton, M., Smith, C., Moreira, A., and Proudfoot, N. (2004). Polypyrimidine tract binding protein modulates efficiency of polyadenylation. Mol Cell Biol 24(10), 4174-83. Cemy, J., Stockinger, H., and Horejsi, V. (1996). Noncovalent associations of T lymphocyte surface proteins. EurJIm munol 26(10), 2335-43. Chazal, N., and Gerlier, D. (2003). Vims entry, assembly, budding, and membrane rafts. Microbiol Mol Biol Rev 67(2), 226-37, table of contents. Cheever, F. S., Daniels J. B., Pappenheimer A. M., Bailey O. T. (1949). A murine vims (JHM) causing disseminated encephalomyelitis with extensive destmction of myelis. Isolation and biological properties of the vims. J. Exp. Med. 90, 121-194. 154 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Choi, K. S., Huang, P., and Lai, M. M. C. (2002). Polypyrimidine-tract-binding protein affects transcription but not translation of mouse hepatitis virus RNA. Virology 303(1), 58-68. Compton, S. R., Rogers, D. B., Holmes, K. V., Fertsch, D., Remenick, J., and Mcgowan, J. J. (1987). In vitro replication of mouse hepatitis virus strain A59. J. Virol. 61, 1814-1820. Corse, E., and Machamer, C. E. (2002). The cytoplasmic tail of infectious bronchitis virus E protein directs Golgi targeting. Journal o f Virology 76(3), 1273-84. Cote, C. A., Gautreau, D., Denegre, J. M., Kress, T. L., Terry, N. A., and Mowry, K. L. (1999). A Xenopus protein related to hnRNP I has a role in cytoplasmic RNA localization. Mol. Cell 4, 431-437. Daya, M., Cervin, M., and Anderson, R. (1988). Cholesterol enhances mouse hepatitis virus-mediated cell fusion. Virology 163(2), 276-83. Dea, S., and Tijssen, P. (1988). Identification of the structural proteins of turkey enteric coronavirus. Archives o f Virology 99(3-4), 173-86. Delmas, B., Gelfi, J., L'Haridon, R., Vogel, L. K., Sjostrom, H., Noren, O., and Laude, H. (1992). Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357(6377), 417-20. Delmas, B., and Laude, H. (1990). Assembly of coronavirus spike protein into trimers and its role in epitope expression. Journal o f Virology 64(11), 5367-75. Del Real, G., Jimenez-Baranda, S., Lacalle, R. A., Mira, E., Lucas, P., Gomez- Mouton, C., Carrera, A. C., Martinez, A. C., and Manes, S. (2002). Blocking of HIV- 1 infection by targeting CD4 to nonraft membrane domains. J Exp M ed 196(3), 293- 301. Deregt, D., Gifford, G. A., Ijaz, M. K., Watts, T. C., Gilchrist, J. E., Haines, D. M., and Babiuk, L. A. (1989). Monoclonal antibodies to bovine coronavirus glycoproteins E2 and E3: demonstration of in vivo virus-neutralizing activity. Journal o f General Virology 70(Pt 4), 993-8. Doyle, L., Hutchings LM. (1946). A transmissible gastroenteritis in pigs. J. Am. Vet. Assoc 108, 257-259. Dveksler, G. S., Pensiero, M. N., Cardellichio, C. B., Williams, R. K., Jiang, G. S., Holmes, K. V., and Dieffenbach, C. W. (1991). Cloning of the mouse hepatitis virus 155 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV. J Virol 65(12), 6881-91. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.[see comment]. Nature 411(6836), 494-8. Escors, D., Camafeita, E., Ortego, J., Laude, H., and Enjuanes, L. (2001). Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core. Journal o f Virology 75(24), 12228-40. Fleming, J. O., Stohlman, S. A., Harmon, R. C., Lai, M. M., Frelinger, J. A., and Weiner, L. P. (1983). Antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to JHM (MHV-4) virus. Virology 131(2), 296-307. Furuya, T., and Lai, M. M. C. (1993). Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA. J. Virol. 67, 7215-7222. Gallagher, T. M., Escarmis, C., and Buchmeier, M. J. (1991). Alteration of the pH dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein. Journal o f Virology 65(4), 1916-28. Gallagher, T. M., Parker, S. E., and Buchmeier, M. J. (1990). Neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino- terminal half of the spike glycoprotein. Journal o f Virology 64(2), 731-41. Ghetti, A., Pinol-Roma, S., Michael, W. M., Morandi, C., and Dreyfuss, G. (1992). hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs. Nucleic Acids Res. 20, 3671-3678. Graham, D. R., Chertova, E., Hilbum, J. M., Arthur, L. O., and Hildreth, J. E. (2003). Cholesterol depletion of human immunodeficiency virus type 1 and simian immunodeficiency virus with beta-cyclodextrin inactivates and permeabilizes the virions: evidence for virion-associated lipid rafts. J Virol 77(15), 8237-48. Grosset, C., Chen, C. Y., Xu, N., Sonenberg, N., Jacquemin-Sablon, H., and Shyu, A. B. (2000). A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex. Cell 103(1), 29-40. Hannon, G. J. (2002). RNA interference. Nature 418(6894), 244-51. 156 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998). Lipid domain structure of the plasma membrane revealed by patching of membrane components. Journal o f Cell Biology 141(4), 929-42. Harris, C. E., Boden, R. A., and Astell, C. R. (1999). A novel heterogeneous nuclear ribonucleoprotein-like protein interacts with NS1 of the minute virus of mice. J. Virol 73(1), 72-80. Harris, T. J., and Siu, C. H. (2002). Reciprocal raft-receptor interactions and the assembly o f adhesion complexes. Bioessays 24(11), 996-1003. Hellen, C. U., Pestova, T. V., Litterst, M., and Wimmer, E. (1994). The cellular polypeptide p57 (pyrimidine tract-binding protein) binds to multiple sites in the poliovirus 5' nontranslated region. J. Virol. 68, 941-950. Hellen, C. U., Withered, G. W., Schmid, M., Shin, S. H., Pestova, T. V., Gil, A., and Wimmer, E. (1993). A cytoplasmic 57-kDa protein that is required for translation of picomavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein. Proc. Natl. Acad. Sci. U S A 90, 7642- 7646. Herold, J., and Andino, R. (2000). Poliovirus requires a precise 5' end for efficient positive-strand RNA synthesis. J. Virol. 74, 6394-6400. Herold, J., and Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Mol. Cell 7, 581-591. Hirano, N., Fujiwara, K., Hino, S., and Matumoto, M. (1974). Replication and plaque formation of mouse hepatitis vims (MHV-2) in mouse cell line DBT culture. Arch. Gesamte Virusforsch. 44, 298-302. Holm, K., Weclewicz, K., Hewson, R., and Suomalainen, M. (2003). Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100. J Virol 77(8), 4805-17. Hresko, R. C., and Mueckler, M. (2002). Identification of pp68 as the Tyrosine- phosphorylated Form of SYNCRIP/NSAP1. A cytoplasmic RNA-binding protein. J. Biol. Chem. 277(28), 25233-8. Huang, P., and Lai, M. M. C. (1999). Polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis vims 3' untranslated region, thereby altering RNA conformation. J. Virol. 73, 9110-9116. 157 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Huang, P., and Lai, M. M. C. (2001). Heterogeneous nuclear ribonucleoprotein al binds to the 3'-untranslated region and mediates potential 5'-3'-end cross talks of mouse hepatitis virus RNA. J. Virol. 75, 5009-5017. Izumi, R. E., Valdez, B., Baneijee, R., Srivastava, M., and Dasgupta, A. (2001). Nucleolin stimulates viral internal ribosome entry site-mediated translation. Virus Res. 76, 17-29. Jackson, R. J. (2002). "Proteins involved in the function of picomavirus internal ribosome entry sites." Molecular biology of picomavirus (B. L. S. a. E. Wimmer, Ed.) ASM press, Washington DC. Kamath, R. V., Leary, D. J., and Huang, S. (2001). Nucleocytoplasmic shuttling of Polypyrimidine Tract-binding protein is Uncoupled from RNA export. Mol. Biol. Cell 12, 3808-3820. Kaminski, A., Hunt, S. L., Patton, J. G., and Jackson, R. J. (1995). Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA. RNA 1, 924-938. Kanai, Y., Dohmae, N., and Hirokawa, N. (2004). Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron 43(4), 513-25. Katja, S., and Hurt, E. (2001). Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yralp. Nature 413, 648 - 652. Kim, J. H., Paek, K.Y., Ha, S.H., Cho, S., Choi, K., Kim, C.S., Ryu, S.H., Jang, S.K (2004). A Cellular RNA-Binding Protein Enhances Internal Ribosomal Entry Site- Dependent Translation through an Interaction Downstream of the Hepatitis C Vims Polyprotein Initiation Codon. Mol Cell Biol 24(18), 7878-7890. Kim, Y. K., Hahm, B., and Jang, S. K. (2000). Polypyrimidine tract-binding protein inhibits translation of bip mRNA. J. Mol. Biol. 304, 119-133. Klumperman, J., Locker, J. K., Meijer, A., Horzinek, M. C., Geuze, H. J., and Rottier, P. J. (1994). Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. Journal o f Virology 68(10), 6523-34. Kovbasnjuk, O., Edidin, M., and Donowitz, M. (2001). Role o f lipid rafts in Shiga toxin 1 interaction with the apical surface o f Caco-2 cells. J Cell Sci 114(Pt 22), 4025-31. Kozak, S. L., Heard, J. M., and Kabat, D. (2002). Segregation of CD4 and CXCR4 into distinct lipid microdomains in T lymphocytes suggests a mechanism for 158 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. membrane destabilization by human immunodeficiency virus. J Virol 76(4), 1802- 15. Kubo, H., Yamada, Y. K., and Taguchi, F. (1994). Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein. Journal o f Virology 68(9), 5403-10. Lai, M. M., and Cavanagh, D. (1997). The molecular biology of coronaviruses. Advances in Virus Research 48, 1-100. Lai, M. M. C. (1998). Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology 244, 1- 12. Lai, M. M. C., and Cavanagh, D. (1997). The molecular biology of coronaviruses. Adv. Virus Res. 48, 1-100. Lai, M. M., Patton, C. D., and Stohlman, S. A. (1982). Replication of mouse hepatitis virus: negative-stranded RNA and replicative form RNA are of genome length. Journal o f Virology 44(2), 487-92. Laude, H., Gelfi, J., Lavenant, L., and Charley, B. (1992). Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus. Journal o f Virology 66(2), 743-9. Lee, A. (2001). Membrane structure. Current Biology 11(20), R811-4. Lee, H. J., Shieh, C. K., Gorbalenya, A. E., Koonin, E. V., La Monica, N., Tuler, J., Bagdzhadzhyan, A., and Lai, M. M. (1991). The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology 180(2), 567-82. Li, B., and Yen, T. (2002). Characterization of the Nuclear Export Signal of Polypyrimidine Tract-binding Protein. J. Biol. Chem. 277, 10306-10314. Li, H. P., Huang, P., Park, S., and Lai, M. M. C. (1999). Polypyrimidine tract- binding protein binds to the leader RNA of mouse hepatitis virus and serves as a regulator of viral transcription. J. Virol. 73, 772-777. Li, H. P., Zhang, X., Duncan, R., Comai, L., and Lai, M. M. C. (1997). Heterogeneous nuclear ribonucleoprotein A l binds to the transcription-regulatory region of mouse hepatitis virus RNA. Proc. Natl. Acad. Aci. U S A 94, 9544-9549. 159 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lin, Y. J., Zhang, X., Wu, R. C., and Lai, M. M. C. (1996). The 3' untranslated region o f coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. J. Virol. 70(10), 7236-40. Liao, C. L., Zhang, X., and Lai, M. M. C. (1995). Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase. Virology 208, 319-327. Lin, Y. J., and Lai, M. M. (1993). Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication. Journal o f Virology 67(10), 6110-8. Lin, Y. J., Zhang, X., Wu, R. C., and Lai, M. M. C. (1996). The 3' untranslated region of coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. J. Virol. 70, 7236-7240. Locker, J. K., Rose, J. K., Horzinek, M. C., and Rottier, P. J. (1992). Membrane assembly of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation. Journal o f Biological Chemistry 267(30), 21911-8. Luytjes, W., Bredenbeek, P. J., Noten, A. F., Horzinek, M. C., and Spaan, W. J. (1988). Sequence of mouse hepatitis virus A59 mRNA 2: indications for RNA recombination between coronaviruses and influenza C virus. Virology 166(2), 415- 22. Luytjes, W., Geerts, D., Posthumus, W., Meloen, R., and Spaan, W. (1989). Amino acid sequence of a conserved neutralizing epitope of murine coronaviruses. Journal o f Virology 63(3), 1408-12. Maeda, J., Repass, J. F., Maeda, A., and Makino, S. (2001). Membrane topology of coronavirus E protein. Virology 281(2), 163-9. Maeda, J. M., A. Makino, S. (1999). Release of coronavirus E protein in membrane vesicles from virus-infected cells and E protein-expressing cells. Virology 263(2), 265-72. Makino, S., Joo, M., and Makino, J. K. (1991). A system for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. J. Virol. 65, 6031-6041. Makino, S., Shieh, C. K., Soe, L. H., Baker, S. C., and Lai, M. M. C. (1988). Primary structure and translation of a defective interfering RNA of murine coronavirus. Virology 166, 550-560. 160 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Marjomaki, V., Pietiainen, V., Matilainen, H., Upla, P., Ivaska, J., Nissinen, L., Reunanen, H., Huttunen, P., Hyypia, T., and Heino, J. (2002). Internalization of echovirus 1 in caveolae. J Virol 76(4), 1856-65. Mcbride, A. E., Schlegel, A., and Kirkegaard, K. (1996). Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells. Proc. Natl. Acad. Sci. U S A 93, 2296-2301. McIntosh, K. (1974). Coronaviruses: a comparative review. Curr. Top. Microbiol. Immunol 63, 85-129. Meerovitch, K., Pelletier, J., and Sonenberg, N. (1989). A cellular protein that binds to the 5'-noncoding region of poliovirus RNA: implications for internal translation initiation. Genes & Dev. 3, 1026-1034. Meerovitch, K., Svitkin, Y. V., Lee, H. S., Lejbkowicz, F., Kenan, D. J., Chan, E. K., Agol, V. I., Keene, J. D., and Sonenberg, N. (1993). La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J. Virol. 67(7), 3798-807. Mizutani, A., Fukuda, M., Ibata, K., Shiraishi, Y., and Mikoshiba, K. (2000). SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms. J. Biol. Chem. 275(13), 9823-31. Moreira, A., Takagaki, Y., Brackenridge, S., Wollerton, M., Manley, J. L., and Proudfoot, N. J. (1998). The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3' end formation by two distinct mechanisms. Genes & Dev. 12, 2522-2534. Mourelatos, Z., Abel, L., Yong, J., Kataoka, N., and Dreyfuss, G. (2001). SMN interacts with a novel family of hnRNP and spliceosomal proteins. EMBO J. 20(19), 5443-52. Nanda, S. K., and Leibowitz, J. L. (2001). Mitochondrial aconitase binds to the 3' untranslated region of the mouse hepatitis virus genome. J. Virol. 75, 3352-3362. Narayanan, K., Maeda, A., Maeda, J., and Makino, S. (2000). Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells. Journal o f Virology 74(17), 8127-34. Neubauer, G., King, A., Rappsilber, J., Calvio, C., Watson, M., Ajuh, P., Sleeman, J., Lamond, A., and Mann, M. (1998). Mass spectrometry and EST-database 161 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. searching allows characterization of the multi-protein spliceosome complex. Nat. Genet. 20(1), 46-50. Nomura, R., Kiyota, A., Suzaki, E., Kataoka, K., Ohe, Y., Miyamoto, K., Senda, T., and Fujimoto, T. (2004). Human coronavirus 229E binds to CD 13 in rafts and enters the cell through caveolae. J Virol 78(16), 8701-8. Oh, Y. L., Hahm, B., Kim, Y. K., Lee, H. K., Lee, J. W., Song, O., Tsukiyama- Kohara, K., Kohara, M., Nomoto, A., and Jang, S. K. (1998). Determination of functional domains in polypyrimidine-tract-binding protein. Biochem. J. 331, 169- 175. Ono, A., and Freed, E. O. (2001). Plasma membrane rafts play a critical role in HIV- 1 assembly and release. Proc Natl Acad Sci U S A 98(24), 13925-30. Panchal, R. G., Ruthel, G., Kenny, T. A., Kallstrom, G. H., Lane, D., Badie, S. S., Li, L., Bavari, S., and Aman, M. J. (2003). In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding. Proc Natl Acad Sci U S A 100(26), 15936-41. Parton, R. G., and Lindsay, M. (1999). Exploitation of major histocompatibility complex class I molecules and caveolae by simian virus 40. Immunol Rev 168, 23- 31. Perez, I., Mcafee, J. G., and Patton, J. G. (1997). Multiple RRMs contribute to RNA binding specificity and affinity for polypyrimidine tract binding protein. Biochemistry 36, 11881-11890. Pfleiderer, M., Routledge, E., Herrler, G., and Siddell, S. G. (1991). High level transient expression of the murine coronavirus haemagglutinin-esterase. Journal o f General Virology 72(Pt 6), 1309-15. Pickl, W. F., Pimentel-Muinos, F. X., and Seed, B. (2001). Lipid rafts and pseudotyping. J Virol 75(15), 7175-83. Popik, W., Alee, T. M., and Au, W. C. (2002). Human immunodeficiency virus type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into CD4(+) T cells. J Virol 76(10), 4709-22. Rawat, S. S., Viard, M., Gallo, S. A., Rein, A., Blumenthal, R., and Puri, A. (2003). Modulation of entry of enveloped viruses by cholesterol and sphingolipids (Review). Mol Membr Biol 20(3), 243-54. 162 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rossoll, W., Kroning, A. K., Ohndorf, U. M., Steegbom, C., Jablonka, S., and Sendtner, M. (2002). Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons? Hum. Mol. Genet. 11(1), 93-105. Scheiffele, P., Rietveld, A., Wilk, T., and Simons, K. (1999). Influenza viruses select ordered lipid domains during budding from the plasma membrane. J Biol Chem 274(4), 2038-44. Scheiffele, P., Roth, M. G., and Simons, K. (1997). Interaction o f influenza virus haemagglutinin with sphingolipid-chole sterol membrane domains via its transmembrane domain. Embo J 16(18), 5501-8. Schnapp, B. J. (1999). A glimpse of the machinery. Current Biology 9, R725-727. Shen, X., and Masters, P. S. (2001). Evaluation of the role of heterogeneous nuclear ribonucleoprotein A l as a host factor in murine coronavirus discontinuous transcription and genome replication. Proc. Natl. Acad. Aci. U S A 98, 2717-2722. Schultze, B., Gross, H. J., Brossmer, R., and Herrler, G. (1991). The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant. Journal o f Virology 65(11), 6232-7. Schultze, B., Gross, H. J., Brossmer, R., Klenk, H. D., and Herrler, G. (1990). Hemagglutinating encephalomyelitis virus attaches to N-acetyl-9-O- acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza C virus. Virus Res 16(2), 185-94. Schultze, B., and Herrler, G. (1992). Bovine coronavims uses N-acetyl-9-O- acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells. Journal o f General Virology 73(Pt 4), 901-6. Shen, X., and Masters, P. S. (2001). Evaluation of the role of heterogeneous nuclear ribonucleoprotein A l as a host factor in murine coronavims discontinuous transcription and genome replication. Proc. Natl. Acad. Sci. USA 98(5), 2717-22. Shi, S. T., Huang, P., Li, H. P., and Lai, M. M. C. (2000). Heterogeneous nuclear ribonucleoprotein A l regulates RNA synthesis of a cytoplasmic vims. EMBO J. 19, 4701-4711. Shi, S. T., Yu, G. Y., and Lai, M. M. C. (2003). Multiple type A/B heterogeneous nuclear ribonucleoproteins (hnRNPs) can replace hnRNP A l in mouse hepatitis vims RNA synthesis. J. Virol. 77(19), 10584-93. 163 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387(6633), 569-72. Simons, K., and Ikonen, E. (2000). How cells handle cholesterol. Science 290(5497), 1721-6. Simons, K., and Toomre, D. (2000). Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1(1), 31-9. Singer, S. J., and Nicolson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175(23), 720-31. Snijder, E. J., Bredenbeek, P. J., Dobbe, J. C., Thiel, V., Ziebuhr, J., Poon, L. L., Guan, Y., Rozanov, M., Spaan, W. J., and Gorbalenya, A. E. (2003). Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J M ol Biol 331(5), 991-1004. Spagnolo, J. F., and Hogue, B. G. (2000). Host protein interactions with the 3' end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication. J. Virol. 74, 5053-5065. Stohlman, S. A., Baric, R. S., Nelson, G. N., Soe, L. H., Welter, L. M., and Deans, R. J. (1988). Specific interaction between coronavirus leader RNA and nucleocapsid protein. J. Virol. 62,4288-4295. Strasser, K., and Hurt, E. (2001). Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yralp. Nature 413(6856), 648-52. Suomalainen, M. (2002). Lipid rafts and assembly of enveloped viruses. Traffic 3(10), 705-9. Suzuki, H., and Taguchi, F. (1996). Analysis of the receptor-binding site of murine coronavirus spike protein. Journal o f Virology 70(4), 2632-6. Taguchi, F. (1993). Fusion formation by the uncleaved spike protein of murine coronavims JHMV variant cl-2. Journal o f Virology 67(3), 1195-202. Tahara, S. M., Dietlin, T. A., Nelson, G. W., Stohlman, S. A., and Manno, D. J. (1998). Mouse hepatitis vims nucleocapsid protein as a translational effector of viral mRNAs. Adv. Exp. Med. Biol. 440, 313-318. Thiel, V., and Siddell, S. G. (1994). Internal ribosome entry in the coding region of murine hepatitis vims mRNA 5. Journal o f General Virology 75(Pt 11), 3041-6. 164 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thorp, E. B., and Gallagher, T. M. (2004). Requirements for CEACAMs and cholesterol during murine coronavirus cell entry. J Virol 78(6), 2682-92. Tooze, J., Tooze, S., and Warren, G. (1984). Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions. European Journal o f Cell Biology 33(2), 281-93. Tsui-Pierchala, B. A., Encinas, M., Milbrandt, J., and Johnson, E. M., Jr. (2002). Lipid rafts in neuronal signaling and function. Trends Neurosci 25(8), 412-7. Tyrrell, D., Almeida JD., Berry DM. (1968). Coronavirus. Nature, 220-650. Tyrrell, D., Bynoe ML. (1965). Cultivation of a novel type of common-cold virus in organ culture. Br. Med. J. 1, 1467-1470. Valcarcel, J., and Gebauer, F. (1997). Post-transcriptional regulation: the dawn of PTB. Curr Biol 7(11), R705-8. Vennema, H., Godeke, G. J., Rossen, J. W., Voorhout, W. F., Horzinek, M. C., Opstelten, D. J., and Rottier, P. J. (1996). Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. Embo J 15(8), 2020-8. Vincent, S., Gerlier, D., and Manie, S. N. (2000). Measles virus assembly within membrane rafts. J Virol 74(21), 9911-5. Waggoner, S., and Samow, P. (1998). Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells. J. Virol. 72, 6699-6709. Wagner, E. J., and Garcia-Bianco, M. A. (2001). Polypyrimidine tract binding protein antagonizes exon definition. Mol. Cell. Biol. 21, 3281-3288. Wang, Y., and Zhang, X. (1999). The nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein A l in vitro and in vivo. Virology 265, 96-109. Weismiller, D. G., Sturman, L. S., Buchmeier, M. J., Fleming, J. O., and Holmes, K. V. (1990). Monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions. Journal o f Virology 64(6), 3051-5. Xu, Y., Liu, Y., Lou, Z., Qin, L., Li, X., Bai, Z., Pang, H., Tien, P., Gao, G. F., and Rao, Z. (2004). Structural basis for coronavirus-mediated membrane fusion. Crystal 165 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. structure of mouse hepatitis virus spike protein fusion core. Journal o f Biological Chemistry 279(29), 30514-22. Yeager, C. L., Ashmun, R. A., Williams, R. K., Cardellichio, C. B., Shapiro, L. H., Look, A. T., and Holmes, K. V. (1992). Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357(6377), 420-2. Yokomori, K., Asanaka, M., Stohlman, S. A., Makino, S., Shubin, R. A., Gilmore, W., Weiner, L. P., Wang, F. I., and Lai, M. M. (1995). Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression, [see comment]. Journal o f Neurovirology 1(5-6), 330-9. Yokomori, K., La Monica, N., Makino, S., Shieh, C. K., and Lai, M. M. (1989). Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus. Virology 173(2), 683-91. Yokomori, K., and Lai, M. M. (1991). Mouse hepatitis virus S RNA sequence reveals that nonstructural proteins ns4 and ns5a are not essential for murine coronavirus replication. Journal o f Virology 65(10), 5605-8. Yokomori, K., and Lai, M. M. (1992). The receptor for mouse hepatitis virus in the resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection. J Virol 66(12), 6931-8. Zang, W. Q., Li, B., Huang, P. Y., Lai, M. M. C., and Yen, T. S. (2001). Role of polypyrimidine tract binding protein in the function of the hepatitis B virus posttranscriptional regulatory element. J. Virol. 75, 10779-10786. Zhang, X., and Lai, M. M. (1996). A 5'-proximal RNA sequence of murine coronavirus as a potential initiation site for genomic-length mRNA transcription. Journal o f Virology 70(2), 705-11. Zhang, X., and Lai, M. M. C. (1995). Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed. J. Virol. 69, 1637-1644. Zhang, X., Liao, C. L., and Lai, M. M. C. (1994). Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis. J. Virol. 68, 4738-4746. Zhou, M., Williams, A. K., Chung, S. I., Wang, L., and Collisson, E. W. (1996). The infectious bronchitis virus nucleocapsid protein binds RNA sequences in the 3’ terminus of the genome. Virology 217(1), 191-9. 166 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator Choi, Keum Sook (author)

Core Title Cellular factors involved in mouse hepatitis virus replication

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Degree Program Molecular Microbiology and Immunology

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Advisor Lai, Michael M.C. (committee chair), Laird-Offringa, Ite A. (committee member), Ou, Jing-Hsiung James (committee member)

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