Vpu mediates depletion of interferon regulatory factor 3 during HIV infection by a lysosome-dependent mechanism.

Abstract

HIV has evolved sophisticated mechanisms to avoid restriction by intracellular innate immune defenses that otherwise serve to control acute viral infection and virus dissemination. Innate defenses are triggered when pattern recognition receptor (PRR) proteins of the host cell engage pathogen-associated molecule patterns (PAMPs) present in viral products. Interferon regulatory factor 3 (IRF3) plays a central role in PRR signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Productive infection of T cells by HIV is dependent upon the targeted proteolysis of IRF3 that occurs through a virus-directed mechanism that results in suppression of innate immune defenses. However, the mechanisms by which HIV controls innate immune signaling and IRF3 function are not defined. Here, we examined the innate immune response induced by HIV strains identified through their differential control of PRR signaling. We identified viruses that, unlike typical circulating HIV strains, lack the ability to degrade IRF3. Our studies show that IRF3 regulation maps specifically to the HIV accessory protein Vpu. We define a molecular interaction between Vpu and IRF3 that redirects IRF3 to the endolysosome for proteolytic degradation, thus allowing HIV to avoid the innate antiviral immune response. Our studies reveal that Vpu is an important IRF3 regulator that supports acute HIV infection through innate immune suppression. These observations define the Vpu-IRF3 interface as a novel target for therapeutic strategies aimed at enhancing the immune response to HIV.

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Published Ahead of Print 16 May 2012.
2012, 86(16):8367. DOI: 10.1128/JVI.00423-12.
J. Virol.
McNevin, M. Juliana McElrath and Michael Gale Jr.
Brian P. Doehle, Kristina Chang, Arjun Rustagi, John
a Lysosome-Dependent Mechanism
Regulatory Factor 3 during HIV Infection by
Vpu Mediates Depletion of Interferon
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Vpu Mediates Depletion of Interferon Regulatory Factor 3 during
HIV Infection by a Lysosome-Dependent Mechanism
Brian P. Doehle,aKristina Chang,aArjun Rustagi,cJohn McNevin,bM. Juliana McElrath,b,d,eand Michael Gale, Jr.a,c
University of Washington School of Medicine, Department of Immunology, Seattle, Washington, USAa; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer
Research Center, Seattle, Washington, USAb; and Departments of Global Health,cMedicine,dand Laboratory Medicine,eUniversity of Washington School of Medicine,
Seattle, Washington, USA
HIVhasevolvedsophisticatedmechanismstoavoidrestrictionbyintracellularinnateimmunedefensesthatotherwiseserveto
controlacuteviralinfectionandvirusdissemination.Innatedefensesaretriggeredwhenpatternrecognitionreceptor(PRR)
proteinsofthehostcellengagepathogen-associatedmoleculepatterns(PAMPs)presentinviralproducts.Interferonregulatory
factor3(IRF3)playsacentralroleinPRRsignalingofinnateimmunitytodrivetheexpressionoftypeIinterferon(IFN)and
interferon-stimulatedgenes(ISGs),includingavarietyofHIVrestrictionfactors,thatservetolimitviralreplicationdirectly
and/orprogramadaptiveimmunity.ProductiveinfectionofTcellsbyHIVisdependentuponthetargetedproteolysisofIRF3
thatoccursthroughavirus-directedmechanismthatresultsinsuppressionofinnateimmunedefenses.However,themecha-
nismsbywhichHIVcontrolsinnateimmunesignalingandIRF3functionarenotdefined.Here,weexaminedtheinnateim-
muneresponseinducedbyHIVstrainsidentifiedthroughtheirdifferentialcontrolofPRRsignaling.Weidentifiedvirusesthat,
unliketypicalcirculatingHIVstrains,lacktheabilitytodegradeIRF3.OurstudiesshowthatIRF3regulationmapsspecifically
totheHIVaccessoryproteinVpu.WedefineamolecularinteractionbetweenVpuandIRF3thatredirectsIRF3totheendolyso-
someforproteolyticdegradation,thusallowingHIVtoavoidtheinnateantiviralimmuneresponse.OurstudiesrevealthatVpu
isanimportantIRF3regulatorthatsupportsacuteHIVinfectionthroughinnateimmunesuppression.Theseobservationsde-
finetheVpu-IRF3interfaceasanoveltargetfortherapeuticstrategiesaimedatenhancingtheimmuneresponsetoHIV.
V
functionstosuppressviralreplicationandspread(35,38).Specific
motifs within viral products, including genomic RNA, DNA, or
nucleic acid replication intermediates, are recognized by the host
cellaspathogen-associatedmolecularpatterns(PAMPs)bycellu-
lar factors termed pattern recognition receptors (PRRs) (35, 38).
Nucleicacidsensors,includingToll-likereceptors(TLRs),RIG-I-
like receptors (RLRs), as well as several classes of DNA sensors,
comprise PRRs that engage viral PAMPs to thereby trigger intra-
cellular processes of innate immunity (35, 38). PRR signaling of
downstream interferon regulatory factor 3 (IRF3) activation is a
central feature of the innate immune response in most cell types,
leading to alpha/beta interferon (IFN-?/?) production; the ex-
ceptionisplasmacytoiddendriticcells(pDCs),whichutilizeIRF7
in this process (17, 38). IFN produced by virus-infected cells and
pDCs drivesautocrineandparacrineIFNsignalingtogenerateare-
sponse in the infected cell and surrounding tissue that induces hun-
dredsofinterferon-stimulatedgenes(ISGs)(38).ISGproductshave
direct antiviral or immune modulator actions that limit virus repli-
cation and spread (35, 38). In order to replicate efficiently, many
viruses direct strategies to disrupt various aspects of innate immune
signaling or response that range from disrupting PRR signaling to
inhibitingISGfunction(22).ViralcontrolofIRF3activationpresents
acentralstrategytopreventtheonsetoftheinnateimmuneresponse,
therebyallowingthevirustoavoidthelimitationsonreplicationim-
posed by IFN-?/?, proinflammatory cytokines, and other IRF3-re-
sponsive gene products (22). Indeed, several studies have linked the
direct or indirect regulation of IRF3 to infection outcome and the
pathogenesisofhumanviruses(22).
HIV-1 is a major human pathogen that has evolved sophisti-
catedmechanismstomodulateintracellularinnateimmuneeffec-
irusinfectionofmammaliancellstriggersanintracellularim-
mune response, termed the “innate immune response,” that
tors and restriction factors that otherwise serve to control acute
retroviralinfectionandvirusdissemination(5,21,27).IRF3plays
acentralroleininductionofinnateimmunityinTcellsandmac-
rophages to drive the expression of IFN and ISGs, including a
varietyofdirectlyanti-HIV-1restrictionfactors,aswellastopro-
gram downstream adaptive immunity (1, 20, 27, 30, 37). How-
ever, a variety of studies have shown that HIV-1-infected periph-
eral blood mononuclear cells (PBMCs) or T cell lines exhibit only
a limited spectrum of ISG expression concomitant with little, if
any, IFN production (3, 12, 28, 33), suggesting that during acute
HIV-1infectionPRRsignalingprogramseitherarenotengagedor
are counterregulated by virus-directed processes. In support of
thisnotion,weandothershaveshownthatproductiveinfectionof
T cells by HIV-1 is accompanied by the specific targeted proteol-
ysis of IRF3 that occurs through a virus-directed mechanism re-
sulting in suppression of innate immune defenses (6, 29). These
studies revealed that IRF3 activation drives an innate immune
response that is highly deleterious to productive HIV-1 infection,
suggesting that targeted viral antagonism of IRF3 by HIV-1 may
provide an additional level of viral control of the innate immune
system.WenowdemonstratethattheHIV-1proteinVpuplaysan
important role in HIV-1 innate immune regulation by targeting
and relocalizing IRF3 for proteolysis. Our studies define the IRF3
Received 17 February 2012 Accepted 8 May 2012
Published ahead of print 16 May 2012
Address correspondence to Michael Gale, Jr., mgale@u.washington.edu.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.00423-12
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regulation to be an additional feature of immune control to sup-
port HIV-1 infection.
MATERIALS AND METHODS
Cell culture, transfections, and treatments. All cells were grown under
standardconditionsasdescribedpreviously(6).SupT1cellswerecultured
in RPMI medium supplemented with 10% fetal bovine serum (FBS), L-
glutamine, and antibiotics. HEK293, 293, 293T, and Tzm-bl cells were
cultured in Dulbecco modified Eagle medium supplemented with 10%
FBS, L-glutamine, and antibiotics. Transfection of cells was performed
using the calcium phosphate method or using Fugene 6 transfection re-
agent (Roche) according to the manufacturer’s suggested protocol. Plas-
mids pcDNA3.1 (Invitrogen), green fluorescent protein (GFP), GFP-la-
beled IRF3, and Flag-labeled IRF3 were used where indicated and have
been described previously (36). All other expression plasmids have been
described previously and were obtained through the AIDS Research and
Reference Reagent Program, as described below. The protease inhibitors
MG115 (50 ?M) and MG132 (5 or 10 ?M), as well as the endolysosomal
inhibitor chloroquine (2, 5, 10, or 50 ?M), were used in the indicated
experiments, with 10 ?M used in single-dose experiments.
Viral stocks and infection. HIV-1LAIwas propagated using standard
proceduresasdescribedpreviously(6),andinfectionsutilizedamultiplic-
ity of infection of 1. Vpu point mutant proviral constructs have been
described previously (YU2, AD8) (32) or were generated via site-directed
mutagenesis (NL4-3, JR-CSF). Generation of point mutant proviral
clones was accomplished using a QuikChange mutagenesis kit (Strat-
agene) following the manufacturer’s recommended protocol, optimizing
for the large size of the plasmids. Thefollowingprimerswereused:JR-CSF
A/C Forward (5=-GCAGTAAGTAGTGCATGTACTGCAACCTTTACAA
ATATTAGCAATAGTAGC-3=) and Reverse (5=-GCTACTATTGCTAAT
ATTTGTAAAGGTTGCAGTACATGCACTACTTACTGC-3=) and NL4-
3 A/C Forward (5=-GCAGTAAGTAGTACATGTAGGGCAACCTATAA
TAGTAGC-3=) and Reverse (5=-GCTACTATTATAGGTTGCCCTACAT
GTACTACTTACTGC-3=). HIV-1 strains NL4-3, JR-CSF, and YU2 and
Vpu-deficientproviralclonesweretransfectedinto293Tcellsasdescribed
previouslytogenerateinfectiousvirus(15,16).Mockinfectionsrepresent
treatment with conditioned medium. The titers of all HIV-1 strains were
determined on Tzm-bl cells to determine the concentration of infectious
virus. Sendai virus (SeV) strain Cantell was obtained from Charles River
Laboratories.
Immunoblot analysis, coimmunoprecipitation, and immunofluo-
rescence imaging. SDS-PAGE, immunoblot analysis, and immunofluo-
rescence were performed using standard procedures as described previ-
ously (6). The following antibodies were used in the study: mouse (M)
anti-p24, goat (Gt) anti-beta-actin (Santa Cruz), rabbit (Rb) total anti-
IRF3(agiftfromMichaelDavid),Rbanti-IRF3-p(CellSignaling),Mtotal
anti-IRF3 (13), Rb anti-HIV-1NL4-3Vpu, Rb anti-HIV-1 Vpr, M anti-
HIV-1 Vif (Santa Cruz), and M anti-human LAMP2 (Abcam). For im-
munoblot detection, the appropriate horseradish peroxidase-conjugated
secondary antibody was used (Jackson ImmunoResearch Laboratories),
followed by treatment of the membrane with ECL-plus reagent (Roche)
and imaging on X-ray film. Densitometry was performed using ImageJ
software (NIH) on unsaturated blots. Coimmunoprecipitation assays
were performed utilizing standard procedures and anti-Flag M2 agarose
(Sigma) to pull down Flag-labeled IRF3. For immunofluorescence imag-
ing,appropriateAlexaFloursecondaryantibodies(Invitrogen)wereused
along with DAPI (4=,6-diamidino-2-phenylindole) during secondary
staining for each slide. All images were photographed on a Nikon
TE2000-E microscope and processed with Nikon EIS-Elements software.
Dual luciferase assays. Dual luciferase assays (Promega) were per-
formed according to the manufacturer’s specifications and as described
previously. The IFN-? promoter plasmid has been described previously
(10, 11).
Statisticalanalysis.Differencesbetweengroupswereanalyzedforsta-
tistical significance by the Student t test.
ThefollowingreagentswereobtainedthroughtheAIDSResearchand
ReferenceReagentProgram,DivisionofAIDS,NIAID,NIH:Tzm-blcells
werefromJohnC.Kappes,XiaoyunWu,andTranzymeInc.,monoclonal
FIG 1 Role for Vpu in disruption of IRF3-dependent signaling. (A) Proviral constructs for HIV strains NL4-3, JR-CSF, and YU2 or control plasmid were
transfectedin293cellsalongwiththeIFN-?promoterluciferaseconstructandchallengedwithSeVtodriveIRF3-dependentsignaling.(B)(Top)Alignmentof
LAI, NL4-3, JR-CSF, and YU2 highlighting the start codon (arrow) for Vpu and the A-to-C transversion mutation found in the YU2 strain. *, upstream,
nonproductive ATG. (Bottom) Amino acid alignment of Vpu from the same strains. (C) Vpu or Vif overexpression constructs tested as described for panel A.
Vpu expression was titrated by expressing 50 ng to 375 ng of expression plasmid within equal DNA dosage transfections using control plasmid as filler; Vif
expression from 375 ng of expression plasmid is shown. (D) Immunoblot analysis of either mock-infected HIV-1LAI-infected SupT1 cells or HIV-1LAI-infected
SupT1cellsat8,24,or48hpostinfection.CelllysateswereprobedforIRF3,Vpu,HIV-1Gag,oractinasaloadingcontrol.Luciferasereportergeneexperiments
were repeated 3 or more times, and representative immunoblot analyses are shown.
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antibody to HIV-1 p24 (AG3.0) was from Jonathan Allan, pNL4-3 was
from Malcolm Martin, pcDNA-HVif and pcDNA-Vphu were from
StephanBourandKlausStrebel,pEGFP-VprwasfromWarnerC.Greene,
HIV-1NL4-3VpuantiserumwasfromKlausStrebel,andHIV-1Vpr(1-50)
antibody was from Jeffrey Kopp.
RESULTS
Role for Vpu in disruption of IRF3-dependent signaling. To
identify viral determinants that direct IRF3 regulation during
acuteHIV-1infection,wescreenedaseriesofHIV-1proviralcon-
structs for their ability to regulate IRF3-dependent IFN-? pro-
moter induction in response to SeV infection, a potent inducer of
IRF3 activation. In contrast to lab strains of HIV-1 such as NL4-3
and JR-CSF, which mediate the degradation of IRF-3 to block its
inductionofIFN-?(6),wefoundthatexpressionoftheHIV-1YU2
strain failed to inhibit virus-induced signaling (Fig. 1A). Interest-
ingly, YU2 is one of only ?1% of all sequenced HIV-1 primary
isolatescarryingamutationintheaccessorygenevpu(4),harbor-
ingasingleA-to-CtransversioninthestartcodonoftheVpuopen
reading frame (ORF) that fails to produce a functional protein
(Fig. 1B and 2A). To test the hypothesis that Vpu may be respon-
sible for the ability of HIV to downmodulate IRF3-dependent
signaling,weexaminedtheeffectofVpuoverexpressionsignaling
of the IFN-? promoter. We found that ectopic expression of Vpu
disruptedIFN-?promotersignalinginadose-dependentmanner
and occurred in a manner similar to that observed with cognate
HIV-1 provirus (Fig. 1C). As we have reported previously (6),
expression of similar levels of Vif did not alter signaling to the
IFN-? promoter (Fig. 1C). Vpu is a late HIV-1 gene product,
being produced off transcripts along with Env. To determine if
Vpu is expressed with kinetics similar to that of the depletion of
IRF3protein,weinfectedSupT1cellsandprobedforVpuexpres-
sion. We observed low levels of Vpu within 8 h postinfection of
cellswithHIV-1LAI(Fig.1D),withpeakexpressionlevelscoincid-
ing with the strongest depletion of IRF3 at 24 h postinfection.
Vpu is necessary and sufficient for IRF3 depletion and dis-
ruption of IRF3-dependent signaling. To determine how the
HIV-1YU2vpu mutation impacts IRF3-dependent signaling, we
characterizedthesamemutationengineeredintothesamecontext
within three otherwise Vpu-positive proviruses (JR-CSF, NL4-3,
andAD8).WhenexaminedforVpuproteinexpression,wefound
that each of the resulting viruses failed to express Vpu protein,
thus releasing the HIV-1-mediated blockade of innate immune
signaling (Fig. 2A). Importantly, placement of a functional start
codon into the vpu ORF of HIV-1YU2restored Vpu expression,
FIG 2 Vpu is necessary and sufficient for disruption of IRF3-dependent signaling and IRF3 depletion. (A) Wild-type and Vpu-deficient proviral mutants of
JR-CSF,pNL4-3,AD8,andYU2weretransfectedandtestedforsignalinginresponsetoSeVasdescribedforFig.1A;constructsweretestedforexpressionofVpu,
Vpr,Vif,HIV-1Gag,oractinbyimmunoblotting.*,P?0.05;**,P?0.01.(B)CellsweretransfectedasdescribedforFig.1AandimmunoblottedforIRF3,Vpu,
and actin. (C) Cells were treated as described for panel A, with Vpu added in trans in increasing doses along with the Vpu-deficient pNL4-3 proviral construct.
Luciferase reporter gene experiments were repeated 3 or more times, and representative immunoblot analyses are shown.
Vpu Antagonism of IRF3
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resulting in a gain of function to disrupt virus signaling of IFN-?
induction (Fig. 2A). Moreover, this gain of function associated
withareductioninIRF3abundancewithincellsexpressingHIV-1
provirus (see Fig. 5C) or Vpu alone (Fig. 2B). In these experi-
ments, all proviruses expressed similar levels of Vpr, Vif, and Gag
proteins, demonstrating that Vpu deficiency does not impact
global HIV-1 protein production or processing (Fig. 2A). Addi-
tionally, when coexpressed in trans along with a vpu mutant pro-
virus, wild-type (wt) Vpu was able to restore disruption of virus
signaling of IFN-? induction in a manner similar to that for wt
HIV-1 provirus (Fig. 2C). These results identify Vpu as an HIV-1
antagonistofIRF3-dependentsignalingthatmayfunctioninpart
to suppress IRF3 protein levels in the host cell.
VpucomplexeswithIRF3,whichisstrengtheneduponvirus-
induced IRF3 activation. To determine how Vpu impacts the
abundance and metabolism of IRF3, we first performed coim-
munoprecipitation analysis to assess a possible Vpu-IRF3 in-
teraction.WetransfectedHIVJR-CSForHIVJR-CSF A/Cproviruses
with Flag-tagged IRF-3 in 293 cells and performed immuno-
precipitation of IRF3 using anti-Flag beads. The recovered
complexeswerethenassessedforthepresenceofbothIRF3and
HIV proteins. In cells expressing HIV-1JR-CSFprovirus, Vpu
formed a stable complex with IRF3 but not with the viral Vpr
protein (Fig. 3A). Neither Vpu nor Vpr was found to be asso-
ciated in the mutant HIVJR-CSF A/C, which fails to express Vpu
but expresses normal levels of Vpr. Moreover, when expressed
in the absence of other viral products, Vpu but not Vpr associ-
atedwithIRF3intransfectedcells(Fig.3B).InadditiontoVpu,
Vpr altered IRF3 levels, possibly explaining previous observa-
tions that implicated Vpr in IRF3 regulation (6, 29). However,
wenotethatdifferencesintheexpressionconstructsusedhereandin
other studies preclude a direct comparison of Vpu and Vpr (6). In-
terestingly, the Vpu-IRF3 interaction was strengthened if IRF3 was
activated by SeV infection, resulting in a 5-fold or more increase in
VpuassociatedwithIRF3comparedtothatforcellsnotinfectedwith
SeV, despite lower levels of recoverable IRF3 in the presence of Vpu
(Fig.3BandC).ThesedatatogethersuggestthatVpucomplexeswith
IRF3 and this interaction may be more robust if IRF3 is activated/
phosphorylated.
IRF3andVpucolocalizewithlysosomalmarkersduringHIV
protein expression. IRF3 normally displays a cytoplasmic local-
ization in resting cells but translocates to the nucleus upon its
activation by virus infection, as demonstrated by SeV stimulation
(Fig.4A).However,immunofluorescencemicroscopyanalysisre-
vealed that during HIV-1 provirus expression but prior to maxi-
maldepletion,IRF3becomesredistributedtocolocalizewithVpu
into punctate bodies (Fig. 4B) that codistribute with LAMP2, a
protein marking the endolysosome (Fig. 4C and E), as previously
described for Vpu localization (19). In contrast, within neighbor-
ing cells that lack detectable levels of Vpu, IRF3 remained nor-
mallydistributedandhighlyabundantthroughoutthecytoplasm.
Image quantification of micrographs from these experiments re-
vealed that over 80% of Vpu-expressing cells exhibited IRF3 lo-
calizedintopunctatestructures(Fig.4D).Incontrast,thispattern
of IRF3 distribution was not observed in Vpu-deficient cells from
thesamefields(Fig.4E).Moreover,IRF3stainingalsocolocalized
with endolysosomal markers in cells expressing HIV-1JR-CSF(Fig.
4E). These results demonstrate that Vpu forms a stable and specific
complex with IRF3 in HIV-1-infected cells, resulting in its specific
sequestration within the endolysosomal compartment. Therefore,
the Vpu-IRF3 interaction may alter the cellular metabolism of IRF3
inHIV-1-infectedcells(Fig.1D)(6).
Lysosomal disruption specifically rescues IRF3 depletion
during HIV infection. To maintain immune homeostasis, active
IRF3istypicallymetabolizedthroughautoregulatoryprocessesof
proteasomal degradation after being marked by ubiquitin at late
times during the innate immune response (5, 6). We therefore
assessed the role of proteasomal degradation in regulating IRF3
levelsduringHIV-1infection.WhileHIV-1infectionsignificantly
reduces IRF3 levels, treatment of HIV-1-infected cells with the
proteasomal inhibitor MG132 resulted in only a slight rescue of
IRF3, indicating that enhanced proteasomal targeting is not the
major process of HIV-1-mediated IRF3 suppression (Fig. 5A).
Considering that Vpu can direct the relocalization of cellular
factors, including the HIV-1 restriction factor tetherin/BST2, to
the endolysosomal compartment for degradation (9, 19, 26), we
assessed if Vpu could similarly target IRF3 to the lysosome for
proteolysis.Treatmentofcellstodisruptendolysosomalacidifica-
tion prevented the degradation of IRF3 that otherwise occurred
during HIV-1LAIinfection (Fig. 5B). Treatment with increasing
concentrations of the inhibitor resulted in rescue of IRF3 levels in
adose-dependentmanner.ThisrecoveryofIRF3peakedat?65%
rescue, after which cell toxicity became apparent in the cultures,
precludingfullrecoveryofIRF3levels.Importantly,HIV-1YU2 C/A
containing a functional Vpu ORF restored the ability of this
HIV-1 strain to suppress IRF3 levels, but treatment of these cells
with lysosome inhibitor rescued IRF3 from degradation in the
presence of Vpu (Fig. 5C). Two key serine residues in Vpu (S52,
FIG 3 Vpu coimmunoprecipitates with IRF3, and the interaction is strength-
ened upon IRF3 activation. (A) 293 cells were transfected with Flag-tagged
IRF3aswellasproviralconstructsforJR-CSFandJR-CSFA/C(Vpu?and?,
respectively).Celllysateswereexposedtoanti-Flagbeadsforbindingandthen
washed extensively. Input and bound fractions were immunoblotted for the
presence of Vpu, Vpr, p24, and IRF3. (B) Vpu, Vpr, and control plasmids
(vector control, GFP alone, GFP-IRF3) were transfected along with Flag-
taggedIRF3in293cells.At24hposttransfection,cellswereeithermockorSeV
treated to activate IRF3. After 12 h, cells were harvested, IRF3 was immuno-
precipitated, and samples were probed for Vpu, GFP, IRF3, Flag, and beta-
actin. A portion of the total cell lysate was saved and probed as input. (C)
Overexpressed Vpu was isolated with Flag-tagged IRF3 during activation of
IRF3 with SeV for increasing times (mock [M], 12 h, or 24 h) posttreatment
(hpi), as described for panel B.
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S56) have been described to be essential for its ability to down-
modulate both CD4 and tetherin/BST2 in ?-TrCP and lysosome-
dependentmechanisms(8,24).Wethereforetestedthesemutants
to determine if the same residues were also important for Vpu to
disrupt IRF3-dependent signaling. We found that the S52 and
56N double mutation of Vpu completely ablated its ability to
block innate immune signaling (Fig. 5D). Taken together, these
resultsindicatethatVpucomplexeswithIRF3andtargetsittothe
lysosomeforproteolyticdegradationinamannermechanistically
similar to antagonism of CD4 and tetherin/BST2.
DISCUSSION
The degradation of IRF3 in HIV-1-infected cells is specific and
occurs rapidly during acute infection of T cells and myeloid
FIG 4 IRF3 and Vpu colocalize with lysosomal markers during HIV protein expression. (A) Tzm-bl cells were mock or SeV infected overnight, stained with
anti-IRF3 (green) and DAPI (blue), and visualized by immunofluorescence microscopy. (B) Tzm-bl cells were transfected with JR-CSF provirus for 24 h and
stained with anti-Vpu (red), anti-IRF3 (green), and DAPI (blue). Cells were visualized with two fields presented for IRF3/Vpu staining. Arrows, areas of strong
colocalization(C)CellstreatedasdescribedforpanelBbutstainedwithanti-Vpu(red),anti-LAMP2(green),andDAPI(blue).(D)Quantificationoftheimages
inpanelBwithatotalof20fieldsfrom3experimentsofcontrolorVpu-positivecellsisdisplayed.(E)AdditionalcellstreatedasdescribedforpanelsBandCbut
stained with anti-LAMP2 (red), anti-IRF3 (green), and DAPI (blue). For all panels, representative cells are shown, with images of individual channels and a
merged image of all three signals shown. Control cells were transfected with vector alone and treated as with their JR-CSF-matched staining panels.
Vpu Antagonism of IRF3
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cells in vitro and within mucosal T cells ex vivo (6, 29). Our
study defines Vpu as an IRF3 antagonist encoded by HIV-1 and
uncovers a novel role for Vpu in directing IRF3 degradation
and control of innate antiviral immunity in HIV-1-infected
cells. Previous work suggested possible roles for the viral pro-
tein Vif or Vpr in targeting IRF3 (29); however, we have not
observed an effect of Vif on IRF3 binding or suppression of
IRF-3 levels (Fig. 1C) (6), and the effects of Vpr on IRF3 regu-
lation have been only modest in cell models of Vpr expression
(6). Here we report a stable interaction between IRF3 and Vpu
that appears to not involve either Vif or Vpr (Fig. 3A and B).
Indeed, our genetic and biochemical evidence indicates that
Vpu is necessary and sufficient for IRF3 degradation by HIV-1.
Of additional note, however, is that by providing Vpu in trans
to otherwise Vpu-deficient proviruses (Fig. 2C), we were able
to impose a reduction of IRF3 more potent than that when
ectopic Vpu was expressed alone in the absence of provirus.
This additional boost in inhibiting the innate immune re-
sponse within the context of full HIV-1 protein expression
could suggest a secondary role in innate immune regulation
facilitated by additional viral proteins (such as Vpr) or possibly
reflect other known HIV-1 strategies to suppress the host cell
response to infection (21, 27, 34, 39).
It is surprising that Vpu, a late HIV-1 gene product and
membrane protein, would be responsible for promoting mod-
ulation of the cellular environment so that it is conducive to
viral growth by preventing the innate sensing of infection.
However, we found that Vpu expression coincides with IRF3
suppression, suggesting that the Vpu-IRF3 interaction repre-
sents a point in the HIV-1 infection cycle where viral products
are sensed by the host cell to otherwise trigger innate immune
signaling and IRF3 activation. Our working model of IRF3 reg-
ulation by HIV-1 therefore suggests that Vpu antagonizes IRF3
at a key point later in the viral life cycle, perhaps as replication
intermediates accumulate and trigger innate immune signaling
that would otherwise suppress HIV-1 infection. This model is
consistent with recent reports describing innate immune acti-
vation in other contexts, including IRF3 activation during in-
FIG 5 Vpu promotes the endolysosomal degradation of IRF3. (A) SupT1 cells were infected with HIV-1LAIor mock treated. At 24 h after infection, HIV-1-
infectedsamplesweretreatedwiththeproteasomeinhibitorsMG115andMG132ormocktreated(HIV-1)foranadditional8h.Cellswereharvestedandprobed
for IRF3 and HIV-1 p24 levels. IRF3 levels are quantified and displayed as a percentage of the IRF3 of the mock-infected sample. (B) HIV-1LAI-infected SupT1
cells or mock-infected cells were treated with increasing doses of chloroquine. *, noticeable cell toxicity was apparent in culture. IRF3 levels were determined by
immunoblotting and quantified as described for panel A. (C) 293 cells were transfected with HIVYU2or HIVYU2 C/Aprovirus in the presence or absence of
chloroquine. Lysates were immunoblotted for IRF3, p24 (HIV-1), and actin as a loading control. (D) IFN-? signaling determined in the presence of control, wt
Vpu,orVpu(S52,56N)mutationplasmids,withcellstreatedasdescribedinthelegendtoFig.1A.ImmunoblotforVpuandactinascontrolsforexpressionand
loading. RLU, relative light units.
Doehle et al.
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fection in TREX-deficient cells (7, 40). HIV-1 PAMP recogni-
tion and signaling by specific but yet to be defined PRRs would
thereby activate IRF3, further promoting its interaction with
Vpu. This process might also explain the ability of Vpu to in-
teract with a cytoplasmic IRF3, which might move in closer
proximity to membranes during innate immune synapse for-
mation on a variety of cellular membranes, as has been recently
described (18). This triggering of innate immune signaling
likely occurs independently of host cell sensing of HIV-1 cap-
sids, where incoming virions are sensed and then trigger innate
immune programs in an AP-1- and NF-?B-dependent mecha-
nism (31), and is likely specific to distinct cell types. More
studies are necessary to delineate the full breadth of these var-
ious sensing pathways and the full context of HIV antagonism
of these programs.
ByinteractingwithIRF3andtargetingittothelysosome(and,
to a lesser extent, the proteasome), Vpu acts to facilitate IRF3
proteolysisandpreventtheexpressionofgenesinvolvedininnate
immune defenses against HIV-1, including type I IFN, ISGs, and
direct IRF3-target genes that mediate antiviral actions. Included
among the genes responsive to IRF3 are known HIV-1 restriction
factors, including APOBEC3G, tetherin, ISG15, and others. In-
deed, HIV-1 suppression of IRF3 may serve to enhance cell per-
missivenessforinfectionbyrelievinginnateimmunerestrictionof
virus replication and cell spread during the critical stage of acute
infection (see the accompanying paper [4a]).
These conclusions are supported by the observations that
many pathogenic viruses have evolved means to suppress IRF3
function (2, 14, 22). Most relevant is that hepatitis C virus,
which, like HIV-1, typically mediates chronic infection, en-
codes its own protease to ablate signaling of IRF3 activation to
suppress innate immunity and support viral persistence (11,
23). Our data underscore a major feature of Vpu to target key
host proteins of the immune response, including tetherin/
BST2 and CD4, for lysosomal destruction, while revealing that
IRF3 is a new target of Vpu control. IRF3 signaling is essential
to promote cell expression of proinflammatory and immuno-
modulatory cytokines and chemokines from the site of infec-
tion that are required for effective adaptive immune responses
(5). IRF3 regulation by HIV-1 is therefore expected to contrib-
ute to early immune dysfunction in HIV-1-infected patients,
thus serving to establish a permissive environment for seeding
the initial infection. Once HIV-1 infection is established, IRF3
suppression by Vpu may also serve to promote the systemic
dissemination of the virus, likely promoting the innate im-
mune dysfunction that is linked to the end stages of the viral
eclipse period (25). Strategies to disrupt the Vpu-IRF3 interac-
tion and/or block Vpu lysosomal targeting actions could serve
as a new therapeutic avenue against HIV-1.
ACKNOWLEDGMENTS
We thank Hilario Ramos and Mehul Suthar for helpful conversation and
review.
This work was supported by grants from the U.S. Public Health Ser-
vice,includingBMGFCAVD38645andNIH/NIAIDU01AI068618.This
work was also supported by funds from the state of Washington, NIH
grants DA024563 (to M.G.), AI078768 (to B.P.D.), 5T32GM007266-34
(to A.R.), and AI07140 (to A.R.), and the Burroughs-Wellcome Fund (to
M.G.).
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