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Characterization of three novel variants of the MAVS adaptor

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Characterization of Three Novel Variants of the MAVS
Adaptor

by

Arlet Minassian

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MAJOR: GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)

December 2016

Copyright 2016 Arlet Minassian

ii

Acknowledgements
The work described in this dissertation would not have been possible without the
help of so many people.
First and foremost, I would like to express my sincere gratitude to my advisor Dr.
Pinghui Feng for his continuous support, encouragement, patience, and
guidance throughout my Ph.D. study. Dr. Feng has been my inspiration as I
hurdle all the obstacles in the completion of this research project. This thesis
would not have been possible without his efforts to guide me.
I would like to express my gratitude to my thesis committee members: Dr. Omid
Akbari, Dr. Chengyu Liang, Dr. Weiming Yuan, and Dr. Ebrahim Zandi for their
insightful comments and suggestions. They have been very supportive and have
motivated me to work with enthusiasm.
I would also like to thank my past and present fellow lab mates for their
invaluable assistance.
Last, but by no means least, I would like to thank my fiancé for always being
there for me with great patience, my parents and my brother for their
unconditional love, support, and the trust they always had in me, without which I
would have not been able to accomplish this dissertation and be who I am today.

iii

Table of Contents
Acknowledgements ............................................................................................... ii
Abstract ............................................................................................................... vii
1.Introduction ........................................................................................................ 1
1.1. Innate Immune Signaling ............................................................................ 1
1.2. RIG-Like Receptors .................................................................................... 1
1.2.1. Pathogen Sensing by RIG-I ................................................................. 2
1.2.2. RIG-I Activation Mechanism ................................................................. 3
1.3. Mitochondrial Antiviral Signaling ................................................................. 3
1.3.1. Subcellular Localization of MAVS ........................................................ 4
1.4. RIG-I-MAVS Signaling Pathway ................................................................. 5
1.5. Herpesviridae ............................................................................................. 6
1.5.1. Classification of Herpesviruses ............................................................ 6
1.5.1. Herpes Simplex virus 1 (HSV-1) .......................................................... 7
1.5.3. HSV-1 Replication Cycle ...................................................................... 8
2. An Internally Translated MAVS Variant Exposes Its Amino-terminal TRAF-
Binding Motifs to Deregulate Interferon Induction .............................................. 11
2.1. Introduction ............................................................................................... 11
2.2. Results ..................................................................................................... 13
2.2.1. Identification of MAVS50 .................................................................... 13

iv

2.2.2. MAVS Activated NF-ĸB, but Not IRF and IFN Induction ................... 16
2.2.3. MAVS50 Interacts with MAVS70 ........................................................ 19
2.2.4. MAVS50 Inhibits IFN-β Induction ...................................................... 21
2.2.5. MAVS50 Competes with MAVS70 for Binding to TRAF Molecules ... 22
2.2.6. N-Terminal TRAF2-Binding Motif Is Crucial for MAVS50 to Interact
with TRAF Molecules and Inhibit IRF Activation ......................................... 24
2.3. Discussion ................................................................................................ 26
3. HSV-1 gene expression is inhibited by two novel variants of MAVS ............. 57
3.1. Introduction ............................................................................................... 57
3.2. Results ..................................................................................................... 60
3.2.1. Identification of MAVS40 and MAVS30 .............................................. 60
3.2.2. Mapping the initiation site of MAVS40 and MAVS30 ......................... 63
3.2.3. MAVS40 and MAVS30 interact with MAVS70 and itself ................... 64
3.2.4. Characterization of roles of MAVS40 and MAVS30 in RIG-I-dependent
signaling ...................................................................................................... 66
3.2.5. MAVS40 and MAVS30 interact with HSV-1 structural components .. 67
3.2.6 MAVS40 and MAVS30 inhibit HSV-1 gene expression ...................... 69
3.3. Discussion ................................................................................................ 70
4. Materials and Methods ................................................................................... 90
5. References ..................................................................................................... 97

v

List of Figures
Figure 1. Identification of the MAVS50 variant ................................................... 34
Figure 2. Characterize the roles of MAVS50 in RIG-I dependent signaling ........ 36
Figure 3. Innate immune signaling of MAVS50 .................................................. 39
Figure 4. MAVS50 expression and its effect on innate immune signaling in 293T
cells .................................................................................................................... 41
Figure 5. MAVS50 interacts with MAVS70 ......................................................... 43
Figure 6. Co-elution of MAVS70 and MAVS50 in 293T cells infected with virus 45
Figure 7. MAVS50 inhibits MAVS70-dependent IFN induction ........................... 46
Figure 8. MAVS50 targets TRAF molecules to inhibit MAVS70-mediated IFN
induction ............................................................................................................. 48
Figure 9. MAVS50 modulates IRF-IFN signaling cascade .................................. 50
Figure 10. The N-terminal TRAF-2 binding motif is critical for MAVS50 to inhibit
IFN induction ...................................................................................................... 52
Figure 11. Characterization of TRAF2- and TRAF6-binding motifs of MAVS50 . 54
Figure 12. A hypothetical model on MAVS50 in modulating MAVS70-dependent
signaling ............................................................................................................. 56
Figure 13. Identification of MAVS40 and MAVS30 variants ................................ 76
Figure 14. Mapping the initiation site of MAVS40 and MAVS30 variants ........... 78
Figure 15. Identification of the cleavage site of MAVS40 and MAVS30 ............. 80
Figure 16. MAVS40 and MAVS30 interact with MAVS70 and itself .................... 81
Figure 17. Co-elution of MAVS variants upon infection with virus ...................... 83

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Figure 18. Characterization of roles of MAVS40 and MAVS30 in RIG-I-dependent
signaling ............................................................................................................. 84
Figure 19. MAVS40 and MAVS30 interact with HSV-1 structural components .. 86
Figure 20. MAVS40 and MAVS30 inhibit HSV-1 gene expression ..................... 88

vii

Abstract
Host innate immunity is the first line of defense against invading pathogens.
Upon viral infection, the cytosolic retinoic-acid-inducible gene I (RIG-I), a major
intracellular pathogen recognition receptor, senses double-stranded RNA and
dimerizes with the mitochondrial antiviral signaling protein (MAVS), which
activates two innate immune kinase complexes to promote the production of
antiviral cytokines and interferons. Previous studies on the MAVS adaptor have
focused on the full-length MAVS of 72 kDa. However, a smaller isoform of
MAVS, with a molecular weight of ~50 kDa (thus, referred to as MAVS50) is
consistently detected in a number of cell lines and its function remains unknown.
We and others reported that MAVS50 was produced via internal translation from
an alternative translation start site [the second AUG (142)]. Unlike full-length
MAVS, which activates both NF-ĸB and interferon (IFN) signaling cascades,
MAVS50 specifically activates NF-ĸB, but not the IRF-IFN induction. Lacking the
N-terminal sequence, including the CARD domain, MAVS50 exposes two key
TRAF-binding motifs within its N-terminus to effectively compete for association
with TRAF molecules, thereby inhibiting the IRF-IFN induction pathway. This
study identifies an example of protein diversity in eukaryotes that can be reached
by internal translation from a single mRNA and highlights a new means by which
innate immune signaling events are differentially regulated via exposing key
internally embedded interaction motifs.

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We recently identified that the MAVS transcript can generate two novel variants
with molecular weights of ~40 and ~30 kDa (thus designated MAVS40 and
MAVS30). Our studies demonstrate that MAVS40 is produced by internal
cleavage within amino acids 227-233, and MAVS30 is generated by internal
translation from the third initiation site [AUG (303)] and proteolytic cleavage
within amino acids 298-302. MAVS40 and MAVS30 lack the N-terminal
sequence, including the CARD domain. Consistent with the lack of N-terminal
CARD, MAVS40 and MAVS30 fail to stimulate IFN induction, when
“reconstituted” in cells depleted with MAVS expression. Interestingly, MAVS40
and MAVS30 demonstrate antiviral activity against Herpes Simplex virus 1 (HSV-
1), a model DNA virus. Molecular virology analysis indicated that MAVS40 and
MAVS30 potently inhibit viral gene expression. Moreover, the biochemical
analysis identified multiple virion components such as major capsid protein
(UL19), primary nuclear envelopment proteins (UL31 and UL34), and teguments
protein (UL37) that were associated with MAVS40 and MAVS30. Our findings
collectively support the model whereby MAVS40 and MAVS30 target herpesvirus
virion particle to block the initiation of viral gene expression. The successful
outcome of my study will likely identify a novel mechanism by which HSV-1
replication is restricted by truncated variants of MAVS, and will provide new
insights into regulatory roles of truncated proteins that arise from internal
translation and proteolytic cleavage.

1

1. Introduction
1.1. Innate Immune Signaling
The innate immune system forms the first line of host defense during infection
and thus plays an important role in the early recognition and subsequent
triggering of pro-inflammatory responses to invading pathogens.

Upon infection, the innate immune system utilizes germline-encoded pattern
recognition receptors (PRRs), such as Toll-like receptors (TLRs) and cytosolic
NOD-like and RIG-I-like receptors (RLRs), to sense evolutionarily conserved
molecular structures that are shared by pathogens, named pathogen-associated
molecular patterns (PAMPs). The transmembrane-anchored toll-like receptors
recognize pathogens at the cell surface or endosome membranes. In contrast,
cytoplasmic pattern recognition receptors (PRRs) such as NOD-like receptors
and RIG-I-like receptors can sense the pathogens which have evaded the cytosol
[1, 2]. Upon PAMP recognition, PRRs initiate a signaling cascade that ultimately
results in transcriptional activation and gene expression of a variety of pro-
inflammatory cytokines and secretion of interferons. Activation of these signaling
pathways and production of pro-inflammatory cytokines and interferons ultimately
constitute an antiviral innate immunity to control the early stages of infection [3,
4].

2

1.2. RIG-I-Like Receptors
The RIG-I-like receptors (RLRs) are a family of ATPase-dependent DExD/H RNA
helicases that function as cytoplasmic sensors to recognize viral RNA as PAMPs
[5, 6].

The RLR family is comprised of three members: RIG-I (retinoic acid-inducible
gene I) [7], MDA5 (melanoma differentiation associated factor 5) [8], and LGP2
(laboratory of genetics and physiology 2) [9]. RLRs are widely expressed in most
tissues and are highly induced by IFN exposure and after virus infection [7, 10,
11]. All three RLRs share a common structure which includes a central helicase
domain and a C-terminal domain known as the repressor domain (RD) [12, 13].
RIG-I and MDA5 also contain two N-terminal tandem caspase activation and
recruitment domains (CARDs) that function to recognize cytosolic RNA during
infection to activate innate immune signaling. LGP2 lacks the N-terminal CARD
domains and is thought to function as a regulator of RIG-I and MDA5 signaling
[6, 14].

1.2.1. PAMP Recognition by RIG-I
RIG-I is localized in the cytoplasm and recognizes the genomic RNA of dsRNA
viruses and dsRNA generated as the replication intermediate of ssRNA viruses.
Recognition of RNA ligands by RIG-I depends on several properties of viral RNA
including PAMP motif length, sequence composition, structure, and the presence

3

of exposed 5’triphosphate or 5’diphosphate. 5’-capped RNA does not trigger
RIG-I signaling. RIG-I preferentially recognizes dsRNA of <300 base pairs with
blunt ends containing a 5’triphosphate (5’ppp) [15-19]. However, recent studies
show that RIG-I recognizes RNA containing 5’-diphosphate (5’pp) moiety and
mediates antiviral responses [20].

1.2.2. RIG-I Activation Mechanism
The signaling activity of RIG-I is regulated by autoregulation mediated by an
intramolecular interaction between the CARDs and the helicase domain of RIG-I
[12, 21]. In resting cells, RIG-I is held in a “closed” conformation with the RD at
the carboxyl terminus covering the two CARDs and the central helicase domain.
Upon recognition of viral RNA by the CTD, RIG-I hydrolyzes ATP and undergoes
a conformational change which releases the CARDs from the RD. RIG-I is then
K63 polyubiquitinated at Lys172 by E3-ligase tripartite motif-containing 25
(TRIM25), which in turn induces the formation of CARD-CARD tetramers [22,
23]. Tetrameric CARD-CARD domains are then able to mediate association with
its adaptor protein, MAVS, and induce its activation [24].

1.3. Mitochondrial Antiviral Signaling
Mitochondrial antiviral signaling protein (MAVS), also named interferon promoter
stimulator-1 (IPS-1), virus-induced signaling adaptor (VISA), and Caspase
recruitment domain (CARD) adaptor inducing IFN-β (Cardif) is an adaptor

4

molecule downstream of RLRs, which relays signal from RLRs to downstream
kinase complexes and mediates the activation of NF ĸB and IRF3 in response to
viral infection [25-28]. MAVS is comprised of 540 amino acids in humans and
contains three functional domains: 1- An N-terminal caspase activation and
recruitment domain (CARD), which interacts with the CARD domain of RIG-I and
MDA5. 2- A proline-rich region (PRR) that is implicated in downstream signaling
by its interaction with TRAF2, TRAF3, and TRF6. 3- A C-terminal
transmembrane (TM) domain that localizes MAVS to a diverse set of membranes
including mitochondrial outer membrane, peroxisomes and mitochondrial-
associated membranes (MAM) [27, 29, 30].

1.3.1. Subcellular Localization of MAVS
MAVS is known to be localized to the mitochondrial outer membrane,
peroxisomes and mitochondrial-associated endoplasmic reticulum (ER)
membranes (MAM) [27, 29, 30]. Localization of MAVS to both mitochondrial
outer membrane and peroxisomes is shown to be necessary for maximal antiviral
response. Upon viral infection, peroxisomal MAVS induces rapid and transient
interferon-independent expression of ISGs, such as viperine, whereas
mitochondrial MAVS works with delayed kinetics and activates an interferon-
dependent ISG expression, which amplifies and stabilizes the antiviral response.
There is no obvious difference reported in the downstream regulators activated
by peroxisomal vs. mitochondrial MAVS [27, 29].

5

1.4. RIG-I – MAVS Signaling Pathway
As noted above, in the presence of RNA ligand binding, RIG-I undergoes a
conformational change, which induces RIG-I activation. Once the CARD-CARD
tetramers of RIG-I are formed, it translocates to the mitochondrial associated
membrane (MAM), which is facilitated by the 14-3-3 ε protein [31]. At the MAM,
RIG-I CARDs form a homotypic protein: protein interaction with the CARD of the
membrane-bound MAVS adaptor, catalyzing prion-like filament formation of
MAVS which leads to the activation of IKKα/IKKβ and TBK1/IKK-ɛ kinase
complexes to initiate downstream IRF and NF ĸB signaling [32].

Once MAVS is activated, it recruits transforming growth factor β-activated kinase
1 (TAK1) and TNF receptor-associated factors (TRAF), through distinct TRAF-
binding motifs [33, 34]. TRAF molecules, such as TRAF6, promote synthesis of
K63-linked polyubiquitin chains which recruit and activate IKK and TBK1 to
trigger IFN production [35-37]. Activated IKK complex (IKKα/IKKβ/IKKγ)
phosphorylates the inhibitor of NF-ĸB (IκBα) and induces its subsequent
degradation by the 26S proteasome [38, 39]. As a result, NF-κB translocates into
the nucleus and activates gene expression of pro-inflammatory cytokines [40].
On the other hand, TBK-1/IKKɛ kinase complex phosphorylates MAVS at its C-
terminal conserved serine and threonine cluster. Phosphorylated MAVS
subsequently recruits and binds to conserved, positively charged surface of
IRF3, allowing IRF3 phosphorylation by TBK1. Phosphorylated IRF3 dissociates

6

from MAVS, dimerizes and translocates into the nucleus to bind to its cognate
site and induce IFN [41]. Activation of these signaling pathways ultimately
results in the expression of pro-inflammatory cytokines and type I interferons and
establishment of an intracellular antiviral state. Secreted interferon then signals
both in the infected cell and surrounding cells in the local tissue through
interferon receptor and stimulates the JAK-STAT pathway, which ultimately
results in the nuclear localization of IFN-stimulated gene factor 3 (ISGF3). ISGF3
then binds to the interferon-stimulated response elements (ISRE) region of the
ISGs and trans-activates their expression. ISGs have antiviral and other
properties that serve to restrict virus replication [42, 43].

1.5. Herpesviridae
Herpesviruses are a large family of enveloped, double-stranded DNA viruses that
cause disease in animals, including humans. All herpesviruses share a common
structure, composed of a large, double-stranded, linear DNA genome within an
icosahedral capsid, which itself is surrounded by an amorphous layer known as
the tegument, which contains a number of viral proteins that are important for
efficient infection. The tegument is enclosed by a lipid bilayer membrane called
envelope. The entire viral particle is called virion.

7

1.5.1. Classification of Herpesviruses
Herpeviruses infecting mammals, birds and reptiles are classified into three
subfamilies: Alphaherpesvirinae [including herpes simplex virus (HSV) and
varicella zoster virus (VZV)], Betaherpesvirinae [including human
cytomegalovirus (HCMV), human herpesvirus (HHV) 6 and 7] and
Gammaherpesvirinae [including Epstein-Barr virus (EBV) and Kaposi’s sarcoma-
associated herpesvirus (KSHV)]. Herpesviruses infecting fish and amphibia are
categorized in the new family Alloherpesviridae. The herpesvirus identified in
oyster is the only member of the Malacoherpesviridae [44-46].

1.5.2. Herpes Simplex Virus 1 (HSV-1)
Herpes simplex virus type 1 (HSV-1) is a human pathogen which is carried by
60-80 % of the population worldwide, with the higher frequencies is developing
countries [47]. HSV-1 can establish lifelong latent infection in sensory neurons.
The virus can be periodically reactivated, which will trigger lytic infection mainly in
epithelial or mucosal cells, causing renewed symptoms of a clinical disease that
enable transmission among the population. Primary infection site of the host is
normally the mucosal surfaces [48-50]. During this initial active infection in
epithelial cells, virus particles enter the neuronal axons and transmit to dorsal
root ganglia and cranial nerve neurons, to establish latent infection. The latent
infection is characterized as maintenance of the HSV-1 genome within the
neuron in the absence of new viral particle production. The viral genome

8

localizes in the nucleus of the host neuron where they assemble into a repressed
chromatinized episomes, which do not integrate into the host cell genome [51,
52]. During latency in human dorsal root ganglia, the viral DNA does not
replicate, majority of the genome is transcriptionally inactive and only restricted
gene expression takes place [53]. Only one group of transcripts are highly
expressed, known as the latency-associated transcripts (LATs) [54]. Since
neuronal cells do not divide, the viral genomes are maintained despite the
absence of replication; therefore, infected individuals carry the virus for life. Once
the virus is reactivated within the latently infected neurons and has been
transported to the periphery, its replication in epithelial cells can lead to
symptomatic disease. The most common clinical signs of HSV-1 infection are the
formation of minor vesicular lesions in the oral region. When the lesions are
ruptured, the virus can spread to neighboring cells or transmit to a new host.
Also, HSV-1 can also cause lesion at other sites, such as on fingers and eyelids,
and it can also replicate in corneal epithelial cells and lead to herpes stromal
keratitis (corneal scarring), which is one of the leading causes of blindness in the
USA [55, 56]. The most serious HSV- associated clinical syndrome is when the
virus enters the central nervous system and causes herpes simplex encephalitis
(HSE). Although this is a rare disease, it has a high mortality rate [57][58, 59]

9

1.5.3. HSV-1 Replication Cycle
As mentioned above, primary infection of the host normally begins with entry into
epithelial cells. The virion enters the cells by fusing its envelope with the cellular
envelope. This fusion is mediated by viral proteins, including glycoprotein-B (gB),
gD and gH-gL [60, 61], which bind to cell surface receptors, including heparan
sulphate glycosaminoglycans [62, 63], herpes virus entry mediator (HVEM) [64]
and nectin-1 [65]. This fusion delivers the viral nucleocapsids into the cytoplasm.
Once released into the cytoplasm, the capsids are carried along microtubules,
which allow the capsids to move to the nuclear envelope, where they bind to
nuclear pores and inject the viral DNA into the nucleoplasm [66, 67]. The
herpesvirus genome can be immediately transcribed to produce early mRNAs,
which are translated to produce viral proteins that promote further transcription
and replication of the viral DNA. Overall, viral gene expression can be divided
into three groups named immediate early (or α), early (or β), and late (or ɣ). The
earliest genes transcribed are the immediate early (IE) genes, by a process that
uses cellular transcriptional machinery. This phase is followed by the expression
of early genes, which directly or indirectly are involved in viral genome
replication, e.g. enzymes that are required for replication of viral genome: DNA
polymerase, single-strand DNA binding protein, helicase-primase, origin-binding
protein, and enzymes involved in DNA repair and deoxynucleotide metabolism.
Once viral DNA replication starts, late genes get transcribed and produce viral
structural proteins. Next, replicated DNAs are packaged into capsids in the

10

nucleoplasm. (Reviewed in detail in [68]). Once the herpesvirus genomes are
packaged into capsids, they have to egress from the nucleus to extracellular
spaces. To egress from nucleus, nucleocapsids approach and contact the inner
nuclear membrane (INM) via nuclear actin filaments which are induced by
infection with HSV-1 [69, 70] followed by budding into perinuclear space. This
process is known as primary envelopment. This primary envelope then
undergoes de-envelopment, by fusion between the virion envelope and the outer
nuclear membrane (ONM) resulting in the translocation of nucleocapsids into the
cytosol, where they are coated with a layer of proteins that form the tegument
[71]. Next, the tegument coated capsids undergo secondary envelopment in the
cytoplasm by budding into cytoplasmic membranes such as the Golgi, trans-
Golgi network (TGN) and endosomes producing mature, infectious virion
particles. Cytoplasmic vesicles carrying mature virions are then transported to
the cell surface, where they fuse with the plasma membrane and release the
virions [67, 71, 72].

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2. An Internally Translated MAVS Variant Exposes Its Amino-
terminal TRAF-Binding Motifs to Deregulate Interferon Induction
2.1. Introduction
The host innate immune system is the first line of defense against invading
pathogens. Upon infection, the innate immune system utilizes pattern recognition
receptors (PRRs), such as toll-like receptors (TLRs) and cytosolic NOD-like and
RIG-I-like receptors (RLRs), to recognize molecular structures that are shared by
pathogens, known as pathogen associated molecular patterns (PAMPs) [2, 3].
The retinoic acid-inducible gene I (RIG-I), is one of the major cytosolic receptors
that senses viral RNA bearing distinct structural elements [16, 17, 73]. Upon
recognition of viral RNA, RIG-I triggers downstream signaling by binding to an
adaptor protein, MAVS, through CARD domain interactions. Dimerization of RIG-
I with mitochondrial antiviral signaling (MAVS) adaptor, in turn, triggers the
activation of downstream IKKα/β/ɣ and TBK-1/IKKɛ kinase complex to promote
cytokine and interferon production [25-28]. Activated IKKα/β/ɣ complex
phosphorylates the inhibitor of NF-ĸB (IκBα) and induces its subsequent
degradation. As a result, NF-κB translocates into the nucleus and activates gene
expression of pro-inflammatory cytokines [38, 39, 74]. On the other hand, TBK-
1/IKKɛ complex phosphorylates the interferon regulatory factors (IRFs) to enable
the expression and secretion of interferons (e.g., interferon β) [40, 75]. As such,
these signaling cascades constitute an effective antiviral state to restrict
pathogen propagation during early stages of infection; therefore, regulation of

12

these pathways is critical to maintain homeostasis and avoid collateral damage in
cells.

One of the common means to regulate signaling pathways is by producing
proteins with diverse functions. Diversification of protein isoforms can be
achieved by alternative splicing and/or alternative translation. Alternative splicing
of precursor messenger RNA (pre-mRNA), during mRNA processing is an
established mechanism by which proteome diversity is generated in eukaryotes
[76-78]. Examples of this form of regulation include alternatively spliced variants
of RIG-I [22], NOD2 [79, 80] and MyD88 [81] that function as negative regulators
of RIG-I, NOD2 and TLR-induced signal transduction, respectively. Thus,
alternative splicing has been identified as an important method of controlling host
immune-signaling activity. In contrast, alternative translation through the internal
AUG start codon within a single transcript is considered to be common in
prokaryotes and is poorly investigated in eukaryotes. However, recent studies
show that thousands of eukaryotic mRNAs are predicted to produce protein
isoforms due to use of alternative translation initiation sites [82-84]. These
studies suggest that contribution of alternative translation may be more common
in eukaryotes than appreciated.

MAVS is a critical adaptor protein in the type I interferon pathway downstream of
RIG-I [25-28]. Original studies identifying the MAVS gene reported that MAVS

13

protein exists as multiple bands on SDS-PAGE. To date, all of the studies on the
MAVS gene have been attributed to the 72 kDa full-length (FL) MAVS (hereafter
referred to as MAVS70), while the lower bands were speculated to be a
degradation product or the processed version of the FL MAVS [27]. Recently, we
identified a 50 kDa MAVS variant (referred to as MAVS50) that arises via internal
translation from the second AUG start site at codon 142, lacking the N-terminal
caspase recruitment domain (CARD) [85]. Notably, the MAVS50 variant was also
reported by Burbaker S.W. et al [86]. Our studies have revealed that MAVS50 is
sufficient to activate NF- ĸB, but not IFN induction. MAVS50 exposes its TRAF-
binding motifs within the N-terminus and inhibits MAVS70-mediated IRF
activation and IFN induction by competing with full-length MAVS for binding to
TRAF molecules. These findings identify a delicate mechanism by which innate
immune signaling events are regulated by truncated proteins that expose their
key protein-interacting domains.

2.2. Results
2.2.1. Identification of MAVS50
We have previously identified gamma herpesviral homologues of glutamine
amidotransferase (referred to as vGAT) activate RIG-I via deamidation [87]. We
noted a remarkable feature of this innate immune activation is the preferential
activation of the NF- B signaling cascade, but not that of IRF and IFN induction.
In an experiment that aims to examine MAVS activation by Sendai virus (SeV)

14

infection or expression of HV68 vGAT, we observed that a smaller isoform of
MAVS, of ~50 kD (designated MAVS50), did not migrate into the Triton X-100-
insoluble fraction in cells infected with SeV or expressing HV68 vGAT (Fig 1A).
In contrast, the full-length MAVS, designated as MAVS70, accumulated in the
Triton X-insoluble fraction, indicative of its activation [27]. This result suggests
that MAVS50 likely possesses function distinct from its kin, MAVS70.

We then set out to determine the nature of the MAVS50 variant. Visual inspection
of MAVS mRNA revealed an internal translation initiation site at codon 142 (Fig
1B). We reasoned that MAVS50, if produced from internal translation from the
second initiation codon, lacks the N-terminal region including the entire CARD
domain. We employed two antibodies, one was raised against a polypeptide of
the first 135 amino acids and the other against an internal sequence
encompassing amino acids 150 to 250, to differentiate these two putative MAVS
isoforms. When whole cell lysates were analyzed by immunoblotting, we found
that only MAVS70 reacted with the antibody against the first 135 amino acids,
whereas both MAVS70 and MAVS50 reacted with the antibody against the
internal region (aa 150-250) (Fig 1C). This result indicates that MAVS50 lacks
the amino-terminal region. We then engineered a MAVS construct that carries an
amino-terminal Flag epitope and a carboxyl-terminal HA epitope to probe MAVS
expression. Such a dually-tagged MAVS construct yielded a single MAVS

15

species of 70 kDa reacting with anti-Flag antibody, and both species of ~70 kDa
and 50 kDa reacting with anti-HA antibody. These two species are of similar
sizes to endogenous MAVS (Fig 1D). To determine whether MAVS50 was
produced from the second initiation codon, we mutated the second ATG
(methionine) into TGC (cysteine) within the cDNA of MAVS70. To exclude that
MAVS50 is a product of MAVS70 due to internal proteolytic cleavage, we deleted
the first ATG of MAVS70. We also generated a construct that contains the cDNA
sequence encoding amino acids 142 to 540 of MAVS70. As shown in Fig 1E,
deletion of the first initiation codon abolished the expression of MAVS70, while
produced more MAVS50 than a construct containing wild-type MAVS. The
increased MAVS50 expression likely stems from the lack of competition of
translation initiation at the first AUG codon. We noted additional species larger
than 50 kDa were produced from the construct missing the first initiation codon,
suggesting that these proteins are produced from internal translation using non-
AUG codons upstream of the second AUG (142) initiation codon. As expected,
mutation of the second initiation codon abolished the expression of MAVS50.
Moreover, the MAVS construct containing the sequence encoding amino acids
142-540 yielded a MAVS protein migrating identically as MAVS50. These results
collectively support the conclusion that MAVS50 is produced from internal
translation initiation using the second AUG codon. Next, we probed a number of
human cell lines for the expression of MAVS50 and found that MAVS50, similar
to MAVS70, was abundantly expressed from HEK 293T, Jurkat T cells, BJAB B

16

cells, HUVEC endothelial cells, HCT116 colorectal cells, although both isoforms
were detected at very low level in HeLa cervical cells (Fig 1F). We further
examined the expression of MAVS70 and MAVS50 expression in cells infected
with SeV and herpes simplex virus type 1 (HSV-1), prototype RNA and DNA
viruses, respectively. Both viruses modestly induced the expression of MAVS50
at late time points post-infection, specifically 24-48 (Fig 1G) and 12 (Fig 1H)
hours post-infection for SeV and HSV-1, respectively.

2.2.2. MAVS50 Activates NF- ĸB, but Not IFN Induction
MAVS serves as an adaptor to relay signaling from RIG-I and MDA5 receptor to
downstream kinases that bifurcate to activate NF- B and IRF transcription
factors [27, 28]. To examine the roles of MAVS50 in these signaling cascades,
we over-expressed MAVS50 and examined signaling events leading to NF- B
and IRF activation. Using reporter assays, we found that MAVS50 expression
activated NF- B (Fig 2A) in a dose-dependent manner. By contrast, MAVS50
expression did not up-regulate the promoter of IFN-β, but MAVS wild-type and
MAVS70 did (Fig 2B). Consistent with the NF- B activation, over-expressed
MAVS50 also up-regulated the kinase activity of IKKβ by an in vitro kinase assay,
in comparison to RIG-I-N and MAVS70 (Fig 2C). TRAF molecules are important
adaptors downstream MAVS and are implicated in specific activation of NF- B
and IRF transcription factors. Thus, we examined MAVS50 interactions with a

17

panel of six TRAF molecules by co-IP assays in transfected 293T cells. While
MAVS70 interacted with all TRAF molecules except TRAF4, MAVS50
demonstrated preferential interaction with TRAF2 and TRAF6 (Fig 3A and 3B).
This result suggests that MAVS50, in comparison to MAVS70, is distinct in
interacting with downstream TRAF adaptors. When signaling events of TBK-1
and IRF activation were examined, we found that MAVS50 expression had no
detectable effect on the kinase activity of TBK-1, nor the dimerization of IRF3
(Fig 3C and 3D), supporting the conclusion that MAVS50 does not activate the
IRF signaling cascades. As controls, RIG-I-N (2CARDs) and MAVS70 potently
activated TBK-1 by kinase assay and induced IRF3 dimerization by native gel
electrophoresis. These results collectively show that MAVS50 preferentially
activates the IKK -NF- B signaling cascade.

MAVS is characterized by a CARD-mediated oligomerization in provoking
downstream signaling events [27, 88]. MAVS50 lacks the CARD domain and we
determined whether MAVS50 forms oligomers by size exclusion
chromatography. When purified from 293T cells, MAVS70 was eluted in fractions
corresponding to ~670 kDa, consistent with the notion that MAVS70 forms large
oligomers (Fig 2D). However, MAVS50 was eluted in fractions corresponding to
~120 kDa, suggesting that MAVS50 forms smaller oligomers, likely dimer or
tetramer. This result is consistent with the critical roles of CARD domain in

18

mediating large signaling-competent oligomers [32, 89]. Given that MAVS50
lacks the CARD for interaction with RIG-I, we determined whether MAVS50 could
relay signal transduction downstream of RIG-I. Thus, we knocked down the
expression of endogenous MAVS (both isoforms) (Fig 2E) and “reconstituted”
MAVS expression with MAVS wild-type, MAVS70 or MAVS50 (Fig. 2F). These
“reconstituted” cells were then used to examine RIG-I-dependent signaling in
response to SeV infection. Upon SeV infection, 293T cells “reconstituted” with
wild-type MAVS and MAVS70 activated TBK-1 as determined by TBK-1
phosphorylation at ser172, a marker for TBK-1 activation (Fig 2G). However,
MAVS50 expression failed to do so. When mitochondrion-enriched fractions were
analyzed for phosphorylated TBK-1 (pS172), we found that phosphorylated TBK-
1 was abundant in the mitochondrion-enriched fraction from cells “reconstituted”
with wild-type MAVS and MAVS70, but not that of cells expressing MAVS50 (Fig
2H). Consistent with this, “reconstituted” expression of wild-type MAVS and
MAVS70 restored robust expression of IFNb, CCL5, ISG56, IL-8 and Viperin,
indicative of activation of IRF and NF- B (Fig 2I and 4A). However,
“reconstituted” expression of MAVS50 failed to up-regulate the transcription of
these anti-viral cytokines. Enzyme-linked immunoassay (ELISA) further show
that IFN and CCL5 were produced from knockdown cells expressing wild-type
MAVS and MAVS70 (Fig 2J). We observed marginal but detectable level of IFNb
and significantly ISG56 in resting cells that were “reconstituted” for the
expression of MAVS wild-type and MAVS70. Similar to MAVS wild-type and

19

MAVS70, MAVS50 localizes to the mitochondrion and peroxisome when
expressed in 293T cells (Fig 4B and 4C), consistent with the notion that MAVS is
targeted to the mitochondrion via a transmembrane tail [27]. Thus, MAVS50 does
not relay signal transduction from RIG-I to downstream molecules, re-enforcing
the critical role of CARD in RIG-I-mediated signaling.

2.2.3. MAVS50 Interacts with MAVS70
MAVS50 lacks the CARD domain and fails to relay signal transduction
downstream of RIG-I. Considering that MAVS50 possesses most of the
sequence of MAVS70, we reasoned that MAVS50 likely regulates MAVS70-
mediated signaling. To test this hypothesis, we first examined whether MAVS50
can interact with MAVS70 and itself. In transfected 293T cells, V5-tagged
MAVS70 and MAVS50 were readily detected in protein complexes precipitated
with anti-Flag antibody against Flag-MAVS50 (Fig 5A), indicating that MAVS50
can interact with MAVS70 and MAVS50. This result is consistent with our
observation that MAVS50 eluted as ~120 kDa in gel filtration, which implies self-
oligomerization of MAVS50 (Fig 2D). Due to the largely overlapping sequence
between MAVS70 and MAVS50, it is technically challenging to probe the
interaction between endogenous MAVS70 and MAVS50. Thus, we established a
stable 293 cell line that expresses Flag-tagged MAVS50 under the control of
doxycycline in a dose-dependent manner (Fig 4D). Precipitation of MAVS50

20

effectively pulled down MAVS70, indicating that MAVS50 physically associates
with endogenous MAVS70 (Fig 5B).

We further analyzed the interaction between MAVS70 and MAVS50 with gel
filtration that is routinely used to assess protein complex formation. Purified
MAVS50 was predominantly eluted in fractions corresponding to proteins of ~120
kDa. However, when MAVS50 and MAVS70 were co-expressed in 293T cells,
purified MAVS50 was eluted in fractions corresponding to ~670 kDa oligomer
(Fig 5C). These results show that MAVS70 can convert MAVS50 into oligomers
of larger sizes. Moreover, MAVS70 was also detected in fractions that were
enriched with oligomerized MAVS50, indicating that MAVS70 is integrated in the
MAVS50 oligomers and vice versa (Fig 5C). Finally, we assessed the elution
pattern of MAVS70 and MAVS50 in lysates of three representative cell lines,
including THP-1 monocyte, 293T fibroblast and HeLa cervical epithelial cells, by
size exclusion chromatography. MAVS70 and MAVS50 co-eluted in fractions
corresponding to ~220-440 kDa in THP-1 monocytes, 293T and HeLa cells (Fig
5D). A notable difference in the elution patterns of MAVS70 and MAVS50 was
observed, i.e., MAVS70 were more evenly distributed in fractions 24 and 26,
whereas MAVS50 was predominantly eluted in fraction 24. Furthermore, upon
Sendai virus infection, MAVS50 increased in fraction 26 that MAVS70 peaked in
elution (Fig 6). Similar result was observed for 293T cells infected with murine

21

gamma herpesvirus 68, a DNA virus (Fig 6). These observations suggest that
MAVS 50 preferentially associate with the larger size of MAVS70 oligomers.
Collectively, these results indicate that MAVS50 physically interacts with
MAVS70.

2.2.4. MAVS50 Inhibits IFN-β Induction
To determine the effect of MAVS50 on MAVS70-mediated signaling, we
assessed activation of NF- B and IFN-β promoter by reporter assays. We found
that MAVS50 inhibited MAVS70-induced transcription of the IFN-β promoter in a
dose-dependent manner (Fig 7A). By contrast, MAVS50 did not significantly
impact the NF- B activation by MAVS70 (Fig 7B). We noted that MAVS50 was
capable of activating NF- B. We then expressed exogenous MAVS50 either by
lentivirus transduction or transient transfection, and examined host cytokine gene
expression and viral replication. When MAVS50 was expressed in 293T cells by
lentivirus transduction (Fig 7C), while ISG56 expression was not significantly
impacted, the expression of IFN  was reduced by 50% in response to SeV
infection (Fig 7D). Conversely, exogenously expressed MAVS50 enhanced the
replication of vesicular stomatitis virus (VSV), a prototype RNA virus, by
fluorescence microscopy (Fig 7E). Plaque assay further showed that MAVS50
expression increased VSV replication by more than 5-fold (Fig 7F). Taken
together, MAVS50 inhibits IFN-β induction in response to viral infection.

22

2.2.5. MAVS50 Competes with MAVS70 for Binding to TRAF Molecules
The cytosolic sensor-mediated IFN induction pathway constitutes of key signaling
molecules, including RIG-I/MDA5, MAVS, TBK-1/IKK , and IRF3 (Fig 8A). Over-
expression of these components is sufficient to activate downstream signaling
events, cumulating in the up-regulation of IFN expression. To identify the point of
inhibition by MAVS50, we employed reporter assay that takes advantage of the
IFN-β promoter as a surrogate and the over-expressed key components outlined
in Fig 8A. While MAVS50 inhibited transcription of the IFN-β promoter induced by
RIG-I-N and MAVS70 (Fig 8B and 5D), MAVS50 had no inhibitory effect on the
IFN-β promoter induced by TBK-1, IKKε and the constitutively active IRF3-5D
mutant (Fig 8C and 9A and 9B). These results suggest that MAVS50 targets a
step between MAVS70 and TBK-1, a link between the common adaptor and
bifurcated downstream signaling events that trigger IRF activation and IFN
induction.

Considering that MAVS50 contains TRAF2- and TRAF6-binding motifs within its
N-terminus and that TRAF molecules serve as link downstream of MAVS, we
reasoned that MAVS50 likely targets TRAF molecules to modulate MAVS70-
mediated signaling of the IRF branch. We then assessed TRAF6 interaction with
MAVS70 and MAVS50 by co-immunoprecipitation. When TRAF6 was
precipitated in transfected 293T cells, MAVS50 was readily detected and

23

MAVS70 was detected at background level (Fig 8D). We noted that MAVS70
expression consistently diminished the interaction between MAVS50 and TRAF6.
We further probed TRAF6 interaction with MAVS50 or MAVS70 in 293T cells that
were “reconstituted” with exogenous MAVS70 and MAVS50. Co-IP assays
demonstrated that MAVS50, but not MAVS70, interacted with TRAF6 in 293T
cells (Fig 8E). Interestingly, although MAVS50 can interact with TRAF3, MAVS50
failed to precipitate with TRAF3 in the presence of MAVS70 (Fig 9C). This result
suggests that MAVS50 weakly interacts with TRAF3.

To determine whether MAVS50 can compete with MAVS70 for binding to
TRAF6, we took advantage of the MAVS knockdown cells that were
“reconstituted” with exogenous V5-tagged MAVS70 to examine MAVS70
interaction with TRAF6 by Co-IP assay. We found that increasing amount of
MAVS50 reduced TRAF6 precipitated with anti-V5 (MAVS70) in a dose-
dependent manner (Fig 8F). Interestingly, MAVS50 expressed at a lower level
increased the amount of TRAF6 precipitated with MAVS70 (compare lane 2 and
3), suggesting that MAVS50 integrates into MAVS70 to bridge an interaction
between MAVS70 and TRAF6. With higher levels of MAVS50, MAVS50
effectively reduced TRAF6 precipitated by MAVS70, indicative of competition
between MAVS50 and MAVS70 for association with TRAF6 (compare lanes 4
and 5 to lanes 2 and 3). In SeV-infected cells, the effect of MAVS50 on

24

interaction between MAVS70 and TRAF6 was reduced, suggesting that SeV
infection partly inhibits MAVS50 action. We also recognized that SeV infection
slightly reduced, rather than increased, MAVS70 interaction with TRAF6. This is
likely due to the “reconstituted” expression of MAVS70 that already activated
downstream signaling in resting cells (Fig 2I) and further activation by SeV
infection likely precipitated MAVS70 out from the Triton X-100-soluble fraction.
Nevertheless, MAVS50 competes with MAVS70 for binding to TRAF6.

2.2.6. N-Terminal TRAF2-Binding Motif Is Crucial for MAVS50 to Interacts
with TRAF Molecules and Inhibit IRF Activation
A major TRAF2-binding motif (PVQE, [P/S/A/T]x[Q/E]E) locates at the very
amino terminus of MAVS50 and a TRAF6-binding motif (PGENSE, PxExx[Ar/Ac];
Ar, aromatic; Ac, acetic)[90, 91] immediately follows the TRAF2-binding motif
(Fig 10A). To probe the contribution of these N-terminal TRAF-binding motifs, we
mutated the critical residues of TRAF2- and TRAF6-binding motifs into alanines,
thus named M2 and M6 of MAVS50 (Fig 11A), and examined their interactions
with TRAF2 or TRAF6 by co-IP assay. As expected, mutations within the TRAF2-
binding motif abolished MAVS50 interaction with TRAF2, while mutations within
the TRAF6-binding motif had no effect on MAVS50 interaction with TRAF2 (Fig
10B). MAVS50 mutant ablated both TRAF2- and TRAF6-binding motifs lost the
interaction with TRAF2. Surprisingly, mutating the TRAF2-binding motif nearly

25

abolished the MAVS50 interaction with TRAF6 (Fig 10C). Mutations within the
immediate downstream TRAF6-binding motif, although reduced MAVS50
interaction with TRAF6, had less effect than mutations within the TRAF2-binding
motif (Fig 10C). Simultaneously mutating the TRAF2- and TRAF6-binding motifs
reduced MAVS50 association with TRAF6 to residual level. The residual level of
TRAF6 interaction of MAVS50 M2,6 mutant is likely due to the transmembrane-
proximal TRAF6-binding motif. Additionally, the MAVS50 M2,6 mutant also
demonstrated reduced interactions with TRAF3 and TRAF5 in transfected 293T
cells (Fig 11B and 11C). Mutations within the TRAF2- and TRAF6-binding motifs
had no significant effect on MAVS50 interaction with MAVS70 by co-IP assay
(Fig 11D), suggesting that these MAVS50 mutants are functionally competent.
These results indicate that the very amino-terminal TRAF2-binding motif is critical
for binding to both TRAF2 and TRAF6, and likely other TRAFs, suggesting that
the TRAF2-binding motif is a functionally degenerate interaction motif for more
than one TRAF molecule.

To determine the role of the N-terminal TRAF-binding motifs of MAVS50, we
examined the effect of MAVS50 mutant that harbors mutations in both TRAF2-
and TRAF6-binding motifs, designated MAVS50-M2,6. Compared to wild-type
MAVS50, MAVS50-M2,6 was significantly impaired to inhibit MAVS70-induced
IFN-β expression by reporter assay (Fig 10D). We then expressed exogenous

26

MAVS50 or MAVS50M2,6 mutant by lentivirus (Fig 11E) and infected these cells
with SeV. Real-time PCR analysis showed that the MAVS50-M2,6 mutant failed
to inhibit IFNb gene expression in response to SeV infection, but wild-type
MAVS50 did (Fig 10E). Consistent with this, wild-type MAVS50 expression
increased VSV replication, but MAVS50-M2,6 mutant completely lost the ability
to promote VSV replication (Fig 10F and 10G). These results collectively
demonstrate the critical role of the TRAF-binding motifs of MAVS50, locating
within the very N-terminal region, in inhibiting IFN induction downstream of RIG-I.

2.3. Discussion
Upon sensing pathogen-associated molecular patterns, pattern recognition
receptors initiate signaling events that bifurcate into NF- B and IRF activation
downstream of common adaptor molecules. While NF- B activation and cytokine
production are important for inflammatory response that attracts other immune
cells to the site of infection, activated IRF and secreted IFN exert immediate
antiviral effect within the site of infection. How NF- B and IRF activation,
triggered by shared upstream signaling molecules such as RIG-I and MAVS, are
differentially regulated is not well understood. We report here that the MAVS50
variant, translated from an internal initiation codon, effectively competes with
MAVS70 for binding to TRAF molecules. In doing so, MAVS50 inhibits MAVS70-
mediated signal transduction, specifically IRF activation and interferon induction.

27

Our work agrees well with a recent study reporting that MAVS50 suppressed
RIG-I-dependent IFN induction [86]. Additionally, linear ubiquitination of NEMO,
the scaffold protein of IKK and TBK-1 kinase complexes, was previously reported
to dampen IFN induction while stimulating NF- B activation [92]. This activity
requires the LUBAC E3 ligase that catalyzes linear ubiquitin chain assembly on
NEMO and disrupts MAVS interaction with TRAF3, which relays signaling from
MAVS to TBK-1 and IRF activation. A more recent study showed that cholera
toxin induced RIG-I-dependent signaling events with a signature of NF- B
activation, although the molecular mechanism underpinning this preferential
activation is not clear [93]. These studies, including our current work, highlight a
recurring theme in differential regulation of the two signaling ramifications
downstream of shared receptors that sense invading pathogens, pointing to a
common shift from IFN induction to a NF- B-dependent inflammatory response.
Under conditions of pathogen infection, it is likely that IFN induction is the
immediate robust response of the innate immune phase, whereas NF- B
activation and cytokine secretion constitute a modest but sustained inflammatory
response. Given the pro-survival roles of NF-B activation, it is not surprising that
viruses often usurp NF- B activation or upstream signaling events to facilitate
their infection, such as HIV and herpesviruses [94, 95]. By contrast, IRF
activation and IFN signaling promote cell death. In contrast to what was reported
by Brubaker et al., we did not observe cell death induced by MAVS70 and

28

MAVS50. This discrepancy may stem from the difference in our experimental
conditions.

Despite of missing the N-terminal CARD domain that mediates hetero-
oligomerization with RIG-I and self-oligomerization of MAVS [32, 89], MAVS50
forms small oligomers that correspond to the size of a dimer or tetramer analyzed
by gel filtration. Consistent with that, MAVS50 can homo-dimerize as determined
by co-immunoprecipitation. This intriguing observation suggests the existence of
unknown sequence that, in addition to CARD, mediates MAVS dimerization. To
define a dimerization domain, we have applied serial truncations from the N-
terminus of MAVS50 and performed co-IP assays. Unfortunately, we failed to
pinpoint a key homo-dimerization sequence, implying that MAVS50 homo-
dimerization requires a structural sequence, rather than the primary linear
sequence. Alternatively, other cellular factors, including mitochondrial membrane,
may scaffold the homo- and hetero-dimerization of MAVS70 and MAVS50.
Nevertheless, MAVS50 is prone to form oligomer and, when it is over-expressed,
is sufficient to trigger NF- B activation. On the other hand, “reconstituted”
expression of MAVS50 in cells that endogenous MAVS isoforms, both MAVS70
and MAVS50, were depleted by shRNA-mediated knockdown failed to trigger the
expression of inflammatory cytokines and IFN-  in response to SeV infection.
This result indicates that MAVS50 cannot relay signal transduction from RIG-I to

29

NF- B and IRF transcription factors and requires MAVS70 to do so, re-enforcing
the critical role of the CARD domain in assembling the RIG-I-MAVS signaling
platform. Indeed, MAVS50 interacts with MAVS70 and MAVS70 expression
converted MAVS50 from oligomers of ~120 to those of ~670 kDa. When purified
MAVS50 was analyzed by gel filtration, MAVS70 was detected in fractions that
were enriched for oligomerized MAVS50. Thus, MAVS70 can incorporate into
MAVS50 oligomers, and vice versa. These activities enable MAVS50 to serve as
a modulator of the MAVS-dependent immune pathways via interaction with
MAVS70 and TRAF molecules, key amplifiers at the crossroad in innate immune
signaling. However, MAVS70 expression diminished the interaction between
MAVS50 and TRAF molecules, including TRAF6 and TRAF3. These results
suggest that the innate immune signaling, activated by MAVS70 expression, is
capable of inactivating MAVS50 and potentially releasing the MAVS50-mediated
inhibition. This hypothesis remains to be examined in the near future.

How does MAVS50 differentially alter MAVS70-dependent signaling, i.e.,
inhibiting IRF activation and IFN induction while weakly stimulating NF- B
activation? Based on our findings, we propose the following hypothetical model
that summarizes the action of MAVS50 in specific inhibiting IFN induction (Fig
12). Upon stimulation such as activated RIG-I, MAVS70 forms large oligomers in
the form of prion-like polymers or fibrils, resulting potent activation of both NF- B

30

and IRF transcription factors. By positioning TRAF-binding motifs at the very N-
terminus, MAVS50 interacts more strongly with TRAF6, and likely TRAF2, than
MAVS70. In doing so, MAVS50 efficiently sequesters TRAF6 from the prion-like
MAVS70 polymers, attenuating the MAVS70-mediated signaling. When activated
MAVS70 undergoes oligomerization, MAVS50 is induced to oligomerize via
interacting with MAVS70. Given its high affinity for TRAF adaptor molecules,
oligomerized MAVS50 is sufficient to induce NF- B activation, but not IRF
activation and IFN induction. Sum of both results is the specific inhibition of IFN
induction and modest NF- B activation by MAVS50. Then, how does MAVS70
activate both NF- B and IRF, while MAVS50 activates only NF- B. In SeV-
infected or vGAT-expressing cells, MAVS70 migrated into the Triton X-100-
insoluble fraction, whereas MAVS50 remained in the soluble fraction. In
transfected 293T cells, MAVS50 forms smaller oligomers than MAVS70. These
findings largely agree with the notion that higher order of MAVS70 oligomers
form fibrils and precipitates out from Triton X-100-containing solution, whereas
MAVS50 does not [27, 32]. Together with TBK-1 that phosphorylates an IRF3-
binding domain, MAVS70 fibrils provide a signaling platform that enables IRF3
activation and IFN induction [96]. Thus, it is conceivable that MAVS70 and
MAVS50 form at least two types of oligomers that are of distinct sizes. The large
oligomeric MAVS70 is capable of activating NF- B and IRF, while the smaller
MAVS50-containing oligomer only activates NF- B. Perhaps, integration of
MAVS50 into the MAVS70 larger oligomers shifts the signaling capacity of the

31

complex from activating both NF- B and IRF to that activating only NF- B. This
possibility remains to be formally tested in the future.

Compared to MAVS70, MAVS50 preserves all three TRAF-binding motifs, i.e.,
TRAF2- and TRAF6-binding motifs. Indeed, MAVS50 demonstrated preferential
interaction with TRAF2 and TRAF6, while MAVS70 interacted with five TRAFs
except TRAF4. This result agrees with the notion that the N-terminally exposed
TRAF-binding motifs are better accessed and endow MAVS50 better interactions
with TRAF2 and TRAF6. Intriguingly, mutations within the putative TRAF2-
binding motif, but not those within the predicted TRAF6-binding motif, abolished
MAVS50 interaction with both TRAF2 and TRAF6. Similarly, the MAVS50 M2,6
mutant was impaired to interact with TRAF3 and TRAF5. These results highlight
the critical role of the very N-terminally exposed TRAF2-binding motif in
interacting with more than one TRAF molecule, implying the degeneracy of the
TRAF2-binding motifs in recruiting TRAF molecules and relaying signal
transduction. It is important to note that, compared to MAVS50, these MAVS50
mutants were expressed well and interacted with MAVS70, suggesting that the
defect in TRAF-binding is not due to overall protein misfolding. Alternatively, it is
possible that TRAF2 recruited by the terminally positioned TRAF2-binding motif
facilitates the interaction of MAVS50 with TRAF6 and other TRAFs [28, 92]. This
is supported by the observation that MAVS recruits multiple TRAFs with non-

32

redundant roles in innate immune signaling [34] and TRAF molecules are prone
to oligomerize [97]. The space between these two TRAF-binding motifs may not
permit two TRAF molecules to dock on the N-terminus of MAVS50, but multiple
TRAFs decorating on oligomerized MAVS50 are possible [90, 91]. Nevertheless,
by positioning the TRAF2-binding motif at the very N-terminus, MAVS50
effectively competes with MAVS70 for recruiting these key signaling molecules,
resulting in attenuation of the MAVS70-mediated signal transduction.

The MAVS50 variant is generated from internal translation initiation, such that
two TRAF2-binding motifs are exposed at the very N-terminus to facilitate protein
interaction. Such a delicate mechanism, crafted by millions of years of evolution,
symbolizes the importance of the tight regulation of MAVS-dependent innate
immune signaling in response to viral infection. Notable regulatory action alike is
exemplified by alternatively spliced variant of RIG-I [22] and TRIF [98], and a
LGP2 allele likely due to gene duplication [12, 99]. While alternative splicing has
been extensively studied, the contribution of internal translation initiation in
regulating fundamental biological processes such as immune response is not
well understood. On the other hand, internal translation initiation is one of the
mechanisms that viruses frequently deploy to maximize the coding capacity of
their limited genomes [100, 101] and that critical cellular genes avoid shutdown
under stressed conditions [102, 103]. It is probable that internal translation

33

initiation of MAVS50 is activated to enhance its expression in virus-infected cells
that the cap-dependent translation is suppressed. Expression of MAVS50 will
alleviate and prevent an overacting antiviral immune response, thereby
promoting cell survival. The roles of MAVS50 under this stressed condition
remain to be determined.

34

Figure 1

35

Figure 1. Identification of the MAVS50 variant.
(A) 293T cells were infected with SeV (100 HA unit/ml) for 2 hours or transfected
with a plasmid containing murine HV68 vGAT for 24 hours. Whole cell lysates
(WCL) were prepared in Triton X-100 buffer to separate into soluble and
insoluble (pellet) fractions, which were analyzed by immunoblotting with indicated
antibodies. (B) Diagram of the mRNA of the full-length MAVS (or MAVS70) and
MAVS50. (B) WCLs of indicated amount were analyzed with antibodies against
the first 135 amino acids ( MAVS
1-135
) or an internal sequence encompassing
amino acids 150-250 ( MAVS
150-250
). (C) The expression of MAVS, carrying a N-
terminal Flag tag and a C-terminal HA tag, in 293T cells was analyzed by
immunoblotting with anti-Flag and anti-HA antibodies, along with endogenous
MAVS (left panel). (D) 293T cells were transfected with plasmids containing wild-
type MAVS and indicated mutants. WCLs were analyzed by immunoblotting with
anti-MAVS antibody ( MAVS
150-250
). , deletion. (E) WCLs of indicated cells were
analyzed by immunoblotting with anti-MAVS ( MAVS
150-250
) and anti- -actin.
Note, antibody against human MAVS does not react with murine MAVS in NIH
3T3 cells. (F) WCLs of indicated cell lines were analyzed by immunoblot with
antibodies against MAVS and -actin. (G and H) 293T cells were infected with
Sendai virus (SeV, 100 HA Unit/ml) (G) and HSV-1 (MOI=5) (H) and cells were
harvested at indicated time points. WCLs were analyzed by immunoblot with
antibody against MAVS.

36

Figure 2

37

Figure 2. Characterize the roles of MAVS50 in RIG-I-dependent signaling.
(A and B) 293T cells were transfected with an NF- B (A) or IFN-  (B) reporter
cocktail and increasing amount of MAVS wild-type (WT), MAVS70 or MAVS50.
Reporter activation was determined by luciferase assay at 30 hours post-
transfection. (C) 293T cells were transfected with plasmids containing indicated
genes. At 48 hours post-transfection, IKK  kinase was precipitated and analyzed
by in vitro kinase assay and immunoblotting with anti-IKK . Whole cell lysates
were analyzed with anti-Flag (MAVS) and anti-GST (RIG-I-N). (D) 293T cells
were transfected with plasmids containing Flag-MAVS70 and Flag-MAVS50.
MAVS70 and MAVS50 were purified by affinity chromatography, eluted and
analyzed by gel filtration chromatography with Superdex 200. Fractions (30 l)
were analyzed by immunoblotting with anti-Flag antibody. V
0
, void volume;
numbers at the top indicate molecular weight in kDa. (E and F) MAVS in 293T
cells was depleted with shRNA and analyzed by immunoblotting (E) and MAVS
expression was “reconstituted” with lentivirus containing MAVS wild-type (WT),
MAVS70 or MAVS50. Whole cell lysates were analyzed with anti-V5 antibody
(F). (G) MAVS knockdown 293T cells “reconstituted” with control lentivirus (Vec)
or lentivirus containing MAVS wild-type (WT), MAVS70 (70) or MAVS50 (50) as
shown in (F), were mock- or infected with Sendai virus (SeV, 100 HAU/ml) for 2
hours, WCLs were prepared and analyzed by immunoblot with indicated
antibodies. (H) Infection of “reconstituted” 293T cells as described in (G).
Mitochondrion-enriched fraction was obtained and analyzed by immunoblot with

38

indicated antibodies. (I and J) MAVS knockdown 293T cells, “reconstituted” with
MAVS expression as described in (F), were infected with SeV (100 HA unit/ml)
for 8 hours, RNA was extracted, cDNA was prepared and real-time PCR with
primers specific for hIFNb and CCL5 were performed (I). Supernatants were
collected and hIFN  and hCCL5 were determined by ELISA (J).

39

Figure 3

40

Figure 3. Innate immune signaling of MAVS50.
(A and B) 293T cells were transfected with plasmids containing V5-tagged
MAVS70 (A) or MAVS50 (B) and those containing Flag-tagged TRAFs. Whole
cell lysates (WCLs) were precipitated with anti-Flag M2 agarose. Precipitated
proteins and WCLs were analyzed by immunoblotting with indicated antibodies.
(C) 293T cells were transfected with plasmids containing indicated genes. WCLs
prepared in kinase lysis buffer were precipitated with anti-TBK-1 antibody and
TBK-1 was subjected to in vitro kinase assay with GST-IRF3C as substrate.
GST-IRF3C was analyzed by PhosphoImager and TBK-1 by immunoblotting.
WCLs were analyzed by immunoblotting with antibodies against MAVS and GST
(RIG-I-N). (D) Transfection was carried out as in (C) and WCLs were analyzed by
native gel electrophoresis for IRF3 dimerization. WCLs were analyzed by
immunoblotting with antibodies against β-actin, MAVS and GST (RIG-I-N).

41

Figure 4

42

Figure 4. MAVS50 expression and its effect on innate immune signaling in
293T cells.
(A) MAVS knockdown 293T cells, “reconstituted” with control lentivirus (Vec) or
lentivirus carrying wild-type MAVS (WT), MAVS70 (70) or MAVS50 (50), were
infected with Sendai virus (100 HA unit/ml) for 8 hours. Total RNA was extracted,
cDNA was prepared and analyzed by real-time PCR with primers specific for
indicated genes. (B and C) MAVS knockdown 293T cells, “reconstituted” with
various MAVS expression as described in (A), were incubated with mitotracker
(B), fixed and stained with corresponding primary and secondary antibodies. For
peroxisome staining, antibody against the 70 kDa peroxisome membrane protein
(PMP70) was used (C). Cells were analyzed with a Nikon E800M microscope
equipped with CCD camera. (D) 293 T-Rex/MAVS50-Flag cells were established
with hygromycin selection (100 μg/ml) and induced with doxycycline at indicated
concentration for 48 hours. Whole cell lysates were analyzed by immunoblotting
with anti-Flag (MAVS50) and anti-β-actin antibodies.

43

Figure 5

44

Figure 5. MAVS50 interacts with MAVS70.
(A) 293T cells were transfected with plasmids containing MAVS wild-type,
MAVS70 or MAVS50. Whole cell lysates (WCLs) were precipitated with anti-Flag
(MAVS50). Precipitated proteins and WCLs were analyzed by immunoblotting
with indicated antibodies. (B) 293T-Rex/MAVS50-Flag cell line was induced with
doxycycline (100 ng/ml) for 24 hours. WCLs were precipitated with anti-Flag
agarose (MAVS50). Precipitated proteins and WCLs were analyzed by
immunoblotting with indicated antibodies. (C) 293T cells were transfected with a
plasmid containing MAVS50-Flag, with or without a plasmid containing MAVS70-
V5. MAVS50 was purified by affinity chromatography, eluted and analyzed by gel
filtration chromatography. Fractions (30 μl) were analyzed by immunoblotting with
anti-Flag and anti-V5 antibodies. (D) Whole cell lysates of 293T, HeLa and THP-
1 macrophage were analyzed by gel filtration chromatography. Fractions (50 μl)
were analyzed by immunoblotting with anti-MAVS antibody. For C and D, V
0
, void
volume; numbers indicate molecule weight in kDa.

45

Figure 6

Figure 6. Co-elution of MAVS70 and MAVS50 in 293T cells infected with
virus.
293T cells were mock-infected, or infected with Sendai virus (SeV, 100 HAU/ml)
(middle three panels) and murine gamma herpesvirus 68 (γHV68, MOI = 1)
(bottom panel) for indicated time. WCLs in Triton x-100-containing buffer were
analyzed by gel filtration with Superose 6 column and fractions were analyzed by
immunoblotting with anti-MAVS antibody.

46

Figure 7

47

Figure 7. MAVS50 inhibits MAVS70-dependent IFN induction.
(A and B) 293T cells were transfected with an IFN-β (A) or NF-κB reporter
cocktail (B), a plasmid containing MAVS70 and increasing amount of a plasmid
containing MAVS50. The promoter activity of IFN-β and NF-κB was determined
by luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (C and
D) 293T cells were infected with control (CTL) lentivirus or lentivirus containing
MAVS50. Whole cell lysates were analyzed with indicated antibodies (C). Stable
293T cells were infected with SeV (100 HA unit/ml) for 8 hours and RNA was
extracted. cDNA was prepared and analyzed by real-time PCR with primers
specific for IFNβ and ISG56 (D). (E and F) 293T cells were transfected with
vector or plasmids containing MAVS wild-type (WT), MAVS70 or MAVS50. At 24
hours post-transfection, cells were infected with VSV-GFP (MOI = 0.01). Cells
were photographed at 24 hours post-infection (E) and VSV in the supernatant
was determined by plaque assay (F).

48

Figure 8

49

Figure 8. MAVS50 targets TRAF molecules to inhibit MAVS70-mediated IFN
induction.
(A) Diagram of key signaling molecules of the RIG-I-dependent IFN induction. (B
and C) 293T cells were transfected with an IFN-β reporter cocktail, a plasmid
containing RIG-I-N (B) or TBK-1 (C), and increasing amount of plasmid
containing MAVS50. The IFN-β promoter activity was determined by luciferase
assay at 30 hours post-transfection.**p<0.01; ***p<0.005. (D) 293T cells were
transfected with plasmids containing MAVS wild-type (WT) or MAVS50 and a
plasmid containing TRAF6. Whole cell lysates (WCL) were precipitated with anti-
Flag (TRAF6). Precipitated proteins and WCLs were analyzed by immunoblotting
with indicated antibodies. (E) 293T cells, depleted with endogenous MAVS and
“reconstituted” with MAVS70 or MAVS50, were precipitated with anti-V5
(MAVS70 or MAVS50). Precipitated proteins and WCLs were analyzed by
immunoblotting with indicated antibodies. (F) MAVS knockdown cells
“reconstituted” with MAVS70-V5 were transfected with increasing amount of
MAVS50-Flag. At 30 hours post-transfection, cells were infected with Sendai
virus (SeV, 200 HA unit/ml) for two hours. WCLs were prepared and precipitated
with anti-V5 (MAVS70) antibody. Precipitated proteins and WCLs were analyzed
by immunoblotting with indicated antibodies. V, vector.

50

Figure 9

51

Figure 9. MAVS50 modulates the IRF-IFN signaling cascade.
(A and B) 293T cells were transfected with an IFN-β reporter cocktail, plasmids
containing IKKε (A) or IRF3-5D (B) and increasing amount of a plasmid
containing MAVS50. At 30 hours post-transfection, whole cell lysates (WCLs)
were analyzed by luciferase assay to determine the activation of the IFN-β
promoter. (C) 293T cells were transfected with plasmids containing indicated
genes. WCLs were precipitated with anti-V5 agarose (MAVS). Precipitated
proteins and WCLs were analyzed by immunoblotting with indicated antibodies.

52

Figure 10

53

Fig 10. The N-terminal TRAF2-binding motif is critical for MAVS50 to inhibit
IFN induction.
(A) Diagram of the TRAF2-binding motif (T2BM) and TRAF6-binding motif
(T6BM) in MAVS50, in relation to MAVS70. (B and C) 293T cells were
transfected with plasmids containing Flag-TRAF2 (B) or Flag-TRAF6 (C) and
plasmids containing MAVS50 wild-type (WT), mutant of TRAF2-binding (M2) or
TRAF6-binding (M6) or both TRAF2- and TRAF6-binding (M2,6). Whole cell
lysates (WCLs) were prepared at 30 hours post-transfection and precipitated with
anti-Flag agarose or anti-HA agarose (as negative control). Precipitated proteins
and WCLs were analyzed by immunoblotting with indicated antibodies. (D) 293T
cells were transfected with an IFN-β reporter cocktail, a plasmid containing
MAVS70 and increasing amount of a plasmid containing MAVS50 wild-type (WT)
or MAVS50 M2,6 mutant. The IFN-β promoter activity was determined by
luciferase assay at 30 hours post-transfection. **p<0.01; ***p<0.005. (E) 293T
cells were infected with control lentivirus (CTL) or lentivirus containing MAVS50
wild-type or MAVS50 M2,6 mutant. At 48 hours, cells were infected with SeV
(100 HA unit/ml) for 8 hours. RNA was extracted and cDNA were prepared for
real-time PCR analysis with primers specific for IFNβ and ISG56. (F and G) 293T
cells were transfected with an empty plasmid (Vector) or a plasmid containing
MAVS50 or MAVS50 M2,6 mutant. At 24 hours post-transfection, cells were
infected with VSV-GFP (MOI = 0.01). Cells were photographed at 24 hours post-
infection (F) and virus in the supernatant was determined by plaque assay (G).

54

Figure 11

55

Figure 11. Characterization of TRAF2- and TRAF6-binding motifs of
MAVS50.
(A) Diagram of MAVS50 carrying N-terminally positioned TRAF-2 and TRAF6-
binding motifs in relation to MAVS70. The key residues of the TRAF2- and
TRAF6-binding motifs were mutated into alanines as indicated. (B and C) 293T
cells were transfected with indicated plasmids. Whole cell lysates (WCLs) were
precipitated with anti-Flag [TRAF3 (B) or TRAF5 (C)]. Precipitated proteins and
WCLs were analyzed by immunoblotting with indicated antibodies. (D) 293T cells
were transfected with plasmids containing indicated genes. Immunoprecipitation
and immunoblotting were carried out as in (B).

56

Figure 12

Figure 12. A hypothetical model on MAVS50 in modulating MAVS70-
dependent signaling.
Upon receiving upstream activation signal, MAVS70 forms large oligomer that
triggers both NF-κB activation and IRF activation and IFN induction. MAVS50
interacts with MAVS70 and integrates into the MAVS70 oligomers. MAVS50
recruits significant portion of TRAF6 and TRAF2 with its N-terminally exposed
TRAF-binding motifs, resulting in the inhibition of MAVS70-dependent NF-κB and
IRF activation. The oligomer containing MAVS70 and MAVS50 activates only
NF-κB, but not IRF-IFN branch. As such, MAVS50 selectively inhibits IFN
induction.

57

3. HSV-1 Gene Expression is Inhibited by Two Novel Truncated
Variants of MAVS
3.1. Introduction
Herpes Simplex virus 1 (HSV-1) is a ubiquitous human pathogen consisting of a
double-stranded DNA genome surrounded by an icosahedral capsid and an
envelope which is associated with tegument proteins. HSV-1 belongs to the
subfamily of Alphaherpesvirinae which is carried by about 80% of the world
population [47] . The virus is capable of causing a broad range of pathologies
ranging from the benign vesicular eruptions around the mouth, called cold sores,
to much more severe pathologies, including blindness and fatal encephalitis [56,
58, 59]. A hallmark of HSV-1 infection is its ability to establish a lifelong latent
infection in neurons and trigger reactivation and lytic infection when the neurons
are stressed. HSV-1 mainly enters the host through mucosal epithelial cells and
causes a lytic infection which is characterized by the expression of viral genes,
including immediate early (IE), early (E), and late (L) genes, which leads to the
production of infectious virions. After the primary infection, HSV-1 spreads to
neurons to establish a latent infection, during which only the Latency Associated
Transcripts (LATs) are produced [48-50].

The HSV-1 replication cycle begins with viral attachment to the host cell and
penetration of the viral nucleocapsid into the cytoplasm. Once released into the
cytoplasm, the capsids are transported to the nucleus, where they dock at the

58

nuclear pores and release viral DNA into the nucleus. Viral entry into the nucleus
is followed by transcription of its genes, DNA replication, and encapsidation of
the replicated DNA. Nucleocapsids then leave the nucleus by budding at the
inner nuclear membrane (INM) into the perinuclear space, which is known as
primary envelopment, followed by the fusion of these primary virions with the
outer nuclear membrane (ONM) resulting in the translocation of nucleocapsids
into the cytoplasm, where they are coated with tegument proteins. Final
maturation of the cytoplasmic capsids occurs during secondary envelopment by
budding into the trans-Golgi network (TGN)-derived vesicles. After transport to
the cell surface, the mature, enveloped virion is ultimately released from the
infected cell [71].

Host innate immunity is the first line of defense against viral infections, including
HSV-1. Viral components, such as nucleic acids, can be sensed by pattern
recognition receptors (PRRs), which ultimately lead to the induction of type 1
interferons (IFNs) and expression of antiviral proteins [2]. Detection of HSV-1
involves a number of PRRs including Toll-like receptor 2, which senses viral
envelope glycoproteins [104]; TLR3, which senses dsRNA generated during
HSV-1 infection [105, 106] [107, 108]; TLR9 [109, 110]; cytosolic RNA receptors
RIG-I [111, 112] and MDA-5 [113], which are triggered by the RNAs generated
during HSV replication [105, 106]; intracellular DNA sensors such as IFI16 [114-
116] and cGAS [117, 118]. However, HSV-1 has developed multiple strategies to

59

evade the host innate immune responses. The virus can target different steps in
the signaling events that lead to the production of IFN-β and therefore facilitate
its infection [119-121], e.g. ICP0, an immediate protein of HSV-1, inhibits the
activation of IRF3 [115, 122-126] and NF-ĸB by its RING finger domain [127,
128]. Us3, a viral protein kinase, blocks the expression of type I IFNs and IFN-
dependent genes by (1) reducing TRAF6 ubiquitination [129], (2)
hyperphosphorylation of IRF3 which inhibits its dimerization and nuclear
translocation [130], and (3) hyperphosphorylation of p65 which abrogates its
nuclear translocation [131]. US11, an RNA binding tegument protein interacts
with endogenous RIG-I and MDA5 in an RNA-independent manner and disrupts
the formation of RIG-I/MAVS and MDA5/MAVS complexes, resulting in reduced
production of IFN-β [132]. UL36, the largest tegument protein of HSV-1, blocks
IFN-β production by deubiquitinating TRAF3 and preventing the recruitment of
TBK-1 [133]. K63-linked polyubiquitination of TRAF3 is required for signaling by
MAVS and recruiting TBK-1 and IKKε kinase [134].

MAVS is an adaptor molecule downstream of RIG-I-like receptors (RLRs), which
relays signal from RIG-I and MDA-5 to downstream cytosolic IKK and TBK-1
kinase complexes and mediates the activation of NF ĸB and induction of IFNs in
response to viral infection [25-28]. We have recently reported a MAVS variant
with a molecular weight of ~50 kDa (MAVS50) that arises by internal translation
from a second initiation codon [AUG (142)]. MAVS50 exposes its TRAF binding

60

motifs and inhibits full-length MAVS-mediated IFN induction by competing with
full-length MAVS for binding to TRAF molecules. Notably, MAVS50 variant was
also reported by Brubaker S.W. et al [86]. MAVS50 provides an example of
protein diversity in eukaryotes that can be achieved by internal translation from a
single mRNA and highlights a delicate mechanism by which signaling events are
differentially regulated by exposing key protein-interacting domains [85].
Moreover, we recently identified that, in addition to full-length MAVS and
MAVS50, the MAVS transcript can produce two novel variants with molecular
weights of ~40 and 30 kDa (hereafter referred to as MAVS40 and MAVS30). We
discovered that MAVS40 and MAVS30 are produced by internal translation
and/or proteolytic cleavage and lack the N-terminal CARD domain. Consistent
with the lack of N-terminal CARD, MAVS40 and MAVS30 fail to stimulate IFN
induction, when “reconstituted” in cells depleted with full-length MAVS
expression. MAVS40 and MAVS30 interact with HSV-1 structural components
including UL19 capsid protein, UL31 and UL34 which are involved in primary
nuclear envelopment, and UL37 tegument protein. MAVS40 and MAVS30 have
antiviral properties against HSV-1 infection and are able to inhibit viral gene
expression with an unknown mechanism that remains to be examined in the near
future. The successful outcome of my studies will identify a new role of truncated
variants of MAVS, in restricting herpervirus replication.

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3.2. Results
3.2.1. Identification of MAVS40 and MAVS30
We have previously identified a MAVS variant with a molecular weight of ~50
kDa (MAVS50), that was produced from internal translation initiation [85]. In an
experiment that aims to examine the expression of MAVS50 produced by
constructs containing wild-type MAVS and MAVS70, we observed two smaller
isoforms of MAVS with molecular weights of ~40kDa and ~30 kDa (thus, referred
to as MAVS40 and MAVS30) that were produced from constructs containing
wild-type(WT) MAVS, MAVS70 and MAVS50 (Figure 13A). We then set out to
characterize the nature of MAVS40 and MAVS30. We hypothesized that
MAVS40 and MAVS30 are produced from internal translation and/or proteolytic
cleavage. To test our hypothesis, we used a WT MAVS construct that carried a
carboxyl-terminal V5 epitope and a WT MAVS construct with an amino-terminal
Flag epitope to probe MAVS expression. The amino-terminally-tagged MAVS
construct yielded a single MAVS species of 70 kDa reacting with anti-Flag
antibody, while the carboxyl-terminal-tagged MAVS construct yielded species of
~70 kDa, ~50 kDa, ~40 kDa, and ~30 kDa when probed with anti-V5 antibody.
The two species of MAVS40 and MAVS30 are of similar size to endogenous
MAVS (Figure 13B). This result indicates that MAVS40 and MAVS30 lack the
amino-terminal region. To examine whether MAVS40 and MAVS30 are produced
by internal translation or proteolytic cleavage, we deleted the first ATG initiation
codon of MAVS70 (full-length MAVS) and MAVS50. As shown in figure 13C,

62

deletion of the first initiation codon of the construct containing MAVS70 abolished
the expression of MAVS70 and MAVS40, while produced MAVS50 and MAVS30.
Similarly, deletion of the first start codon of MAVS50 construct (50∆ATG)
abolished the expression of MAVS50 and MAVS40, while yielded MAVS30.
These results suggest that MAVS40 in generated by proteolytic cleavage from
full-length MAVS, therefore when we delete the first ATG codon of MAVS70
construct, MAVS40 is no longer produced. Moreover, these data suggests that
MAVS30 is produced from internal translation using an initiation codon
downstream of Methionine 142. Visual inspection of MAVS mRNA revealed an
internal translation site at codon 303. To determine whether MAVS30 was
produced from this initiation codon, we mutated ATG (methionine) 303 into TGC
(cysteine) within the cDNA of MAVS50 (Figure 13D). Mutation of methionine 303
significantly reduced the expression of MAVS30; however, it did not completely
abolish its expression. This data suggested that MAVS30 is not solely produced
by internal translation. One possible explanation is that MAVS30 is generated by
both internal translation and internal cleavage. We then probed a number of
human cell lines for the expression of MAVS40 and MAVS30. We found that
MAVS40 and MAVS30 were abundantly expressed from BJAB B cells, HOK
keratinocytes, and HCT116 colorectal cells. Moreover, MAVS40 was highly
expressed in Jurkat T cells, HeLa cervical cells, LEC endothelial cells, and
MAVS30 was highly expressed in HEK293T cells (Figure 13E).

63

3.2.2. Mapping the initiation site of MAVS40 and MAVS30 variants
To probe the initiation site of MAVS40 and MAVS30, we used Edman
degradation sequencing method, which determines the amino acid sequence at
the amino-terminus of the protein. Based on the Edman sequencing data,
MAVS40 is produced from Arginine 230 and MAVS30 in produced from
Methionine 303 (Figure 14A). We then generated a construct that contains the
cDNA sequence encoding amino acids 230 to 540 of wilt-type MAVS and a
construct encoding amino acids 303 to 540 of wild-type MAVS. These expression
vectors yielded proteins that migrated identically as MAVS40 and MAVS30
generated by wild-type MAVS on SDS-PAGE (Figure 14B). Next, we analyzed
the sequence homology at the initiation site of MAVS40 (Figure 14C) and
MAVS30 (Figure 14D), between different species from fish to human. We noted
that Arginine 230 and the upstream amino-acid sequence FQPL (amino acids
227-230) are highly conserved between different species (Figure 14C). However,
the Methionine 303 was only conserved in monkey and human (Figure 14D). To
determine the cleavage site of MAVS40 and MAVS30, we generated a number of
deletion mutants and examined the expression of MAVS40 and MAVS30 in this
constructs. Deletion of amino acids 227-233 (FQPLARS) of wild-type MAVS
abolished the expression of MAVS 40 (Figure 15A, compare lane 2 and 7), and
deletion of amino acids 298-302 (VPTTL) together with mutation of ATG
(methionine) 303 into TGC (cycsteine) significantly reduced the expression of
MAVS30 (Figure 15B, compare lane 2 and 5). It is important to note that when

64

we only deleted amino acids 298-302 (VPTTL) and did not mutate methionine
303, it did not abolish the expression of MAVS30 (Figure 15B, lane 4). Moreover,
when we only mutated methionine 303 into cysteine (Figure 13D) it also did not
completely abolish the expression of MAVS30, supporting the idea that MAVS30
is generated by a combination of both internal translation and internal cleavage.
We then generated HEK 293T stable cell lines expressing wild-type MAVS,
MAVS70, MAVS WT and MAVS70 with the deletion of amino acids 227-233,
298-302, and mutation of methionine 303 to cysteine. As shown in Figure 14E,
such deletion constructs abolished the expression of MAVS40 and MAVS30.

3.2.3. MAVS40 and MAVS30 interact with MAVS70 and itself
We have previously shown that MAVS50, which lacks the amino-terminal CARD
domain, is able to interact with its full-length counterpart (MAVS70) [85].
Therefore, we were curious to determine whether MAVS40 and MAVS30 can
interact with MAVS70 and itself. Since MAVS70 and the smaller variants have a
large overlapping sequence, it is technically challenging to probe the interaction
between endogenous MAVS70 and MAVS40/MAVS30. Therefore, we used
MAVS knock down 293T cells “reconstituted” with lentivirus containing MAVS
wild-type which carries a carboxyl-terminal V5 epitope. We then expressed
exogenous Flag-tagged MAVS40 or MAVS30 by transient transfection and
examined the interaction between MAVS variants. Precipitation of MAVS40
effectively pulled down MAVS70 and MAVS40 (Figure 16A), indicating that

65

MAVS40 can interact with MAVS70 and MAVS40. Similarly, MAVS70 and
MAVS30 were detected in protein complexes precipitated with anit-Flag antibody
against MAVS30 (Figure 16B), indicating that MAVS30 can associate with
MAVS70 and MAVS30. Moreover, infection with Sendai virus (SeV) had no effect
on homo-dimerization of MAVS40 and MAVS30. However, it increased the
interaction between MAVS70 and MAVS40/MAVS30. We noted that SeV
infection induced a smear on MAVS70. The formation of this smear was
abolished when the samples were treated with phosphatase inhibitor (data not
shown). Collectively these results indicate that MAVS40 and MAVS30 physically
interact with MAVS70 and itself.

Oligomerization of MAVS is a hallmark of its activation [27, 32]. Therefore, we
examined whether MAVS40 and MAVS30 form oligomers by size exclusion
chromatography. Our previous data show that MAVS70, when purified from 293T
cell, forms large oligomers and was eluted in fractions corresponding to ~670
kDa. However, MAVS50 forms smaller oligomers corresponding to ~120 kDa,
which was expected since MAVS50 lacks the amino-terminal CARD domain.
Surprisingly, MAVS40 and MAVS30, when purified from 293T cells, formed large
oligomers and were eluted in fractions corresponding to ~670 kDa (Figure 16C).
This data suggests that although MAVS40 and MAVS30 lack the CARD domain,
they can form large oligomers when transfected in 293T cells. Moreover, when
MAVS40 and MAVS30 were co-expressed with either MAVS70 or MAVS50,

66

there was no apparent change in the elution pattern of MAVS40 and MAVS30
(Figure 16D). Finally, we examined the elution pattern of MAVS40 and MAVS30
in lysates of Hela cervical epithelial (Figure 17A) and 293T fibroblast (Figure
17B) cells, by size exclusion chromatography. Endogenous MAVS70, MAVS40,
and MAVS30 co-eluted in fractions corresponding to ~67-220 kDa in 293T and
Hela cells. However, there were differences in elution pattern of MAVS40 and
MAVS30. MAVS40 were more evenly distributed in fractions 20 and 22 in 293T
cells and fractions 18-24 in Hela cells, whereas MAVS30 was predominantly
eluted in fraction 22 in both cell lines. Furthermore, upon Herpes Simplex virus-1
(HSV-1) infection in Hela cells MAVS40 was mainly eluted in fractions 20 and 22
(Figure 17A) and upon Sendai virus (SeV) infection in 293T cells, MAVS40 was
eluted with a peak at fraction 22 (Figure 17B). There was no significant change in
the elution pattern of MAVS30 upon virus infection; however, MAVS30
expression is reduced upon both HSV-1 and SeV infection.

3.2.4. Characterization of the roles of MAVS40 and MAVS30 in RIG-I-
dependent signaling
MAVS is an adaptor protein downstream of the cytosolic receptor RIG-I and
MDA5, which relays signaling from these receptors to downstream kinases to
activate NF- ĸB and IRF transcription factors [25, 27, 28]. To determine whether
MAVS40 and MAVS30 have any function in these signaling cascades, we over-
expressed MAVS40 and MAVS30 and examined the activation of NF- ĸB and IRF

67

signaling pathways. Using reporter assay, we found that MAVS30, but not
MAVS40, marginally activated the promoter of IFN-β in a dose-dependent
manner (Figure 18A). MAVS40 and MAVS30 expression did not activate NF- ĸB
reporter (Figure 18B). “Reconstituted” expression of wild-type MAVS in cells that
endogenous MAVS isoforms were depleted by shRNA restored robust
expression of IFN- β, indicative of activation of IRF. However, “reconstituted’
expression of MAVS40 and MAVS30 failed to up-regulate the transcription of this
antiviral cytokine in response to SeV infection (Figure 18C). Thus, MAVS40 and
MAVS30 do not relay signal transduction from RIG-I to downstream molecules,
highlighting the critical role of CARD domain in RIG-I-mediated signaling. Our
previous data shows that MAVS50 inhibits MAVS70-mediated IFN signaling [85].
Therefore, we examined the effect of MAVS40 and MAVS30 on MAVS70-
mediated IFN signaling, by reporter assay. In contrast to MAVS50, MAVS40 and
MAVS30 did not significantly impact MAVS70-induced transcription of the IFN-β
promoter (Figure 18D). Taken together, these data indicated that MAVS40 and
MAVS30 did not have a significant role in innate immune activation and
modulation; however, these proteins may have other anti-viral functions
independent of innate immune activation.

3.2.5. MAVS40 and MAVS30 interact with HSV-1 structural components
To determine whether MAVS variants are involved in viral replication, we used
Herpes Simplex virus 1 (HSV-1) as a model DNA virus, to examine the roles of

68

MAVS variants during viral infection. HSV-1 is a double-stranded DNA virus
belonging to the subfamily of Alphaherpesvirinae that is carried by about 80% of
the world population [47]. To identify the HSV-1 protein(s) that associate with
MAVS50, MAVS40, and MAVS30, we purified MAVS variants from mock-infected
or HSV-1 infected 293T cells by affinity purification and analyzed proteins co-
purified with MAVS variants by mass spectrometry. The mass spectrometry
analysis identified UL19, UL37, UL47, UL34, and UL31 as the most abundant
MAVS-interacting HSV-1 proteins (Figure 19A and 19B). UL19 is a major capsid
protein which is essential for the construction of the icosahedral capsid that
encloses the genome [135]. UL37 tegument protein has been reported to be
required for active transport of HSV-1 nucleocapsids to the site of secondary
envelopment [136, 137]. UL47 is an RNA-binding protein [138] that has been
suggested to play a positive role in the regulation of viral replication [139, 140].
UL31 and UL34 have been reported to interact together, and this interaction is
required for viral egress from the nucleus during primary envelopment [141-143].
Based on the mass spectrometry data, the interaction between MAVS variants
and HSV-1 proteins was further analyzed using immunoprecipitation. Indeed, all
HSV-1 components including UL31, UL34 (Figure 19C), UL19, and UL37 (Figure
19D) co-precipitated with MAVS50, MAVS40, and MAVS30 from extracts of
transfected 293T cells. UL19, UL37, and UL31 had similar binding affinity to
MAVS wild-type, MAVS50, MAVS40, and MAVS30 (Figure 19E, 19G, and 19H,

69

respectively). UL34 demonstrated strong interaction with full-length MAVS, when
compared to its interaction with MAVS50, MAVS40, and MAVS30 (Figure 19F).

3.2.6. MAVS40 and MAVS30 inhibit HSV-1 gene expression
The interaction between MAVS40/MAVS30 and HSV-1 proteins provided a hint
that these MAVS variants have an essential function in viral infection. To study
the effect of MAVS40 and MAVS30 on HSV-1 infection, we first examined the
efficacy of HSV-1 proliferation by plaque assay. Compared with the titers in the
control infected group, “reconstituted” expression of MAVS40 and MAVS30 in
293T MAVS knocked down cells reduced the average intracellular (Figure 20A)
and extracellular (Figure 20B) virus titers at 12-36 hours post infection. These
results suggest that MAVS40 and MAVS30 have a potential antiviral activity
against HSV-1. To dissect the molecular mechanism of HSV-1 inhibition, we
analyzed multiple steps of the viral life cycle. Expression of MAVS40 and
MAVS30 had no effect on viral entry into the cells (Figure 20C). However, it
potently inhibited expression of HSV-1 immediate early (Figure 20D), early
(Figure 20E), and late genes (Figure 20F). These observations collectively
suggest that MAVS40 and MAVS30 target HSV-1 virion particles and block its
gene expression, which leads to the restriction of viral proliferation.

70

3.3. Discussion
Mitochondrial antiviral signaling protein (MAVS) is a critical adaptor in type I IFN
pathway which relays signal from cytosolic RNA sensors, RIG-I and MDA-5, to
downstream innate immune kinase complexes and promotes the production of
antiviral cytokines and interferons. We previously reported that MAVS50, a
truncated variant of MAVS translated from an internal initiation codon, exposes
two key TRAF-binding motifs within its N-terminus and efficiently competes with
full-length MAVS for binding to TRAF2 and TRAF6, thereby inhibiting the IRF-IFN
induced by full-length MAVS. Although internal translation is considered to be
more common in prokaryotes, MAVS50 provides an example of protein diversity
that can be achieved by internal translation from a eukaryotic mRNA. This is
consistent with recent studies showing that thousands of eukaryotic mRNAs are
predicted to produce protein isoforms due to the use of alternative translation
initiation sites [82-84]. We report here that MAVS40 and MAVS30 variants,
generated by internal translation and/or proteolytic cleavage, block initiation of
HSV-1 gene expression, unveiling a mechanism by which HSV-1 proliferation is
inhibited by two novel variants of the MAVS adaptor.

MAVS40 is generated via internal cleavage at amino acids 227-233, while
MAVS30 is generated by a combination of proteolytic cleavage at amino acids
298-302 and internal translation from the third initiation site [AUG (303)].
Therefore, both MAVS40 and MAVS30 lack the N-terminal sequence, including

71

the CARD domain. Despite missing the N-terminal CARD domain that mediates
self-oligomerization of MAVS and it’s hetero-dimerizaiton with RIG-I [32, 89],
MAVS40 and MAVS30 form large oligomers by gel filtration analysis. Consistent
with that, MAVS40 and MAVS30 can interact with full-length MAVS and can
homo-dimerize as shown by co-immunoprecipitation. This observation suggests
that an unknown sequence, in addition to CARD, may be responsible for MAVS
dimerization. Notably, our previous work showed that MAVS50 can also homo-
and hetero-dimerize with full-length MAVS. However, we failed to identify the
dimerization sequence for MAVS50, indicating that other factors such as the
structural sequence of MAVS may be responsible for its homo- and hetero-
dimerization [85]. Moreover, we note that MAVS40 fails to activate both NFĸB
activation and interferon (IFN) production, while MAVS30 is sufficient to
marginally trigger the production of IFN by reporter assay. On the other hand,
“reconstituted” expression of MAVS30 in cells that endogenous MAVS isoforms
were depleted by shRNA, failed to trigger the expression of IFN-β in response to
SeV infection. This result suggests that MAVS30 requires full-length MAVS to
relay signal transduction from RIG-I to downstream IRF transcription factor and
highlights the critical role of the CARD domain in mediating activation of the RIG-
I-MAVS signaling pathway.

Herpes Simplex virus 1 (HSV-1) is a double-stranded DNA virus that is encased
in an icosahedral capsid, a layer of proteins known as the tegument, and a host-

72

derived envelope containing viral glycoproteins. HSV-1 can be sensed by
different pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs)
[104-110], RIG-I-like receptors (RLRs) [111-113], and intracellular DNA sensors
such as cGAS [117, 118], which ultimately induce the production of type I
interferon and expression of pro-inflammatory cytokines to counteract viral
infection [2]. On the other hand, HSV-1 has also evolved several strategies to
evade the host antiviral responses and facilitate its infection [119-121, 144];
however, it is still subject to regulation by cellular factors. E.g. the DNA sensor
IFI16, which is involved in the innate immune response against HSV-1 [114-116],
was recently shown to inhibit HSV-1 replication by repressing viral gene
expression independent of its roles in innate immune response [145]. Another
study showed that cellular transmembrane protein 140 (TMEM140) inhibits HSV-
1 proliferation by impeding viral nucleocapsid egress from the nucleus [146].
Here we demonstrate that “reconstituted” expression of MAVS40 and MAVS30 in
cells that endogenous MAVS was knocked down inhibits HSV-1 proliferation.
Biochemical analysis identified multiple HSV-1 proteins including major capsid
protein (UL19), tegument proteins (UL37 and UL47), and primary nuclear
envelopment proteins (UL31 and UL34) associate with MAVS40 and MAVS30.
These interactions were confirmed using co-immunoprecipitation assay. UL37
has also been implicated to increase the efficiency of capsid transportation to the
nucleus, although it is not essential for this process. UL47 is an RNA-binding
protein [138] that has been suggested to play a positive role in the regulation of

73

viral replication and pathogenicity [139, 140]. UL31 is a nuclear phosphoprotein
[147] that is recruited to the nuclear membrane through interaction with UL34
[148]. The interaction between UL31 and UL34 is required for viral egress from
the nucleus during primary envelopment [141-143]. Whether the interaction
between MAVS40/MAVS30 and these viral proteins plays a direct role in
restriction of HSV-1 proliferation remains unclear and needs to be further
investigated.

To define the mechanism by which MAVS40 and MAVS30 inhibit HSV-1
proliferation, we dissected different steps of the HSV-1 life cycle. Molecular
virology analysis indicated that MAVS40 and MAVS30 had no effect on the entry
of viral genome into the host cell. However, MAVS40 and MAVS30 significantly
inhibited transcription of viral genes. These results suggest two possible models
for inhibition of HSV-1 gene expression: (1) MAVS40 and MAVS30 block viral
trafficking into the nucleus, consequently inhibiting the initiation of viral gene
expression: Once HSV-1 enters the host cell it is transported to the nucleus,
where they dock at the nuclear pores and release the viral DNA into the nucleus.
Nuclear targeting of many viruses is mediated by microtubules and cytoplasmic
dynein [149]. Various HSV-1 structural proteins have been reported to interact
with the dynein motor and promote trafficking of viral particles to the nucleus. For
example, HSV-1 inner tegument proteins, such as UL36 and UL37, were
reported to promote transportation of capsids along microtubules. Efficient

74

movement of capsids along microtubules in vitro requires UL36 and UL37 [150-
152]. HSV-1 major capsid protein UL19 was reported to interact with the dynein
light-chain; however, this interaction was not confirmed inside infected cells
[153]. Moreover, HSV-1 UL34 was shown to interact with dynein intermediate
chain. It has been suggested that after entry into cells, UL34 interacts with dynein
motor and uses the microtubular network to transport viral particles to the nuclear
pore [154]. Considering the roles of these viral components in transport
machinery, one model is that MAVS40 and MAVS30 target HSV-1 virion particles
by interacting with UL19, UL34, and UL37. MAVS40 and MAVS30 serve as a
physical barrier and interfere with the function of these proteins in transporting
HSV-1 to the nucleus. (2) MAVS40 and MAVS30 directly inhibit transcription of
viral mRNA: Initiation of transcription of HSV-1 immediate early (IE) genes is
mediated by HSV-1 VP16 (also referred to as UL48) and depends on
dissociation of VP16 from capsid during viral entry and subsequent formation of
VP16-induced transcriptional regulatory complex in the nucleus, with two cellular
proteins Oct-1 and HCF-1 [155-160]. It is possible that MAVS40 and MAVS30
associate with VP-16 and either directly or indirectly interfere with the function of
VP-16 in initiation of viral gene expression. These two hypotheses remain to be
formally tested in the near future.

In conclusion, we show that the MAVS transcript generates two novel truncated
variants, MAVS40 and MAVS30, which are generated from internal translation

75

initiation and/or proteolytic cleavage. MAVS40 and MAVS30 demonstrate
antiviral activity against HSV-1 and potently inhibit viral gene expression. Further
investigation will elucidate the mechanism by which MAVS40 and MAVS30
restrict HSV-1 replication and will highlight an important role of truncated
proteins, generated by alternative translation and internal cleavage, against
herpesviral infections.

76

Figure 13

77

Figure 13. Identification of MAVS40 and MAVS30 variants.
(A) 293T cells were transfected with empty vector or plasmids containing wilt-
type MAVS, MAVS70 and MAVS50. WCLs were analyzed by immunoblotting
with anti-V5 antibody. (B) The expression of MAVS, carrying a C-terminal V5 tag
(left panel) and a N-terminal Flag tag (right panel), were analyzed in 293T cells
by immunoblotting with anti-V5 and anti-Flag antibodies, along with MAVS
antibody (right panel). (C and D) 293T cells were transfected with empty vector
or plasmids containing MAVS WT, MAVS70, MAVS50 and indicated mutants.
WCLs were analyzed by immunoblotting with anti-V5 antibody. ∆, deletion. (E)
WCLs of indicated cells were analyzed by immunoblotting with anti-MAVS
(αMAVS
377-513
) and anti-β-actin.

78

Figure 14

79

Figure 14. Mapping the initiation site of MAVS40 and MAVS30 variants.
(A) MAVS40 and MAVS30 were purified from transfected 293T cells and
analyzed by edman degradation for N-terminal amino acid sequence. (B) 293T
cells were transfected with empty vector or plasmids containing MAVS WT,
MAVS40 and MAVS30. WCLs were analyzed by immunoblotting with anti-V5
antibody. (C and D) Diagram of the mRNA of MAVS40 (C) or MAVS30 (D)
(Upper panel), sequence homology of MAVS40(C) or MAVS30(D) between
indicated species. The red arrow indicates the start site of MAVS40 and
MAVS30. (E) 293T cells were infected with control lentivirus (Vec) or lentivirus
containing MAVSWT, MAVS70 and indicated mutants. Whole cell lysates were
analyzed with anti-V5 antibody. ∆, deletion.

80

Figure 15

Figure 15. Mapping the cleavage site of MAVS40 and MAVS30 variants.
(A and B) 293T cells were transfected with empty vector or plasmids containing
full-length MAVS and indicated mutants. WCLs were analyzed by immunoblotting
with anti-V5 antibody. The sequence highlighted in red indicates the amino acids
deleted from full-length MAVS construct. ∆, deletion.

81

Figure 16.

82

Figure 16. MAVS40 and MAVS30 interact with MAVS70 and itself.
(A and B) MAVS knockdown 293T cells “reconstituted” with lentivirus containing
MAVS wild-type (WT) were transfected with empty vector or plasmid containing
Flag.MAVS40 (A) and Flag.MAVS30 (B), followed by mock or Sendai virus
infection (SeV, 100 HAU/ml) for 30 hours. Whole cell lysates (WCLs) were
precipitated with anti-Flag. Precipitated proteins and WCLs were analyzed by
immunoblotting with indicated antibodies. (C) 293T cells were transfected with a
plasmid containing MAVS70.Flag, MAVS50.Flag or MAVS40.Flag and
MAVS30.Flag. MAVS proteins were purified by affinity chromatography, eluted
and analyzed by gel filtration chromatography. Fractions (40μL) were analyzed
by immunoblotting with anti-Flag antibody. (D) 293T cells were transfected with
plasmids containing Flag.MAVS30 and Flag.MAVS40, with or without a plasmid
containing MAVS70.V5 or MAVS50.V5. MAVS40 and MAVS30 were purified and
analyzed by gel filtration chromatography. Fractions (40μL) were analyzed by
immunoblotting with anti-Flag antibody. For C and D, V0, void volume; numbers
indicate molecular weight in kDa.

83

Figure 17

Figure 17. Co-elution of MAVS variants upon infection with virus.
(A) Hela cells were mock-infected, or infected with Herpes Simplex Virus-1 (HSV-
1, MOI=2) for indicated time. Whole cell lysates in Triton x-100-containing buffer
were analyzed by gel filtration. Fractions were analyzed by immunoblotting with
anti-MAVS antibody. (B) 293T cells were mock-infected, or infected with Sendai
virus (SeV, 100 HAU/ml) for indicated times. WCLs were analyzed by gel
filtration and fractions were analyzed by immunoblotting with anti-MAVS
antibody.

84

Figure 18

85

Figure 18. Characterization of the roles of MAVS40 and MAVS30 in RIG-I-
MAVS signaling.
(A and B) 293T cells were transfected with an IFN-  (A) or NF- B (B) reporter
cocktail and increasing amount of MAVS wild-type (WT), MAVS40 (40) or
MAVS30 (30). Reporter activation was determined by luciferase assay at 30
hours post-transfection. (C) MAVS knockdown 293T cells “reconstituted” with
control lentivirus (Vec) or lentivirus containing MAVS wild-type (WT), MAVS40
(40), or MAVS30 (30) were mock- or infected with Sendai virus (SeV, 100
HAU/ml) for 8 hours, RNA was extracted, cDNA was prepared and real-time PCR
with primer specific for IFNβ were performed (D) 293T cells were transfected with
an IFN-β reporter cocktail , a plasmid containing MAVS70 and increasing amount
of a plasmid containing MAVS50, MAVS40 or MAVS30. The promoter activity of
IFN-β was determined by luciferase assay at 30 hours post-transfection.

86

Figure 19

87

Figure 19. MAVS40 and MAVS30 interact with HSV-1 capsid protein,
primary nuclear envelopment and tegument proteins.
(A) 293T cells were transfected with plasmids containing MAVS50.Flag,
Flag.MAVS40 and Flag.MAVS30, followed by Herpes Simplex virus-1 infection
(HSV-1, MOI=1). MAVS variants and its interacting proteins were purified from
transfected 293T cells, analyzed by SDS-PAGE, and identified by mass
spectrometry. (B) Top candidates for MAVS50, MAVS40, and MAVS30 binding
partners were identified by mass spectrometry. (C and D) 293T cells were
transfected with plasmids containing UL31.V5, UL34.V5 (C) or UL19.V5,
UL39.V5 (D) and plasmids containing MAVS50.Flag, Flag.MAVS40 and
Flag.MAVS30. Whole cell lysates (WCLs) were prepared at 30 hours post-
transfection and precipitated with anti-Flag agarose, Precipitated proteins and
WCLs were analyzed by immunoblotting with indicated antibodies. (E, F, G and
H) 293T cells were transfected with plasmid containing UL31.V5 (E), UL34.V5
(F), UL19.V5 (G), UL37.V5 (H) and plasmids containing MAVS wild-type (WT),
MAVS50, MAVS40 or MAVS30. Whole cell lysates (WCLs) were prepared at 30
hours post-transfection and precipitated with anti-Flag agarose, Precipitated
proteins and WCLs were analyzed by immunoblotting with indicated antibodies.

88

Figure 20

89

Figure 20. MAVS40 and MAVS30 inhibit HSV-1 gene expression.
(A and B) MAVS knockdown 293T cells “reconstituted” with control lentivirus
(Vec) or lentivirus containing MAVS wild-type (WT), MAVS40, or MAVS30 were
mock- or infected with Herpes Simplex virus 1 (HSV-1, MOI=0.05) for indicated
times, HSV-1 in the cells (A) and in the supernatant (B) was determined by
plaque assay. (C) MAVS knockdown 293T cells “reconstituted” with MAVS
expression as described in (A), were infected with Herpes Simplex virus 1 (HSV-
1, MOI=10) for 1.5 hours, DNA was extracted and real-time PCR with a primer
specific for HSV-1 gene was performed. (D, E, and F) “Reconstituted” cells as
described in (A) were infected with Herpes Simplex virus 1 (HSV-1, MOI=0.05)
for 12 hours, RNA was extracted, cDNA was prepared and real-time PCR with
primers specific for UL54 (D), UL30 and UL29 (E), and gC and UL27 (F) were
performed.

90

4. Materials and Methods
4.1. Plasmids
Unless otherwise specified, all genes were cloned into pcDNA5/FRT/TO
(Invitrogen) for transient expression, and pCDH-EF-puro-CMV-MCS (System
Bioscience) for lentiviral expression. All cloned cDNAs were confirmed by DNA
sequencing.

4.2. Cell Lines and Viruses
HEK 293T, HEK 293T-Rex, BHK21, THP-1, HeLa, Vero cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL).
Human Jurkat T lymphoid cells were maintained in RPMI 1640 supplemented
with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100
μg/mL). All cells were cultured at 37°C in an atmosphere of 5% CO
2
. VSV-GFP
virus was amplified in BHK-21 cells. HSV-1-GFP virus was amplified in Vero
cells. Sendai virus was purchased from Charles River Laboratories.

4.3. Antibodies
The following antibodies were used in this study: Anti-human MAVS
1-135
(Santa
Cruz Biotechnology), anti-human MAVS
150-250
(Abcam), anti-human MAVS
150-200
(Bethyl group), anti-β-actin (Abcam), anti-Flag (Sigma), anti-V5 (Bethyl Group),
anti-HA (Covance). Antibodies against IKKβ and IKK  were kindly provided by

91

Dr. Ebrahim Zandi (University of Southern California). The antibody against
MAVS40 and MAVS30 was generated by immunizing rabbits with the 6×His
recombinant protein containing amino acids 377-513 of MAVS produced in E.
coli. The antibody was affinity purified using the same protein as antigen

4.4. Luciferase Reporter Assay
Luciferase reporter assays were performed as previously described [161]. Briefly,
HEK293T cells (1 x 10
5
cells/well) were seeded in 24-well plates 16 hours prior to
transfection. Cells were transfected with NF-ĸB or IFN-β reporter plasmid cocktail
(including 50 ng of NF-ĸB or IFN-β promoter luciferase reporter plasmid and 100-
ng of pGK-β-GAL plasmid) and an expression plasmid, by calcium phosphate
transfection method. At 30 hours post-transfection, cell lysates were used to
measure the firefly luciferase activity and β-galactosidase activity.

4.5. Immunoprecipitation and Immunoblotting
Immunoprecipitation and immunoblotting were carried out as previously
described [162-164]. Briefly, cells were harvested, rinsed with ice-cold PBS, and
lysed with NP40 buffer (50 mM Tris-HCL [pH 7.4], 150 mM NaCl, 5 mM EDTA,
1% NP40) supplemented with protease inhibitor cocktail. Centrifuged cell lysates
were then pre-cleared with Sepharose 4B beads, and subjected to precipitation
with antibody-conjugated agarose (Sigma) at 4°C for 4-6 hours. Precipitated

92

proteins were extensively washed with NP40 buffer and eluted with 1x SDS-
PAGE loading buffer by boiling at 95 °C for 5-10 minutes.

For immunoblotting analysis, whole cell lysates (WCL) or precipitated proteins
were resolved by SDS-PAGE, and transferred to nitrocellulose membrane.
Immunoblotting analysis was performed with indicated primary antibodies and
proteins were visualized with IRDye800- or IRDye680-conjugated secondary
antibodies (Licor) using an Odyssey infrared imaging system (Licor).

4.6. Gel Filtration
Gel filtration was performed as previously described by Zandi et al [39]. Briefly,
WCL or purified proteins were applied to superpose 6 or superdex 200 column
(GE Bioscience) and subjected to gel filtration analysis with buffer B (1 mM
EDTA, 0.5 mM EGTA, 150 mM NaCl, 20 mM Tris-HCl [pH 7.6], 0.5% Triton X-
100, 20 mM NaF, 20 mM β-glycerolphosphate, 1 mM Na
3
VO
4
, 5 mM
benzamidine, 2.5 mM metabisulphite). Elutions were collected in 0.5 ml fractions
and were analyzed by immunoblotting.

4.7. Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR was performed as previously described [162, 163]. Briefly, total RNA
was extracted from HEK293T cells using TRIzol reagent (Invitrogen). To remove
genomic DNA, total RNA was digested with RNase-free DNase I (New England

93

Biolab). First-strand cDNA was synthesized from 1 μg total RNA, using reverse
transcriptase (Invitrogen). The abundance of cytokine mRNA was assessed by
qRT-PCR, using SYBR Green Master Mix (Applied Biosystems). Human β-Actin
was used as an internal control.

4.8. Enzyme-linked Immunosorbent Assay (ELISA)
Commercial ELISA kits used in this study include: Human IFNβ (Thermo
Scientific) and human CCL5 (R&D Systems). The supernatants from cultured
cells were collected at the indicated time points after Sendai Virus infection.
Cytokine levels in the supernatants were assessed according to manufacturer’s
instruction.

4.9. In Vitro Kinase Assay
HEK293T cells were transfected with the indicated plasmids. Cells were lysed
and anti-IKK  antibody was used to precipitate endogenous IKKα/β/  complexes.
The precipitated complexes were subjected to in vitro kinase assay as previously
described [164]. The kinase reaction mixture consisted of GST-IĸBαNT as the
substrate, [ -
32
P]ATP, and precipitated kinase complex in 20 μl kinase buffer (25
nM HEPES [pH 7.5], 50 mM KCL, 2 mM MgCl
2
, 2 mM MnCl
2
, 1 mM DTT, 10 mM
NaF, 20 mM β-glycerolphosphate, and 1 mM sodium orthovandate). The reaction
mixture was incubated at room temperature for 40 minutes. Phosphoryation of
IĸBα was analyzed by autoradiography.

94

4.10. Lentivirus-mediated Stable Cell Line Construction
Lentivirus production was performed as previously described [161, 162]. Briefly,
HEK293T cells were transfected with packaging plasmids (DR8.9 and VSV-G)
and the pCDH lentiviral expression plasmids or shRNA plasmids. At 72 hours
post-transfection, supernatant was harvested and, if necessary, concentrated by
ultracentrifugation. HEK293T cells were then infected with lentivirus in the
presence of polybrene (8 μg/ml). Cells were selected and maintained in complete
media.

4.11. Subcellular Fractionation
Cells were lysed and homogenized using hypotonic buffer solution (20 mM Tris-
HCl, pH 7.4, 10 mM NaCl, 3mM MgCl
2
). The homogenates were centrifuged at
500xg for 5 minutes. The supernatant (S1) was centrifuged at 5000xg for 10
minutes to precipitate crude mitochondria. The crude mitochondria fraction (P5)
was then lysed and analyzed by immunoblotting.

4.12. Mass Spectrometric Analysis
Samples were denatured, reduced with DTT, alkylated with Iodacetamide, and
digested with trypsin as described previously (Zhou et al., 2011). Samples were
analyzed using an LC/MS system consisting of an Eksigent NanoLC Ultra 2D

95

(Dublin, CA) and Thermo Fisher Scientific LTQ Orbitrap XL (San Jose, CA).
Briefly, peptides were separated in a 10 cm column (75 μm inner diameter)
packed in-house with 5 μm C18 beads on a Eksigent NanoLC Ultra 2D system
using a binary gradient of buffer A (0.1% formic acid) and buffer B (0.1% formic
acid and 80% ACN). The peptides were loaded directly without any trapping
column with buffer A at a flow rate of 300 nL/min. Elution was carried out at a
flow rate of 250 nL/min, with a linear gradient from 10% to 35% buffer B in 95 min
followed by 50% buffer B for 15 min. At the end of the gradient, the column was
washed with 90% buffer B and equilibrated with 5% buffer B for 10 min. The
eluted peptides were sprayed into the LTQ Orbitrap XL. The source was
operated at 2.1-2.25 kV, with no sheath gas flow, with the ion transfer tube at
250°C. MS spectra in the range of m/z 350–2000 were acquired in the orbitrap at
a FWHM resolution of 30,000 after accumulation to an AGC target value of
500,000 in the linear ion trap with 1 microscan.

For peptide sequencing and modification site localization, the same precursors
selected for fragmentation by CID, and fragment ions were analyzed in the liner
ion trap. The five most abundant precursor ions were selected for fragmentation
by CID. The instrument was operated in data-dependent acquisition mode,
whereby five CID data-dependent MS/MS scans succeeded the high resolution
MS scan. For all sequencing events, dynamic exclusion was enabled to minimize
repeated sequencing. Peaks selected for fragmentation more than once within 60

96

s were excluded from selection (10 ppm window). Proteome Discoverer 1.4
(Thermo Fisher Scientific) was used for protein identification using Sequest
algorithms. The following criteria were followed. For MS/MS spectra, variable
modifications were selected to include N,Q deamination, M oxidation and C
carbamidomethylation with a maximum of four modifications. Searches were
conducted against Uniprot or in-house customer database. Up to two missed
cleavages were allowed for protease digestion and peptide had to be fully tryptic.
MS1 tolerance was 10 ppm and MS2 tolerance was set at 0.8 Da. Peptides
reported via search engine were accepted only if they met the false discovery
rate of 1%. There is no fixed cutoff score threshold, but instead spectra are
accepted until the 1% FDR rate is reached. Only peptides with a minimum of six
amino acid lengths were considered for identification. We also validated the
identifications by manual inspection of the mass spectra.

4.13. Statistical Analysis
The statistical significance (P-value) was calculated using unpaired two
tailed.Student’s t test. A P-value of <0.05 was considered statistically significant.

97

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Abstract (if available)

Abstract Host innate immunity is the first line of defense against invading pathogens. Upon viral infection, the cytosolic retinoic-acid-inducible gene I (RIG-I), a major intracellular pathogen recognition reeptor, senses double-stranded RNA and dimerizes with the mitochondrial antiviral signaling protein (MAVS), which activates two innate immune kinase complexes to promote the production of antiviral cytokines and interferons. Previous studies on the MAVS adaptor have focused on the full-length MAVS of 72 kDa. However, a smaller isoform of MAVS, with a molecular weight of ~50 kDa (thus, referred to as MAVS50) is consistently detected in a number of cell lines and its function remains unknown. We and others reported that MAVS50 was produced via internal translation from an alternative translation start site [the second AUG (142)]. Unlike full-length MAVS, which activates both NF-ĸB and interferon (IFN) signaling cascades, MAVS50 specifically activates NF-ĸB, but not the IRF-IFN induction. Lacking the N-terminal sequence, including the CARD domain, MAVS50 exposes two key TRAF-binding motifs within its N-terminus to effectively compete for association with TRAF molecules, thereby inhibiting the IRF-IFN induction pathway. This study identifies an example of protein diversity in eukaryotes that can be reached by internal translation from a single mRNA and highlights a new means by which innate immune signaling events are differentially regulated via exposing key internally embedded interaction motifs. ❧ We recently identified that the MAVS transcript can generate two novel variants with molecular weights of ~40 and ~30 kDa (thus designated MAVS40 and MAVS30). Our studies demonstrate that MAVS40 is produced by internal cleavage within amino acids 227-233, and MAVS30 is generated by internal translation from the third initiation site [AUG (303)] and proteolytic cleavage within amino acids 298-302. MAVS40 and MAVS30 lack the N-terminal sequence, including the CARD domain. Consistent with the lack of N-terminal CARD, MAVS40 and MAVS30 fail to stimulate IFN induction, when “reconstituted” in cells depleted with MAVS expression. Interestingly, MAVS40 and MAVS30 demonstrate antiviral activity against Herpes Simplex virus 1 (HSV-1), a model DNA virus. Molecular virology analysis indicated that MAVS40 and MAVS30 potently inhibit viral gene expression. Moreover, the biochemical analysis identified multiple virion components such as major capsid protein (UL19), primary nuclear envelopment proteins (UL31 and UL34), and teguments protein (UL37) that were associated with MAVS40 and MAVS30. Our findings collectively support the model whereby MAVS40 and MAVS30 target herpesvirus virion particle to block the initiation of viral gene expression. The successful outcome of my study will likely identify a novel mechanism by which HSV-1 replication is restricted by truncated variants of MAVS, and will provide new insights into regulatory roles of truncated proteins that arise from internal translation and proteolytic cleavage.

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Asset Metadata

Creator Minassian, Arlet (author)

Core Title Characterization of three novel variants of the MAVS adaptor

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 09/27/2017

Defense Date 09/01/2016

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

Tag herpes simplex virus 1,HSV-1,IFN,innate immunity,interferon,MAVS,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zandi, Ebrahim (committee chair), Akbari, Omid (committee member), Feng, Pinghui (committee member), Liang, Chengyu (committee member), Yuan, Weiming (committee member)

Creator Email arlet.minassian@yahoo.com,arletmin@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-307789

Unique identifier UC11280397

Identifier etd-MinassianA-4831.pdf (filename),usctheses-c40-307789 (legacy record id)

Legacy Identifier etd-MinassianA-4831.pdf

Dmrecord 307789

Document Type Dissertation

Format application/pdf (imt)

Rights Minassian, Arlet

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA