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Mapping arenavirus glycoprotein determinants that correlate to cell entry and virus pathogenicity

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Mapping arenavirus glycoprotein determinants that correlate to cell entry and virus pathogenicity

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Content MAPPING ARENAVIRUS GLYCOPROTEIN DETERMINANTS THAT
CORRELATE TO CELL ENTRY CHARACTERISTICS AND VIRUS
PATHOGENECITY

by

Vanessa Kimiko Martin

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fullfillment of the
Requirements of the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2008

Copyright 2008 Vanessa Kimiko Martin

ii
Dedication

This manuscript is dedicated in memory to my father; whose encouragement and sense of
humor will always be the voice in my head.

iii
Acknowledgements

Thank you to Dr. Stefan Kunz for GTOV GP construct, and FLAG-tagged GP constructs.
Thank you to Dr. Paula Cannon for her invaluable advice and direction given to me
throughout this research project. Thank you to my committee members, Dr. Joseph
Landolph and Dr. Minnie McMillan. And thank you to the entire Cannon Lab and
Children’s Hospital Los Angeles Division of Research BMT and Immunology for helpful
discussions, advice, and support.

iv
Table of Contents

Dedication ii

Acknowledgements iii

List of Figures v

Abstract vi

Introduction 1

Materials and Methods 8

Results 11

Discussion 26

References 31

v

List of Figures

Figure 1: Phylogenetic relationship of Old World and 4
New World arenaviruses based on sequence analysis of GPC

Figure 2: Tropism of pathogenic (GTOV) versus 6
non-pathogenic (AMAV) arenaviruses

Figure 3: Alignment of clade B2 arenaviruses GP1 12

Figure 4: A representation of AMAV and GTOV chimeras generated 13

Figure 5: Tropism of AMAV chimeras 14

Figure 6: A(G5) is able to use hTfR1 and fTfR1 for cell entry; 16
thus exhibiting pathogenic tropism

Figure 7: Tropism of GTOV chimeras 17

Figure 8: Ability of GTOV chimeras to transduce hTfR1 or fTfR1 19
transfected CHO-K1 cells

Figure 9: Sequence alignment of domain 5 of clade B New World 20
Arenaviruses

Figure 10: Tropism and TfR1 use for cell entry by A(G5V-Y) and 21
AMAV Y-V mutants

Figure 11: Western blots of chimera GP-pseudotyped vectors 24
probed with anti-Junin Ab

Figure 12: Western blots of FLAG-tagged GP-pseudotyped vectors 25

vi

Abstract.

Five arenaviruses can cause hemorrhagic fever in humans. They are classified by
the CDC as category A pathogens because of the possibility to be manipulated into
biological weapons. Of interest, the New World clade B group of the arenaviruses
contains both pathogenic and non-pathogenic members, including the closely related
Amapari (AMAV) and Guanarito (GTOV) viruses. In our recent study, we determined
that pathogenic viruses have a specific tropism and are able to transduce human T-
lymphocytes and use the human transferrin receptor 1 for cell entry. By dividing the GP1
protein, the subunit that promotes cell binding and entry, into five domains based upon
conserved cysteine residues, we generated a series of AMAV and GTOV chimeras to
map the region of the GP1 subunit that correlates to pathogenic tropism. We have found
a valine in the domain five of the GTOV GP1 that is necessary for pathogenic tropism in
arenavirus.
1
Introduction.

History of arenaviruses.
Arenaviruses are classified under the family Arenaviridae. “Arena” is derived
from the Latin word arenosus, meaning sandy, which is the appearance the viruses have
when viewed under ultrathin section due to ribosomes within the virion (3, 24, 26). The
first arenavirus discovered, lymphocytic choriomeningitis virus (LCMV) in 1933, is a
causative agent of aseptic meningitis. It was isolated from spinal fluid samples during a
St. Louis encephalitis epidemic. The study of LCMV has led to many innovative and
important advances in the field of virology, immunology, and cell biology, and thus its
discovery has been valuable for many fields. For example, the study of LCMV infection
in mice has led to the first description of T cell-mediated killing, virus-induced
immunopathologic disease, and of the two separate signals needed for recognition and
lysis of virus infected targets by cytotoxic T lymphocytes (CTLs).
Since the discovery of LCMV, 24 arenaviruses in total have been discovered.
Seven have been found to cause diseases in humans: LCMV, Lassa (LASV), Machupo
(MACV), Junin (JUNV), Guanarito (GTOV), Sabia (SABV), and White Water Arroyo
(WWAV). MACV, JUNV, GTOV and SABV are the pathogenic agents that cause
Bolivian, Argentine, Venezuelan and Brazilian hemorrhagic fevers respectively; LASV is
the cause of Lassa fever. A name has not been given to the disease state caused by
WWAV, which is still controversial as a pathogen. MACV, JUNV, SABV and GTOV
are found in South America, WWAV is found in North America, LCMV can be found
worldwide, and LASV infections are localized in Africa (3).
2
Because of their highly pathogenic nature, ease of transmission to humans, and
possible manipulation to be used in a bioterrorist attack, arenaviruses have been classified
as BSL4 and Category A agents by the Centers for Disease Control. These viruses are
now being studied more intensely, to develop possible vaccines and therapeutic
treatments against the possibility that they might be used in a terrorist attack, or emerge
more widely as natural pathogens.

Genomics and structure of arenaviruses.
The arenavirus genome consists of two segments of ambisense RNA, designated
large (L) segment and small (S) segment, which are 7.2 kb and 3.5 kb respectively. Each
segment consists of two opposing open reading frames (ORF) separated by a noncoding
intergenic region (IGC) with a predicted folding of a stable hairpin structure(3). The
pseudopositive sense ORF of the L segment codes for an 11kDa RING finger protein Z.
The negative sense ORF codes for the viral RNA-dependent RNA-polymerase, RdRp
(200kDa), or L protein. The S segment codes for the 75kDa viral glycoprotein (GP) on
the pseudopositive sense strand, and the 63 kDa nucleoprotein (NP) on the negative sense
strand(3). The S and L RNA segments are not able to serve as mRNAs and be directly
translated upon entering the cell, hence their designation as pseudopositive sense(23).
The arenaviridae have a spherical to pleomorphic shape, 50-300 nm in diameter,
with club-shaped projections extending 5-10 nm from the virion surface. Arenaviruses
have a host membrane-derived envelope that is obtained upon budding from the host cell
surface(32). The club-shaped projections are the viral GP, which are arranged in a
tetrameric fashion.
3
The arenavirus GP is a type-1 fusion protein(3, 9, 11, 12, 15). The glycoprotein
precursor, GPC, includes a signal peptide (SP), which is cleaved and removed prior to
glycoprotein transport from the endoplasmic reticulum, and two subunits GP1 (40-46
kDa) and GP2 (35 kDa). GP1 is the virion attachment protein that mediates virus
interaction with host cell surface receptors and subsequent virus cell entry via receptor-
mediated endocytosis. GP2 is the transmembrane unit involved in host membrane
fusion(2, 3, 5, 10, 17, 18, 25). Cleavage of the GPC into GP1 and GP2 occurs between
the medial and the trans-Golgi network (36). Cleavage occurs at the dibasic residues
conserved among all arenaviruses at the GP1/GP2 junction consisting of RRxL(3, 4, 16).
This is performed by SKI-1/S1P cellular protease. GP1 and GP2 are covalently bound
and then transported to the cytoplasmic membrane by vesicles to prepare for virion
budding.

Arenavirus phylogeny and potential experimental use to map determinants of
pathogenicity.
The phylogeny of arenaviruses has been classified according to GPC and NP
sequences from the S segment of the arenavirus genome(1, 7, 8). Five lineages have been
identified: New World (Tacaribe complex) viruses grouped into clades A, B, C, a small
recombinant lineage (Rec), and separate Old World (OW) viruses. Our laboratory has
focused upon viruses within the clade B lineage of New World arenaviruses because New
World arenaviruses causing viral hemorrhagic fever are all contained within lineage B
(Fig. 1). Of note are sequence similarities between pathogenic and non-pathogenic
viruses. Specifically, AMAV is a non-pathogenic virus and is closely related to GTOV,
4
the etiological agent of Venezuelan hemorrhagic fever. They have a sequence identity of
approximately 31.1% in the GPC(7). These two viruses are included in the clade B2
lineage of the New World arenaviruses. Because of this similarity in GPC amino acid
sequence, we hypothesize we can utilize this sequence homology and map the region of
viral GP1 that confers pathogenicity to GTOV.

Fig. 1. Phylogenetic relationship of Old World and New World arenaviruses based on
sequence analysis of GPC. Viruses that cause hemorrhagic fever in humans (boxed) are
distributed throughout all three sublineages of clade B in New World viruses, and one,
LASV, in the Old World complex (7). Of note is the close relationship of AMAV (non-
pathogenic) and GTOV (pathogenic).

Differences between pathogenic and non-pathogenic clade B arenaviruses.
In a recent study, we transduced an expanded panel of clade B arenaviruses, both
pathogenic and non-pathogenic, analyzing tropism on various cell lines and the use of
cellular receptors(13). In these assays we found a distinction in lymphocyte cell line
tropism between pathogenic and non-pathogenic clade B arenaviruses. We transduced
MACV
JUNV
TCRV
CPXV
GTOV
AMAV
SABV
}
B1
}
B2
} B3
B
WWAV
BCNV
TAMV
LATV
OLVV
PICV
PIRV
PARV
ALLV
FLEV
LCMV
LASV
MOPV
Rec
C
A
OW
5
GP-pseudotyped retroviral vectors on human (CEM) and mouse (T1B27) T-lymphocyte
cell lines. We found that only pathogenic viruses, such as GTOV, were able to transduce
the human cell line, while non-pathogenic viruses, such as AMAV, were only able to
transduce the mouse cell line (Fig 2A).
Recently, it has been found that transferrin receptor 1 (TfR1) is the receptor for
arenaviruses(28). In our study, we compared the use of human transferrin receptor 1
(hTfR1) and other orthologs by pathogenic and non-pathogenic arenaviruses. HTfR1 has
been found to be an excellent receptor for arenavirus cell entry(13, 28, 29). We also
found that all clade B arenaviruses are able to transduce the hamster cell line, CHO-K1,
albeit at significantly lower levels than human cells. Based upon this data, we transfected
CHO-K1 cells with either hTfR1, feline transferrin receptor 1 (fTfR1), murine transferrin
receptor 1 (mTfR1), or canine transferrin receptor 1 (cTfR1), and challenged the
transfected cells with pseudotyped retroviral vectors. We looked for an increase in viral
titer indicating that the TfR1 is being used by the virus to enter the cell. We observed a
difference between the pathogenic and non-pathogenic viruses in receptor use for cell
entry. Specifically shown in Figure 2B is a comparison of tropism of AMAV and
GTOV. Only the GTOV was found to use hTfR1 and fTfR1 for cell entry. This implies
that use of hTfR1 and fTfR1 for entry is a characteristic of pathogenic clade B
arenaviruses. Similar results have been seen for comparisons between pathogenic
MACV and JUNV versus non-pathogenic TACV GP(13).

6
A

B

Fig. 2. Tropism of pathogenic (GTOV) versus non-pathogenic (AMAV) arenaviruses.
(A) Titers of GP-pseudotyped retroviral vectors on human (CEM) and murine (T1B27)
T-lymphocyte cell lines. The values shown are means ±standard errors from two to eight
independent experiments. All vector-cell combinations that were below detection level
(*) were confirmed with 10X concentrated stocks of vectors (adapted from ref (13)). (B)
GP-pseudotyped retroviral vectors transduced on mock-transfected CHO-K1 cells or cells
transiently transfected with expression plasmids for hTfR1 or fTfR1. Vector titers are
displayed as relative titers (%), where each experimental value was made relative to the
value obtained on the mock-transfected cells (adapted from ref (13)).

In this study, we generated a series of chimeras from GTOV and AMAV GP1
based upon a sequence analysis that divided the GP1 into five domains. We challenged
AMAV and GTOV GP1 chimeras in the assays designed in our previous study. We
looked for AMAV chimeras that have gained the ability to infect human lymphocytes and
293A CEM TIB27
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
VSV-g
GTOV
AMAV
Titer (TU/ml)
C H O -K 1
VSV GTOV AMAV
1
1 0
1 0 0
1 0 0 0
M oc k
hTfR1
fTfR1
Relative Titer (%)
* *
7
utilize hTfR1 with the addition of one or multiple domain swaps, or GTOV chimeras that
have lost their pathogenic characteristics. This enabled us to map the region of GP1 that
confers pathogenicity.

8
Materials and Methods.

Cell lines.
293A, 293T, CHO-K1, and T1B27 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% fetal
bovine serum (FBS) (HyClone, Logan, UT) and 2 mM glutamine (Gemini Bio-Products,
West Sacramento, CA). CEM cells were maintained in RPMI (Mediatech, Herndon, VA)
supplemented with 10% FBS and 2 mM glutamine. All cells were maintained in 5% CO
2

atmosphere except T1B27 cells, which were maintained at 10% CO
2
.

GP-pseudotyped retroviral vectors.
The expression plasmid for the GTOV GP (INH-95551 strain), FLAG GTOV GP
(INH-95551 strain), and FLAG AMAV GP (BeAn 70563 strain) were kindly provided by
Stefan Kunz (The Scripps Research Institute)(19, 31). A human codon-optomized form
of AMAV GP (BeAn 70563 strain) was chemically synthesized and cloned into the
pCAGGS expression vector(30). Chimeras were generated using gene splicing by
overlap extension(35). Pseudotyped retroviral vectors displaying GPs were generated by
cotransfection of 293T cells with expression plasmids for the specific GP, together with
the plasmids expressing murine leukemia virus Gag-Pol and an enhanced green
fluorescent protein (EGFP)-expressing retroviral genome, pMND-eGFP(30).
The efficiency of entry of the GP-pseudotyped retroviral vectors into cells was
measured as previously described(30). Briefly, vectors were incubated with target cells
for 4 h and then replaced with fresh medium. After 48 h, the cells were trypsinized,
9
washed in phosphate-buffered saline, and analyzed for EGFP expression using a
FACScan flow cytometer (BD, Franklin Lakes, NJ). The efficiency of entry (titer) was
determined by multiplying the percentage of EGFP-positive cells by the number of cells
initially incubated with the vectors. Titers were expressed as transducing units (TU) per
ml of vector-containing supernatant. In experiments with control and experimental arms
(e.g. antibody blocking), the titers were made relative to the control (mock treatment)
cells and were expressed as a percentage of the control titer.

Transient transfection of CHO-K1 cells with TfR1.
Expression plasmids for murine, human, and feline TfR1 orthologs were
generously provided by Susan Ross (University of Pennsylvania). CHO-K1 cells were
transiently transfected with the expression plasmids using Lipofectamine 2000
(Invitrogen). Briefly, CHO-K1 cells were plated to be 95-100% confluent in 10-cm
plates at the time of transfection and were transfected with 24 μg of TfR1 expression
plasmids. Cells were incubated in transfection mixture for 4 h and then cultured
overnight in DMEM plus 10% FBS. The following day, cell were scraped, plated into
six-well plates at 40% confluence and incubated overnight. Cells were then transduced
with GP-pseudotyped vectors or chimeric GP-pseudotyped vectors for 4 h and the titers
were determined 48 to 72 h later by FACS analysis for EGFP expression, as described
above. Cell samples were also collected at time of transduction and analyzed by Western
blotting to determine levels of TfR1 expression.

10
Immunoblotting.
TfR1 expression was detected by Western blotting of cell lysates using a 1:500
dilution of mouse anti-TfR1 antibody, clone H68.4 (Invitrogen). This antibody
recognizes a conserved epitope in chicken, mouse, rat, Chinese hamster, and human
TfR1. Specific bands were detected using horseradish peroxidase-conjugated goat anti-
mouse immunoglobulin G (IgG) (Pierce, Rockford, IL), diluted 1:10,000, followed by
incubation with the ECL-enhanced chemiluminescence detection reagent (Amersham
Biosciences, Pistcataway, NJ) and exposure to Kodak BIOMAX XAR film (Sigma). To
ensure equal protein loading, membranes were also probed with mouse anti-actin
monoclonal antibody (Sigma), diluted 1:10,000.
Expression plasmids generously donated by Stefan Kunz (The Scripps Research Institute)
(31)expressing the GTOV or the AMAV GPC with a FLAG epitope at the C-terminus
were used in virion production for the purpose of being detected by Western blot analysis
due to the lack of specific antibody to GTOV or AMAV GPC. Viral GPs in retroviral
particles were detected by Western blot analysis of concentrated viral supernatants,
essentially as described (30). The antibody used was rabbit anti-FLAG polyclonal
antibody (GeneTex, Inc., San Antonio, TX), diluted 1:1000. Other antibodies used in
other Western blots were anti-JUNVGP monoclonal antibody QD04 AF03 (34), diluted
1:2000, and two anti-MACV GP1 antibodies (Sigma Genesys), diluted 1:1000. To
ensure equal protein loading, membranes for virions were detected using goat anti-
Rauscher MLV CA antiserum (Quality Biotech, Camden, NJ), with a 1:10,000 dilution.

11
Results.

Sequence analysis divided GP1 into five domains according to position of conserved
cysteine residues.
In order to map GP1 for a region that corresponds to pathogenic tropism, we
generated chimeras from GTOV and AMAV GP1. To create these chimeras we analyzed
the amino acid composition of the GP1 portion of GPC and designed a sequence directed
analysis of GP1 that preliminarily divided the protein into five domains based upon
placement of cysteine residues (Fig. 3). Domain 1 of the GP1 is approximately 26-35 aa.
This is considered the most conserved part of the GP amongst all arenaviruses. Domain 2
contains residues that are highly glycosylated and so has been designated the
immunoprotective domain. This domain is approximately 31-50 aa. Domain 3 of the
GP1 contains conserved aromatic residues, suggesting that this is an internal part of the
protein. The rest of the amino acid composition of this domain is variable amongst the
arenaviruses. Domain 3 is 29-37 aa. The largest domain is domain 4, which is 45-60 aa.
Domain 4 contains many extra cysteine residues in the B1, A, and C strains that may
contribute to structure. This is also the most variable domain in GP1 across the
arenavirus phylogeny. Domain 5 is at the C-terminus of GP1. It contains the cleavage
site for GPC processing during virion assembly. This domain contains the two amino
acids associated with tropism changes in LCMV and is thought to influence receptor
usage(17).

12

Fig. 3. Alignment of clade B2 arenaviruses GP1. The GP1 sequence was divided into
five domains based upon the placement of cysteine residues that are conserved in all
arenavirus GPs.

Construction and initial titer characterization of chimeric GPs
Seven GP chimeras were generated with AMAV GP as the parental backbone and
GTOV GP1 domains cloned in (Fig. 4A). GP-pseudotyped vectors were made with the
chimera expression plasmid and transduced on human kidney epithelial 293A cells to
examine titer. VSV-G pseudotyped vectors were used as a control. Titer was measured
by FACS analysis for GFP expression. We found that two out the seven AMAV
chimeras were able to transduce 293A cells: AMAV GP with GTOV GP1 domain 3
(A(G3)) and domain 5 (A(G5)) (Fig. 5). The unconcentrated titer of chimeras was
approximately half a log to a log lower than their parental counterpart AMAV
(approximately 10
4
TU/ml). Concentrated 10X stocks of the chimera GP-pseudotyped
retroviral vectors were used for later assays in order for titer analysis to be at detection
level by FACS analysis.
Eight GP chimeras were made with GTOV GP as the parental backbone and
AMAV GP1 domains cloned in (Fig. 4B). These chimeras were made into GP-
Guanarito
Amapari
D4
EPKKTTNAEFTFQLNLTDSPETHHYRSKIEVGIRHLFGNYITNDSYSKMSVVMRNTTWEGQC
QPKRSTNAEFTLQLNISRRHTNDHYRERIETGIRHMFGPFKILTKEGKDCVILRNTTWKEQC
D5
SNSHVNTLRFLVKNAGYLV-GRKPL
VKSHYNTLAFLLKNTANSLPKRRPL
D1
FKVGHHTNFESFTVKLGGVFHELPSLC
FRIGHHTTFESVTMSVGGVFHELPALC
D2
RVNNSYSLIRLSHNSNQALSVEYVDVHPVLCSSSPTILD--NYTQC
RINNSHSLIQLSHNSSLALSVEYVDLC-VVLESDQYLVAGD-YSNC

D3
IKGSPEFDWILGWTIKGLGHDFLRDPRICC
TGEATGYNWVIDWTLKGLGHGLEGDPKLHC
13
pseudotyped vectors and transduced on 293A cells. Four GTOV GP1 chimeras were able
to give titer on 293A cells, GTOV GP with AMAV GP1 domain 1 (G(A1)), domain 4
(G(A4)), domain 5 (G(A5)), and domains 3,4, and 5 (G(A345)) (Fig. 7). Unconcentrated
titers of these chimeras on 293A cells were half a log to one log lower then their parental
counterpart GTOV (approximately 10
4
TU/ml). Again, 10X concentrated stocks of virus
were used for later assays.
A

B

Fig. 4. A representation of AMAV and GTOV chimeras generated. (A) AMAV
chimeras. At the top are wild type AMAV and GTOV GPC divided into the five
domains and GP2. Two chimeras were found to be able to transduce 293A cells (boxed).
(B) GTOV chimeras. Four chimeras were able to transduce 293A cells (boxed).

D1 D2 D3 D4
D5
GP2
WT AMAV
WT GTOV
G(A1)
G(A12)
G(A123)
G(A2)
G(A3)
G(A5)
G(A4)
G(A345)

WT AMAV
WT GTOV
A(G1)
A(G12)
A(G2)
A(G3)
A(G4)
A(G5)
A(G345)
D1 D2 D3 D4 D5
GP2
14
Tropism of functional AMAV chimera GPs on lymphocytes
Functional AMAV chimeras were transduced on two cells lines with concentrated 10X
vector stock: CEM and T1B27 cells. Our recent study has shown that pathogenic viruses
are able to transduce CEM, but not T1B27 cells. Non-pathogenic viruses have been
found to transduce T1B27, but not CEM cells(13). In this study GTOV is the pathogenic
GP and AMAV is the non-pathogenic GP. Because of this difference in tropism between
pathogenic and non-pathogenic viruses, we were interested to determine whether there
was a change in tropism of the non-pathogenic parental GP, AMAV, in either functional
chimera GP, to that of a pathogenic tropism.
The functional chimeras each displayed different tropisms. We found that A(G3)
was unable to enter both CEM or T1B27 cells lines. This behavior of A(G3) suggests
that the chimera may not be folded properly and so may be unable to bind lymphocytes.
A(G5) was only able to transduce CEM cells. Remarkably A(G5) is no longer behaving
like its parental backbone GP, non-pathogenic AMAV. A(G5) has made a tropism
switch, where it is now showing a tropism similar to a pathogenic GP. This is
suggestive that domain 5 of GTOV may confer pathogenic cellular tropism.

15

Fig. 5. Tropism of AMAV chimeras. Chimera GP-pseudotyped retroviral vectors were
first transduced on 293A cells to test for functionality. The two functional chimeras
(A(G3) and A(G5)) were then transduced on CEM and T1B27 cells. Wild type GP-
pseudotyped retroviral vectors were transduced unconcentrated, while chimera GP-
pseudotyped retroviral vectors were transduced with 10X concentrated vector stock.
Values shown are means ± standard errors represented by 3-5 independent experiments.
(*) indicates value was below detection level by FACS analysis.

Use of hTfR1 and fTfR1 for cell entry by AMAV chimera GPs.
In our recent study we found that pathogenic arenaviruses use hTfR1 for cell
entry whereas non-pathogenic arenaviruses do not. We determined that this corresponds
to the pathogenicity of the virus(13). To determine whether the replacement with a
pathogenic arenavirus GP1 domain, GTOV, into a non-pathogenic arenavirus GP1
domain, AMAV, could confer the ability to use hTfR1 for cell entry, we transduced the
two functional chimeras onto CHO-K1 cells transiently transfected with hTfR1or fTfR1.
A(G3) did not significantly increase titer compared to the mock transduced CHO-
K1 cells on the hTfR1 or fTfR1 transfected CHO-K1 cells indicating that it does not use
either receptor for cell entry (Fig. 6A). A(G5) however did increase titer on hTfR1
transfected CHO-K1 by 1 log compared to mock transduced CHO-K1 cells (Fig. 6A).
This is result mirrors tropism exhibited by pathogenic GTOV GP pseudotyped retroviral
vector in this assay. A(G5) also significantly increased in titer by 2 logs when transduced
293A CEM T1B27
1 0
0
1 0
1
1 0
2
1 0
3
1 0
4
1 0
5
1 0
6
1 0
7
V S V G
A M A V
C A (G 3 )
C A (G 5 )
G TO V
Titer (TU/ml)
* * * *
16
on fTfR1 transfected CHO-K1 cells with respect to mock transfected CHO-K1 cells.
This is suggestive that A(G5) has changed to a pathogenic tropism and additionally
supports that domain 5 of GTOV GP1 confers pathogenicity.

A

B

Fig. 6. A(G5) is able to use hTfR1 and fTfR1 for cell entry; thus exhibiting pathogenic
tropism. (A) Wild type and chimera GP-pseudotyped retroviral vectors were transduced
on mock transfected CHO-K1 cells or cells transfected with hTfR1 or fTfR1 expression
plasmids. Wild type GP-pseudotyped retroviral vectors were transduced as
unconcentrated stocks. Chimeras were transduced with 10X concentrated vector stocks.
Vector titers are shown as relative titers (%), where each experimental value was made
relative to the value obtained on the mock transfected cells. Values shown are
represented from 4 independent experiments. (B) Western blot confirming of level of
expression of hTfR1 and fTfR1.

Tropism of GTOV chimera GPs.
The four functional chimeras were transduced on CEM and T1B27 cell lines
using 10X concentrated virus stocks. G(A1) was able to transduce CEM cells only,
which is similar to GTOV tropism on lymphocytes. The reciprocal chimera of A(G5),

CHO-K1
VSVG
GTOV
AMAV
C A(G3)
C A(G5)
1
10
100
1000
10000
mock
huTfR
feTfR
Relative titer (%)
100kD
75kD
actin
mock hTfR1 fTfR1
CHO-K1
17
G(A5), was able to transduce both CEM and T1B27 cells lines. We expected that if
GTOV GP1 domain 5 corresponded to pathogenic tropism, GTOV GP1 with AMAV
GP1 domain 5 would lose its ability to transduce CEMs, however it appears that GTOV
GP1 is has not changed tropism despite this finding. Interestingly, G(A345) is no longer
able to transduce CEM cells, nor is it able to transduce T1B27 cells. It is possible that the
folding of this particular chimera has disturbed the binding ability of the GP to bind
lymphocytes, and thus we are unable to detect transduction. Currently we have no data
on the tropism of G(A4) on CEM or T1B27 cells.

Fig. 7. Tropism of GTOV chimeras. Four chimeras were found to transduce 293A cells
and so were transduced on CEM and T1B27 cell lines. Wild type GP-pseudotyped
retroviral vectors were transduced as unconcentrated stocks. Chimeras were transduced
with 10X concentrated vector stocks. Values shown are means ± standard errors
represented by 3-5 independent experiments. (*) indicates value was below detection
level by FACS analysis. (ND) indicates no data yet.

GTOV chimeras ability to transduce CHO-K1 cells transiently transfected with
TFR1 expression plasmids.
CHO-K1 cells were transiently transfected with expression plasmids for hTfR1
and fTfR1. Transfected CHO-K1 cells were then transduced with 10X concentrated
stocks of GTOV chimeras and titer was measured by FACS analysis. As expected from
293A CEM T1B27
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7 VSVG
AM AV
G TO V
C G (A1)
C G (A5)
C G (A345)
C G (A4)
Titer (TU/ml)
* *
ND ND
*
ND
18
its tropism on CEM cells, G(A1) increased titer on CHO cells transiently transfected with
hTfR1 and fTfR1, displaying a similar response to transfection with these receptors as
parental GP, GTOV; half a log and greater than one log respectively (Fig. 8A). Recall
that we were able to see a change in GP affinity for hTfR1 and fTfR1 when domain 5 of
GTOV GP1 was cloned into AMAV GP1, clone A(G5). If domain 5 is the determinant
domain for hTfR1 usage, the opposite clone should decrease in affinity to hTfR1 to enter
cells. However, G(A5) titer on hTfR1 and fTfR1 transfected CHO-K1 cells was
approximately half a log and one log, respectively, above the mock transfected CHO-K1
cells titer confirming that G(A5) has not changed to a non-pathogenic tropism(Fig. 8).
Interestingly G(345) pseudotyped retroviral vectors did not increase in titer on the two
transfected CHO-K1 cell types with respect to mock transfected CHO-K1 cells indicating
it was not using hTfR1 or fTfR1 for cell entry. The introduction of two additional
consecutive domains of AMAV into GTOV GP1 has seemed to abolish GTOV GP1’s
pathogenic trait. This result still needs to be confirmed with repeat analysis. G(A4)
pseudotyped retroviral vectors have not yet been tested on transferrin transfected CHO-
K1 cells.

19
A

B

Fig. 8. Ability of GTOV chimeras to transduce hTfR1 or fTfR1 transfected CHO-K1
cells. Wild type and chimera GP-pseudotyped retroviral vectors were transduced on
mock transfected CHO-K1 cells or cells transfected with hTfR1 or fTfR1 expression
plasmids. Methods used are same as those used for AMAV chimeras. Values shown are
represented from 1-2 independent experiments. (B) Western blot confirming expression
of hTfR1 and fTfR1 in transfected CHO-K1 cells.

Analysis of requirement for a valine present only in domain 5 of pathogenic clade B
GPs.
Because A(G5) exhibited a change in tropism and TfR1 usage to that of GTOV,
we decided to explore the domain further. After a closer analysis of an alignment of the
clade B arenaviruses in the domain 5 of GP1, we noticed that the pathogenic viruses
within this clade all contained a valine at the fifth position in the domain, while the non-
pathogenic viruses did not (Fig. 9). AMAV has a tyrosine at the fifth residue of domain 5
and TACV, the other non-pathogenic clade B arenavirus, has a threonine. To determine
whether this particular amino acid contributed to binding of hTfR1 and transduction of
CHO-K1
VSVG
GTOV
AMAV
C G(A1)
C G(A5)
C G(A345)
1
10
100
1000
10000
mock
huTfR
feTfR
Relative titer (%) 100kD
75kD
actin
CHO-K1
mock hTfR1 fTfR1
20
CEM cells, we mutated the valine at the fifth residue in A(G5) to tyrosine (A(G5V-Y))
and the tyrosine in Amapari to valine (AMAV Y-V), and tested the resulting GP
pseudotyped-vectors for their tropism in lymphocytic cells and hTfR1 and fTfR1
transiently transfected CHO-K1 cells.
Junin PLDHVNTLHFLTRG-KN-IQPRRSL
Machupo QFDHVNTLHFLVRS-KTHLF-ESRL
Tacaribe KADHTNTFRFLSRSQKSIAV-GTRL
Guanarito SNSHVNTLRFLVKNAGY-LVGRKPL
Amapari VKSHYNTLAFLLKNTANSLPKRRPL
Sabia EMNHVNSMHLMLANAGRS-GSRRPL

Fig. 9. Sequence alignment of domain 5 of clade B New World Arenaviruses.
Pathogenic arenaviruses contain a valine (red) as the fifth residue while non-pathgenic
arenaviruses contain a threonine (Tacaribe) or a tyrosine (Amapari) (residue in blue)
.

Tropism of A(G5V-Y) and AMAV Y-V GPs.
A(G5V-Y) and AMAV Y-V pseudotyped retroviral vectors were first standardized on
293A cells. Unconcentrated titers for A(G5V-Y) on 293A cells were found to be
approximately a log higher than that of A(G5), while concentrated titers were almost
equivalent (Fig. 10A). AMAV Y-V unconcentrated titer on 293A cells was
approximately equivalent to wild type AMAV (10
4
TU/ml), while the concentrated titer
was about two logs higher (Fig. 10A). The two mutant GP pseudotyped retroviral vectors
were then transduced on CEM and T1B27 cells. A(G5V-Y) was unable to transduce
CEM cells (Fig. 10A). This was confirmed with a concentrated stock. We are able to
show that altering the valine in fifth position of the 5
th
domain of A(G5) has abolished its
pathogenic tropism on human lymphocytes. A(G5V-Y) was also unable to transduce
T1B27 cells. It is possible that A(G5) would not gain the ability to transduce T1B27
cells because the same amino acid may not used for binding both species type of T-
21
lymphocytes. AMAV Y-V was not able to transduce CEM cells. By changing the
tyrosine in domain 5 of AMAV to valine did not confer pathogenic tropism of T-
lymphocytes to a non-pathogenic virus. AMAV Y-V was still able to transduce T1B27
cells supporting that a different amino acid may confer binding to mouse T-lymphocyte
cells versus human lymphocytes.
A

B

Fig. 10. Tropism and TfR1 use for cell entry by A(G5V-Y) and AMAV Y-V mutants.
(A) Mutants were
transduced on 293A cells to be standardized and then transduced on CEM and T1B27
cells. Concentrated values shown. Values shown are means ± standard errors represented
by 2-6 independent experiments. (*) indicates value was below detection level by FACS
analysis. ND indicates no data collected yet. (B) Wild type and mutant chimera GP-
pseudotyped retroviral vectors were transduced on mock transfected CHO-K1 cells or
cells transfected with hTfR1 or fTfR1 expression plasmids. AMAV Y-V transduced with
unconcentrated vector, while all others were transfected with 10X concentrated stocks.
Expression confirmed by Western blotting. Methods used are same as those used for
AMAV chimeras. Values shown are represented from 1-2 independent experiments

293A CEM T1B27
10
0
10
1
10
2
10
3
10
4
10
5
10
6
10
7
VSVG
AMAV
A(G5)
GTOV
A(G5V-Y)
AMAV Y-V Titer (TU/ml)
VSVG
GTOV
AMAV
A(G5)
A(G5V-Y)
AMAV Y-V
1
10
100
1000
10000
mock
huTfR
feTfR
Relative titer (%)
* * * * ND
22
Both A(G5V-Y) and AMAV Y-V do not use hTfR1 for cell entry of CHO-K1 cells.
Because A(G5V-Y) lost its ability to transduce CEM cells, we were interested in
confirming its loss of pathogenicity by testing its use of hTfR1 and fTfR1 to enter cells.
We transduced CHO-K1 cells that were transiently transfected with either hTfR1 or
fTfR1 with 10 X concentrated stock of both the A(G5V-Y) and AMAV Y-V mutant
pseudotyped retroviral vectors. Relative to the mock transfected CHO-K1 cells, both
mutants did not exhibit a significant increase in titer when transduced on CHO-K1 cells
transfected with hTfR1 (Fig. 10B). A(G5V-Y) displayed a titer increase of
approximately half a log, while the initial chimera A(G5) when transduced on hTfR1
transfected CHO-K1 cells increases approximately 1 log above the mock. When
transduced on fTfR1 transfected CHO-K1 cells, A(G5V-Y) increased in titer by
approximately 1 log with respect to transduction on mock transduced CHO-K1 cells (Fig.
10B). This titer is approximately 1 log less than the titer given by A(G5) suggesting that
A(G5V-Y) has lost some affinity to using fTfR1 for cell entry. This may also correlate to
loss of pathogenicity since pathogenic arenaviruses use this receptor as well.
AMAV Y-V GP-pseudotyped retroviral vectors did not significantly increase in
titer on either the hTfR1 or fTfR1 transfected CHO-K1 cells. Its titer profile did reflect
that of wild type AMAV GP-pseudotyped retroviral vectors. We did not see a switch to
pathogenic tropism with this mutant suggesting that sufficient pathogenic tropism switch
may include more than one amino acid for wild type AMAV GP.

23
Western blotting unable to properly visualize chimeric proteins possibly due to
abnormal protein folding.
Presently, there is no antibody that recognizes GTOV or AMAV GPC. In order to
confirm by Western blot that certain chimeras were non-functional due to non-
incorporation into virions because of misfolding, we tested antibodies from a previous
study that recognize JUNV to see if the recognize AMAV or GTOV. Only one antibody
was able to visualize AMAV by Western blot (Figure 11A and B), however it was not
sufficient enough to visualize the chimeras. We also tested antibodies we have generated
against regions of GP1 in a related arenavirus, MACV, which did not recognize either
virus (data not shown).
We obtained FLAG-tagged AMAV and GTOV GP constructs from Dr. Stefan
Kunz at the Scripps Research Institute and was able to clone in chimeras into the FLAG
GP vectors. The FLAG protein is cloned into the C-terminus of the GPC. Ordinarily by
Western blot we are able to see processing of the GPC into its two subunits GP1 and
GP2(2-4, 16, 30).However because of the location of the FLAG protein on the GPC, we
would only be able to visualize GPC and GP2 in the Western blots performed in this
study. We visualized bands at approximately 60kD for the GTOV GPC and
approximately 35kD for the AMAV GP2. Western blots were performed with an anti-
FLAG, which weakly recognized the functional and non-functional chimera GPCs. We
were not able to strongly visualize processing of the chimera GPCs. The antibody was
able to recognize wild type FLAG GTOV GPC and FLAG AMAV GPC and GP2 in
virions (Fig. 12A and B). We have not yet made to be tested FLAG chimera GPs for
A(G4), A(G345), G(A4), G(A345), and G(A123). FLAG wild type and FLAG chimera
24
GPs were not able to be visualized in lysate Western blots (data not shown). Due to more
than likely abnormal folding of the chimeric proteins, the FLAG antibody may not be
able to recognize the FLAG epitope and so we are unable to properly visualize the
chimeras and make a conclusion as to why non-functional chimeras are unable to
transduce our standard cell line for titer, 293A cells.

A

B

Figure 11. Western blots of chimera GP-pseudotyped vectors probed with anti-Junin
Ab. (A) Western blot of functional chimera GP-pseudotyped vectors. (B) Western blot of
non-functional chimera GP-pseudotyped vectors.

75kD
50kD
37kD
25kD
GTOV AMAV A(G3) A(G5) G(A1) G(A4) G(A5) G(A345)
GPC
GP1
anti-MLV CA
75kD
50kD
37kD
25kD
GTOV AMAV A(G1) A(G12) A(G2) A(G4) A(G345) G(A12) G(A2) G(A3)
GPC
GP1
anti-MLV CA
25
A

B

Figure 12. Western blots of FLAG-tagged GP-pseudotyped vectors. (A) Western blots
of indicated functional chimera GP-pseudotyped vectors, probed with anti-FLAG
antibody. The positions of GPC and GP2 subunits are indicated. (B) Western blot of
non-functional chimera virions, probed with anti-FLAG antibody.

50kD
37kD
25kD
anti-MLV CA
GTOV AMAV A(G3) A(G5) G(A1) G(A5)
75kD
75kD
50kD
37kD
25kD
anti-MLV CA
GTOV AMAV A(G1) A(G12) A(G2) G(A12) G(A2) G(A3)
GPC
GP2
GPC
GP2
26
Discussion.

The arenavirus GP is made up of two subunits GP1 and GP2 both arranged as
tetramers. GP1 is the subunit of the GP that binds to the cell receptor and promotes cell
mediated endocytosis, while GP2 promotes fusion of the host-virus membrane upon pH
drop within the endosome(3, 10). The arenaviruses have been phylogenetically mapped
according to their GPC sequence and divided into two complexes: Old World and New
World arenaviruses(6-8). Within the clade B viruses, we have observed that pathogenic
GTOV and non-pathogenic AMAV are very close in GPC sequence homology.
In our recent study, we determined that clade B pathogenic and non-pathogenic
arenaviruses have different tropisms(13). On lymphocytic cell lines, pathogenic
arenaviruses displayed a different tropism than non-pathogenic arenaviruses, and were
therefore able to transduce human T-lymphocytic cells. Additionally pathogenic
arenaviruses are able to use hTfR1 and fTfR1 for enhanced cell entry on CHO-K1 cells
that are transfected with expression plasmids for either transferrin receptor, while non-
pathogenic do increase titer on transfected CHO-K1 cells(13, 28, 29).
In this study, we used the arenavirus phylogenetic GPC relationships and
differences in tropisms of pathogenic versus non-pathogenic arenaviruses to map the
region that correlates to pathogenicity of the virus GP1. We generated a series of
chimeras between closely related AMAV and GTOV that have been designed according
to a sequence analysis of the GP1 that has divided GP1 into five domains based on upon
the position of conserved cysteine residues. Chimeras were standardized on 293A cells.
Functional chimeras were then tested for tropism on CEM and T1B27 cells. The
27
chimeras were also transduced on hTfR1 or fTfR1 transfected CHO-K1 cells to test for
use of these transferrin receptors for cell entry. We looked for AMAV chimeras that
gained pathogenic tropism, or GTOV chimeras that lost pathogenic tropism.
From these chimeras, we have identified the amino acids that can confer
pathogenic tropism to AMAV GP1 (A(G5)), specifically domain 5 of GTOV GP1.
A(G5) is able to only transduce CEM cells, and displays an increase in titer when
transduced on hTfR1 or fTfR1 transfected CHO-K1 cells with respect to mock
transfected cells. However, the reciprocal chimera, domain 5 of AMAV GP1 in GTOV
GP1 (G(A5)) has not lost pathogenic tropism of parental GTOV GP. It is still able to
transduce CEM cells; however, it can now transduce T1B27 cells. Further it is still able
to use hTfR1 and fTfR1 for enhanced entry CHO-K1 cells. From this data we can
conclude that the residues in domain 5 of GTOV GP1 are able to confer pathogenicity,
however because we could not abolish pathogenicity in GTOV GP1 with AMAV GP1
domain 5, it is possible that other domains or residues participate in binding to
pathogenic receptors.
We further explored domain 5 by analyzing a sequence alignment of domain 5 of
the clade B arenaviruses. Interestingly, we found that only the pathogenic arenaviruses
have a valine in fifth residue in domain 5. AMAV contains a tyrosine. We then made
single amino acid mutations in the pathogenic A(G5) and non-pathogenic wild type
AMAV GP1s. A single mutation in domain 5 of A(G5) from valine to tyrosine abolished
its ability to transduce CEM cells and use of hTfR1 to enhance entry of CHO-K1 cells,
thus removing its pathogenicity. However, the reciprocal mutation in wild-type AMAV,
mutated tyrosine in domain 5 to valine, has not changed its tropism to that of a
28
pathogenic arenavirus. Therefore we concluded that a valine in domain 5 is necessary,
but not sufficient, for pathogenic tropism.
The evolution of viral GPs is a subject of constant study. Viral genomes are able
to recombine or gain mutations that allow for the formation of viruses that can infect new
hosts. Lefeuvre et al. proposed that in viral recombination, there is an avoidance of
disruption in protein folding(22). From a computational analysis of the perturbation of
folding in chimeric proteins in begomaviruses, they determined there are viral genome
‘hotspots’ where recombination is more likely to occur in synonymous genomes. The
only viable, reciprocal chimeras we were able to generate in this study were domain 5
chimeras. It is possible that this region is a ‘hotspot’ where mutation and recombination
of the genome would be most tolerated and it is possibly the most likely area where the
evolution of pathogenic tropism would begin. However this is merely speculation.
The structure of the GP1 is still unknown and would be indispensable to the study
of arenaviruses. GTOV GP1 does not lose its pathogenic tropism with the replacement of
domain 5 with non-pathogenic AMAV GP1 domain 5, yet AMAV GP1 gains
pathogenicity in the reciprocal chimera. Without knowing the structure of GP1 we are
unable to determine how GTOV GP1 domain 5 in AMAV GP affects the structure of
AMAV GP1, or if there are multiple domain interactions in GTOV GP1 that we are
unable to reproduce in AMAV GP1. Currently the crystal structures of hTfR1 interacting
with a ligand have been made for hTfR1 binding transferrin and HFE, the hereditary
hemochromatosis protein(14, 20, 21). It has been found that both of these ligands bind
to hTfR1 in the helical domain(14). Recently, Radoshitzky et al. have discovered that
amino acids in the apical domain of hTfR1 bind with the arenavirus GP1; therefore
29
confirming a previous hypothesis that transferrin does not compete with arenaviruses for
the cellular receptor giving possible clues as to where on GP1 is the receptor binding
site(29). Yet, until the crystal structure is determined, we are still limited in our
understanding arenavirus cellular binding and entry.
The data in this study has implications for future use in the analysis of
pathogenicity of arenavirus GPs. We can explore whether valine in domain 5 of other
pathogenic arenaviruses, such as JUNV or MACV, is needed for pathogenic tropism as
well. Additionally, identification of a common binding domain of pathogenic
arenaviruses has future therapeutic uses as well. An antibody can be generated against
this domain to block viral entry of cells. Finally, future developments of an attenuated
virus because of the knowledge of the arenavirus GP binding domain would be extremely
valuable for vaccine purposes.
The simplicity of viral pathogenicity is astounding and quite elegant. Humans are
always in a constant battle against emerging new infectious viruses. A virus can go from
innocuous to suddenly virulent by as simple as an amino acid change in its surface
glycoprotein(3). Influenza virus is unable to infect humans until it accumulates a small
amino acid change that confers it the ability to bind sialic acid and enter human
endothelial cells causing rampant disease. Additionally viruses are able to combat our
antiviral therapies with ease. HIV is constantly mutating and bypasses almost any novel
antiretroviral therapies being produced(27). Hemorrhagic fever viruses are no exception
in effortless virulence. Ebola virus can burn through human victims so fast that it runs
out of hosts to infect(33). Because of the high virulence and ease of transmission of
hemorrhagic fever viruses, the pathology of the disease is incredibly hard to study. An
30
insight into the first step of infection, glycoprotein binding and entry into the host cell,
might be our best and possibly only source for antiviral therapy against these deadly
viruses.

31

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Abstract (if available)

Abstract Five arenaviruses can cause hemorrhagic fever in humans. They are classified by the CDC as category A pathogens because of the possibility to be manipulated into biological weapons. Of interest, the New World clade B group of the arenaviruses contains both pathogenic and non-pathogenic members, including the closely related Amapari (AMAV) and Guanarito (GTOV) viruses. In our recent study, we determined that pathogenic viruses have a specific tropism and are able to transduce human T-lymphocytes and use the human transferrin receptor 1 for cell entry. By dividing the GP1 protein, the subunit that promotes cell binding and entry, into five domains based upon conserved cysteine residues, we generated a series of AMAV and GTOV chimeras to map the region of the GP1 subunit that correlates to pathogenic tropism. We have found a valine in the domain five of the GTOV GP1 that is necessary for pathogenic tropism in arenavirus.

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Human papillomavirus type 16 entry via the annexin A2 heterotetramer leads to infection and immune evasion

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Creator Martin, Vanessa Kimiko (author)

Core Title Mapping arenavirus glycoprotein determinants that correlate to cell entry and virus pathogenicity

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology

Publication Date 04/16/2008

Defense Date 03/13/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag arenavirus,glycoprotein,human transferrin,OAI-PMH Harvest

Language English

Advisor Cannon, Paula (committee chair), Landolph, Joseph Jr.. (committee member), McMillan, Minnie (committee member)

Creator Email vkmartin@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1133

Unique identifier UC1461871

Identifier etd-Martin-20080416 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-57334 (legacy record id),usctheses-m1133 (legacy record id)

Legacy Identifier etd-Martin-20080416.pdf

Dmrecord 57334

Document Type Thesis

Rights Martin, Vanessa Kimiko

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu