Imaging of the Alphavirus Capsid Protein during Virus Replication

Imaging of the Alphavirus Capsid Protein during Virus Replication

Yan Zheng, Margaret Kielian

Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA

Alphaviruses are enveloped viruses with highly organized structures. The nucleocapsid (NC) core contains a capsid protein lat-tice enclosing the plus-sense RNA genome, and it is surrounded by a lipid bilayer containing a lattice of the E1 and E2 envelopeglycoproteins. Capsid protein is synthesized in the cytoplasm and particle budding occurs at the plasma membrane (PM), butthe traffic and assembly of viral components and the exit of virions from host cells are not well understood. To visualize the dy-namics of capsid protein during infection, we developed a Sindbis virus infectious clone tagged with a tetracysteine motif.Tagged capsid protein could be fluorescently labeled with biarsenical dyes in living cells without effects on virus growth, mor-phology, or protein distribution. Live cell imaging and colocalization experiments defined distinct groups of capsid foci in in-fected cells. We observed highly motile internal puncta that colocalized with E2 protein, which may represent the transport ma-chinery that capsid protein uses to reach the PM. Capsid was also found in larger nonmotile internal structures that colocalizedwith cellular G3BP and viral nsP3. Thus, capsid may play an unforeseen role in these previously observed G3BP-positive foci,such as regulation of cellular stress granules. Capsid puncta were also observed at the PM. These puncta colocalized with E2 andrecruited newly synthesized capsid protein; thus, they may be sites of virus assembly and egress. Together, our studies providethe first dynamic views of the alphavirus capsid protein in living cells and a system to define detailed mechanisms during alpha-virus infection.

Enveloped virus budding reactions can take place at a variety ofcellular membranes and may be dependent on the viral nu-

cleocapsid, envelope proteins, and/or matrix proteins (reviewedin references 1 and 2). The alphaviruses are small enveloped plus-sense RNA viruses with highly organized structures (reviewed inreferences 3–5). Alphaviruses contain an internal core composedof the �11-kb RNA genome enclosed in an icosahedral capsidprotein shell. This nucleocapsid (NC) is enveloped by the viruslipid bilayer containing a lattice of the E1 and E2 membrane gly-coproteins. Alphavirus budding takes place at the plasma mem-brane (PM) and requires both the NC and the envelope proteins(6). The completed viral particle contains 240 copies of each ofthese structural proteins, with each capsid protein interacting 1:1with the cytoplasmic domain of an E2 protein (7–9).

During infection, the alphavirus genomic RNA is translated toproduce the four nonstructural proteins (nsP1 to nsP4) that me-diate RNA replication, while the structural proteins are producedas a polyprotein from a subgenomic RNA (reviewed in references3, 4, and 10). The N-terminal capsid protein contains a proteasedomain. Once it is translated it rapidly folds, autocleaves itselffrom the polypeptide, and is released into the cytoplasm. The restof the polyprotein contains the viral membrane proteins, whichare translocated into the endoplasmic reticulum and transportedthrough the secretory pathway to the PM.

Two models have been proposed for alphavirus nucleocapsidassembly (reviewed in reference 11). One model predicts that theNC is preassembled in the cytoplasm and then drives virus bud-ding by binding to the glycoproteins at the PM. This model issupported by the presence of abundant NC in the cytoplasm ofinfected cells (12) and by the efficient in vitro assembly of NC inthe absence of glycoproteins (13). Microinjection of such pre-formed NCs into cells expressing the viral envelope proteins cangenerate infectious virus-like particles, albeit at a relatively lowefficiency (14, 15).

An alternative model postulates that a capsid-RNA complex

binds the E2 cytoplasmic domain at the PM, where the lateralinteractions of the glycoproteins drive formation of the icosahe-dral NC and subsequent virus budding. In support of this model,particle production for capsid mutants defective in cytoplasmicNC formation is only mildly reduced compared to that of the wildtype (WT), indicating that preformed NCs are not strictly re-quired for virus budding (9, 16–18). A common feature of bothmodels is that the cytoplasmic NC or the capsid-RNA complexmust be transported to the PM. Based on its high protein concen-tration and extensive cytoskeletal network, the cytoplasmic milieuwill greatly restrict the free diffusion of the capsid/NC (19), butpotential transport mechanisms are undefined.

Early studies of the kinetics of alphavirus particle production in-dicated that only a fraction of the cellular pool of capsid protein isultimately released in virus particles (20). Nascent capsid protein canassociate at least transiently with ribosomes in infected cells (21–24).Later in infection, some capsid proteins associate with the cellularadaptor protein p62, which mediates capsid targeting to autophago-somes (25, 26). It is not clear how or where the remaining capsidproteins might accumulate in the host cell, whether they associatewith specific cellular proteins or trigger host cell responses, or whatother additional functions the capsid protein may have.

Here, we set out to characterize the dynamics of the alphaviruscapsid protein during virus replication by visualizing the proteinin its cellular context in live infected cells. We took advantage ofthe tetracysteine (TC) motif, a 12-amino-acid sequence that canbe labeled with high affinity and specificity by biarsenical dyes

Received 14 May 2013 Accepted 14 June 2013

Published ahead of print 19 June 2013

Address correspondence to Margaret Kielian, [email protected].

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.01299-13

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such as FlAsH and ReAsH (27). Using a Sindbis virus (SINV)infectious clone, we defined a favorable insertion site for the TCmotif on the viral capsid protein and optimized the system tomonitor FlAsH- or ReAsH-labeled capsid in real time. Our studiesidentified three categories of capsid foci in SINV-infected Verocells. One group was comprised of small, round, internal punctathat were highly motile and colocalized with the E2 protein, sug-gesting a role in transport. A second group consisted of larger,more irregular internal structures that colocalized with G3BP andnsP3, two proteins previously shown to interact and regulate stressgranule formation. The third group was composed of capsidpuncta that colocalized with the E2 envelope protein at the PM,suggesting their involvement in the assembly/exit of progeny vi-ruses.

MATERIALS AND METHODSCells. BHK-21 cells were cultured at 37°C in complete BHK medium(Dulbecco’s modified Eagle’s medium [DMEM] containing 5% fetal calfserum, 10% tryptose phosphate broth, 100 U penicillin/ml, and 100 �gstreptomycin/ml). Vero cells from ATCC were kindly provided by KartikChandran and cultured in complete Vero medium (Dulbecco’s modifiedEagle’s medium containing 10% fetal bovine serum, 100 U penicillin/ml,and 100 �g streptomycin/ml) at 37°C.

Construction of SINV infectious clones. The WT SINV and mCherrySINV used in our studies were derived from the WT dsTE12Q infectiousclone (28) and a dsTE12Q clone containing mCherry at the N terminus ofthe capsid (25), both kindly provided by Beth Levine. We constructedgreen fluorescent protein (GFP)-dsTE12Q and Toto1101 (29) clones con-taining superfolder GFP (sfGFP) at the N terminus of the capsid. Thisinsertion was generated using a three-step overlap-extension PCR withsfGFP-C1 (30) (kindly provided by Erik Snapp) to amplify sfGFP and withdsTE12Q or Toto1101 to amplify the SINV capsid region. The fragmentcontaining sfGFP-capsid was subcloned into the dsTE12Q or Toto1101infectious clone using HpaI/AatII restriction endonuclease sites.

TC tag insertions in capsid protein were generated by overlappingPCR using primers encoding the optimized TC sequence FLNCCPGCCMEP (27). The fragment containing the TC insertion at the N terminusof the capsid was subcloned into the dsTE12Q infectious clone using theHpaI/AatII restriction sites, and fragments with TC inserted at other siteswere subcloned using the HpaI/BclI sites. TC-LL (capsid Q94-TC pluscapsid L108A/L110A) was constructed using overlapping PCR usingQ94-TC dsTE12Q as a template and primers containing the capsid L108Aand L110A mutations (9, 16). The fragment containing the Q94-TC in-sertion and capsid L108A/L110A mutation was subcloned into dsTE12Qusing the HpaI/BclI sites. TC Y400K (capsid Q94-TC plus E2 Y400K) wasconstructed by subcloning the E2 Y400K-containing PmlI/BclI fragmentfrom Y400K-dsTE12Q (G. Martinez, unpublished data) into Q94-TCdsTE12Q.

Sequence analysis (Genewiz Inc., North Brunswick, NJ) of the entireHpaI/AatII or HpaI/BclI PCR region confirmed that the TC insertionswere in place without additional mutations introduced during PCR am-plification. For each insertion mutant, two independent clones weretested to confirm the phenotype.

Production and titration of virus stocks. Viral RNAs were in vitrotranscribed from the XhoI-linearized SINV infectious clone and wereelectroporated into BHK-21 cells to generate virus stocks (31). All mutantvirus stocks were prepared by incubation of electroporated cells for 12 h at37°C to prevent possible generation of revertants during extended culturetime.

Virus titers were measured as indicated by plaque titration on BHK-21cells or by infectious center (IC) assays in BHK-21 cells or Vero cells. Forinfectious center assays, cells were infected with serial dilutions of SINVvirus for 1.5 h at 37°C. Media were replaced with complete growth me-dium containing 20 mM NH4Cl to prevent secondary infection and were

incubated at 28°C overnight to allow viral protein expression. Infectedcells were quantitated by immunofluorescence (IF) using monoclonal an-tibodies (MAbs) R2 and R6 to the E1 and E2 proteins, respectively (kindlyprovided by William Klimstra) (32).

Virus assembly assay. The assembly of mutant viruses was evaluatedby pulse-chase analysis (33). The viral RNAs were introduced intoBHK-21 cells by electroporation. After 6 h of incubation at 37°C, cellswere pulse labeled with 50 �Ci/ml [35S]methionine/cysteine (Express la-beling mix; PerkinElmer Life and Analytical Sciences) at 37°C for 30 minand chased for 0 to 3 h at 37°C in the absence of radiolabel. The mediumsamples were collected, immunoprecipitated with MAb R6 against E2(32) in the absence of detergent, and analyzed by SDS-PAGE. Cell lysateswere collected in the presence of 40 �M N-ethylmaleimide to alkylate freecysteine in the TC tag, and aliquots of the lysates were analyzed directly bySDS-PAGE.

Electron microscopy of virus-infected cells. BHK-21 cells were elec-troporated with WT or mutant RNA, plated on 35-mm-diameter dishes,and incubated at 37°C for 12 h before fixing with 2.5% glutaraldehyde, 2%paraformaldehyde in 0.1 M sodium cacodylate buffer for 30 min at roomtemperature. Samples were then processed by the Einstein Analytical Im-aging Facility by postfixing with 1% osmium tetroxide followed by 2%uranyl acetate, dehydration, and embedding in LX112 resin (LADD Re-search Industries, Burlington, VT). Thin sections were stained with uranylacetate followed by lead citrate and examined on a JEOL 1200EX or aJEOL 100CXII electron microscope at 80 kV. Images were recorded at amagnification of �20,000 and assembled with the software Adobe Pho-toshop CS.

Tetracysteine labeling with biarsenical dyes. Vero cells were platedon 8-well Lab-TekTM II chambered cover glass (Thermo Scientific), in-cubated at 37°C overnight, and then infected with WT or TC-tagged SINVat a multiplicity of 0.5 IC/cell. After 2 h at 37°C, the medium was replacedwith Vero complete medium and the incubation continued for another 5h at 37°C to allow the expression of capsid proteins before labeling withbiarsenical dyes. For the budding-negative Y400K mutant, Vero cells weretransfected with viral RNA and incubated at 37°C for 7 h. Labeling pro-tocols were modified from those published previously (34, 35), with ad-ditional helpful suggestions from Brett Lindenbach and Eric Freed. Cellswere incubated in 2.5 �M FlAsH (or ReAsH) for 5 min at 37°C andwashed three times at 37°C with DMEM containing 1 mM 2,3-di-mercapto-1-propanol (British anti-Lewisite [BAL]; Sigma) (34). Live cellimaging was then performed as described below. Alternatively, cells werefixed with 3% paraformaldehyde for immunofluorescence experiments.The time course of infection of the Vero cell culture (and the cellularstaining patterns) were not fully synchronous, even if the viral inoculumwas prebound to the cells in the cold (data not shown). This is likely due tothe effects of the local population context, including cell density (as de-scribed in reference 36).

For FlAsH/ReAsH pulse-chase labeling, cells were labeled with 5 �MFlAsH for 20 min at 37°C. After one wash with DMEM, cells were incu-bated with Vero growth media for 0 to 1 h at 37°C and then labeled with2.5 �M ReAsH as described above.

Immunofluorescence analysis. To stain for the capsid or cellularmarker proteins, Vero cells were infected or transfected as describedabove, incubated at 37°C for 7 h, fixed with 3% formaldehyde, and per-meabilized with 0.2% Triton X-100 for 5 min at room temperature. An-tibodies used for immunofluorescence included mouse anti-capsidmonoclonal antibody (MAb) C12-1 (kindly provided by Irene Greiser-Wilke) (37), rabbit antibodies to SINV nsP1 and nsP3 (kindly provided byMargaret MacDonald and Charles Rice) (38), rabbit anti-LC3 (PM036;MBL International Corporation), rabbit anti-G3BP (anti-G3BP1; G6046;Sigma), mouse anti-dsRNA MAb J2 (English & Science Consulting Kft.),and goat anti-eIF3 (sc-16377; Santa Cruz Biotechnology, Inc.). SINV E2was detected using mouse anti-E2 MAb R6, and E1 was detected usingMAb R2 (32). Preliminary studies showed that R6 recognized the ectodo-main of the E2 protein on intact virus but not in cell lysates or detergent-

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solubilized viruses (data not shown), suggesting that the epitope corre-lates with trimer or lattice assembly. Secondary antibodies were Alexa 488or 568 conjugated (Molecular Probes). For live cell staining, Vero cellswere incubated for 30 min at 37°C with the R6 MAb, placed on ice,washed, incubated with secondary antibody on ice, and fixed with para-formaldehyde on ice for 30 min before imaging. Images were capturedusing a confocal microscope system (Duoscan; Carl Zeiss MicroImaging,Inc.) with a 63�, 1.4-numeric-aperture (NA) oil objective and a 489-nm,100-milliwatt diode laser with a 500 to 550-nm band pass filter for FlAsHor Alexa 488 and a 40-milliwatt 561-nm diode laser with a 580-nm longpass filter for ReAsH or Alexa 568. All images were prepared with AdobePhotoshop CS4 software (Adobe System, San Jose, CA).

Live cell microscopy. Live cells were imaged in imaging media(DMEM without phenol red or bicarbonate [D2902; Sigma], 10 mMHEPES, pH 7, 10% fetal bovine serum, 100 U penicillin/ml, and 100 �gstreptomycin/ml) using the 37°C environmentally controlled chamber ofthe Duoscan confocal microscope system described above. Images wereprepared with Volocity 3D image analysis software (PerkinElmer) andAdobe Photoshop CS4 and Illustrator CS4 software (Adobe System, SanJose, CA).

RESULTSDevelopment of functional SINV capsid protein with a fluores-cent tag. To visualize the movements of the alphavirus capsidprotein during virus replication and assembly, we developedmethods to fluorescently label and image capsid protein. SINVwas used as a model alphavirus, and our constructs were based onthe SINV infectious clone dsTE12Q.

We first tested a previously described virus containingmCherry at the N terminus of the capsid protein (mCherry-dsTE12Q) (25, 26). We also constructed a similar virus in whichsuperfolder GFP was inserted at the capsid N terminus (GFP-dsTE12Q). BHK cells were electroporated with in vitro-tran-scribed viral RNAs, and progeny virus titers were measured. Thetiters of both mCherry-dsTE12Q and GFP-dsTE12Q were about 2logs lower than those of the unlabeled WT virus at 24 h postelec-troporation (data not shown). Transmission electron microscopy(EM) of virus-infected BHK cells showed WT SINV budding atthe cell surface as spherical particles containing a dense core in thecenter (as shown below). In contrast, cells infected with GFP-dsTE12Q (as shown below) or mCherry-dsTE12Q (data notshown) produced larger aberrant particles containing multiplepatches of dense core material. In addition, immunostaining withan antibody against the capsid protein showed that cells infectedfor 7 h with GFP- or mCherry-dsTE12Q virus contained uniquecytoplasmic patches of fluorescence that were not observed in cellsinfected with WT SINV (data not shown). Given that the sizes ofboth GFP and mCherry are similar to those of the capsid protein(247, 250, and 264 amino acids, respectively), it seemed likely thatGFP or mCherry interfered with the assembly of viral nucleocap-sid and/or caused capsid protein aggregation.

As an alternative strategy, we tested insertion of the TC motifand its labeling with biarsenical dyes (27, 39). This system has beensuccessfully used to study the trafficking of the human immuno-deficiency virus (HIV) GAG protein, the hepatitis C virus coreprotein, and the vesicular stomatitis virus M protein (34, 35, 40–42). A TC motif introduced at the N terminus of the SINV capsidprotein appeared to be rapidly lost during virus infection as de-tected by migration on SDS-PAGE (data not shown). Insertion ofa flexible linker between the TC tag and the N terminus of thecapsid protein stabilized the insertion, but the TC-tagged capsidprotein was not successfully labeled by biarsenical dye treatment

of infected cells (data not shown). This presumably reflects lim-ited accessibility of the TC motif in the folded/assembled capsidprotein. The alphavirus capsid protein assembles into NC, with itsN-terminal region located internally in association with the viralRNA (43) and its C-terminal protease domain arrayed on the NCsurface (44). We therefore narrowed our target site selection to theC-terminal domain and the region close to it in the hope of ob-taining better dye accessibility. Within those regions of the capsidprotein, we selected specific sites (Table 1) that appeared morelikely to tolerate insertions based on the results of a whole-genometransposon-mediated insertion screen of the alphavirus Venezu-elan equine encephalitis virus (45). Based on the structure of theSINV capsid protease domain (46), our selected sites were all lo-calized at the tips of flexible loops, in keeping with their potentialto tolerate a small insertion (data not shown). A series of TC-tagged SINV strains was constructed, and the viral growth kineticswere compared to those of WT SINV (Table 1). Most viruses witha TC tag inserted within the C-terminal capsid protease domainproduced viral titers at least 2 logs lower than that of WT SINV.The single exception was D113-TC, which grew to a titer only 1 loglower than that of WT virus. D113 is located at the junction be-tween the N-terminal and C-terminal regions of the capsid pro-tein. Insertion sites just N-terminal to this junction (E89, Q94,K97, and G101) proved more amenable to the TC tag, with all ofthe resultant viruses showing growth comparable to that of WTSINV (Table 1). All of the TC-tagged viruses that grew to within 1log of the WT titer (D113-TC, E89-TC, Q94-TC, K97-TC, andG101-TC) were then tested for their labeling intensity with the

TABLE 1 Summary of TC motif insertion sites tested in SINV capsidprotein

Insert region on capsid proteinand insertion Virus growthd Labeling efficiencye

Capsid N terminus(�1)-TCa WT Loses the TC tag(�1)-TC-Lb WT �

Protease domainc

D113-TC 1 log lower ��E176-TC 2 logs lower NAS182-TC 5 logs lower NAE186-TC 6 logs lower ����S199-TC �7 logs lower NAE259-TC �7 logs lower NA

N terminal to protease domainc

E89-TC WT ��Q94-TC WT ���K97-TC WT ���G101-TC WT ��

a The TC motif plus an N-terminal ATG codon was introduced just before the firstamino acid (methionine) of the capsid protein.b The TC motif plus an N-terminal ATG codon and a C-terminal GSSGGSSGGSSGflexible linker region was introduced before the first amino acid of the capsid protein.c The TC motif was introduced directly after the indicated residue.d The growth of all TC insertion mutants was compared to that of WT SINV at 24 hpostelectroporation (see Materials and Methods). The log of the WT SINV titer was�8.6 as measured by plaque assay on BHK-21 cells. The titer of all mutants designatedWT was �8.e The labeling efficiency of virus-infected Vero cells with biarsenical dyes. NA, notapplicable; �, barely detectable fluorescence; ��, low fluorescence intensity; ���,strong fluorescence intensity; ����, very bright fluorescence intensity.

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biarsenical dye FlAsH or ReAsH. Among these, K97- and Q94-TCshowed the brightest signals. Q94-TC was selected for furtherstudies.

Characterization of Q94-TC virus. BHK-21 cells were electro-porated with WT or Q94-TC viral RNA and the kinetics of prog-eny virus production determined (Fig. 1A). Efficient productionof both viruses was observed by 6 h postelectroporation, and titersreached maximal levels by �14 h. Similar results were observedwhen the titers of progeny viruses were determined using an in-fectious center assay on Vero cells, the cell line used for our imag-ing studies (data not shown). Therefore, the growth kinetics ofQ94-TC were comparable to those of WT SINV.

The assembly of virus particles was evaluated by pulse-chaseanalysis of BHK-21 cells 6 h after RNA electroporation. Cells in-fected with Q94-TC or WT virus produced comparable amountsof viral proteins (Fig. 2, lysate samples). The E2 precursor pE2 wasclearly detected at the 0-h chase time, and maturation comparableto that of E2 was observed in WT- versus Q94-TC-infected cellsduring a 3-h chase. Analysis of medium samples indicated that thebudding efficiencies of Q94-TC and WT SINV were comparable.The capsid protein in both Q94-TC media and lysate samplesmigrated as a single band that ran slightly above the position of theWT SINV capsid (Fig. 2), consistent with the stable incorporationof the 12-amino-acid TC motif in Q94-TC.

The morphology of Q94-TC virus particles was evaluated bytransmission EM of infected BHK cells at 12 h postelectroporation(Fig. 3). Abundant Q94-TC virus particles were observed buddingat the plasma membrane. Their morphology was comparable tothat of WT virus, with a particle diameter of about 70 nm and adense NC core in the center (compare Fig. 3A and C). In contrast,the GFP- or mCherry-capsid viruses both produced aberrant par-ticles (Fig. 3E and data not shown). Typical membranous replica-tion structures, termed CPVI (3, 47), were detected within WT-and Q94-TC-infected cells (data not shown). In addition, bothWT- and Q94-TC-infected cells contained CPVII with associatedNC (Fig. 3B and D). Together, all of the evidence indicated thatthe introduction of a TC tag at the capsid Q94 position did notcause detectable effects on virus replication or progeny virus pro-duction.

ReAsH labeling specificity and effects of ReAsH labeling onvirus assembly. We next tested the specificity of ReAsH labelingand its effects on virus production. Vero cells were infected withQ94-TC or WT virus for 7 h and stained with ReAsH as describedin Materials and Methods. Vero cells were chosen for imaging,given their efficient infection by SINV and flat morphology. Afterlabeling, the cells were fixed, permeabilized, and stained with aMAb against the capsid protein (Fig. 4). No ReAsH signal wasdetected in WT SINV-infected cells, while in Q94-TC-infectedcells clear intracellular staining was observed. The distributionpattern of the ReAsH staining in Q94-infected cells colocalizedwith the signal from the MAb against capsid protein. Thus, theQ94-TC motif was specifically labeled by the ReAsH dye withminimal background and accurately reflected the distribution ofcapsid protein in infected cells. Similar results were obtained withFlAsH dye labeling (data not shown).

We then measured the titers of Q94-TC progeny virusescollected at different time points following ReAsH labeling ormock treatment. Similar titers were detected in the ReAsH-versus mock-treated samples (Fig. 1B), indicating that theReAsH treatment and the binding of ReAsH dye to the capsidprotein had minimal effects on the production of infectiousvirus particles.

Distribution of the capsid protein in infected cells. We thenused dye labeling to monitor the distribution of the capsid protein

FIG 1 Growth properties of Q94-TC SINV. (A) Growth kinetics of Q94-TCversus WT virus. BHK-21 cells were electroporated with WT or mutant virusRNA and incubated at 37°C for the indicated times. Media were collected, andtiters of progeny viruses were determined by plaque assay. (B) The effect ofReAsH labeling on Q94-TC virus production. Vero cells were infected withQ94-TC at a multiplicity of 0.5 IC/cell, cultured for 7 h at 37°C, and mocktreated or treated with ReAsH using the conditions described in Materials andMethods. The incubation was continued at 37°C for the indicated times, andprogeny virus production was quantitated by infectious center assays on Verocells. Data are averages from two independent experiments with ranges indi-cated.

FIG 2 Assembly properties of Q94-TC SINV. BHK-21 cells were electropo-rated with Q94-TC (TC) or WT virus RNA, incubated at 37°C for 6 h, pulselabeled with [35S]methionine/cysteine, and chased for 0 or 3 h at 37°C. (Left)Cell lysates were collected and an aliquot analyzed directly by SDS-PAGE. Thepositions of TC-capsid (TC-C) and WT capsid (C) proteins are indicated, withTC-C migrating slightly slower than WT C. The pE2 protein is visible in the 0-hlysate sample, but E1 and E2 are obscured by the host cell proteins. (Right)Samples of the chase media were immunoprecipitated with an antibodyagainst SINV E2 in the absence of detergent to recover intact virus particles andwere analyzed by SDS-PAGE. The E1 and E2 proteins migrate as a doublet.

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after various times of infection. No significant staining of Q94-TCcells was detectable during the first �5 h of infection (data notshown). After 5.5 to 6 h of infection, Q94-TC-infected cells showedrelatively weak ReAsH staining that was diffusely distributed in thecytoplasm (data not shown). By 7 to 8 h postinfection, some cells stillshowed weak ReAsH staining, but the majority of the cells containeda much brighter ReAsH capsid signal. This staining was distributeddiffusely in the cytoplasm as before but also showed distinct foci, asdiscussed in detail below and depicted in Fig. 5. The absence of clearcapsid foci at earlier times of infection could reflect their formation ata certain point in the infection cycle or could simply be due to theirintensities lying below the detection limit compared to the surround-ing cytoplasmic capsid signal.

We performed a detailed analysis of the intracellular capsid foci byconfocal microscopy of Vero cells infected for 7 to 8 h, approximately

at the start of exponential virus production (as shown in Fig. 1). Q94-TC-infected cells were labeled with ReAsH at 7 h postinfection, fixed,permeabilized, and stained with MAb R6 against the SINV E2 protein(32). The cytoplasmic capsid foci were separated into three groupsbased on their size, shape, approximate cellular location, and colocal-ization with E2. The first category contained capsid foci (termed“small internal capsid puncta”) (Fig. 5A) that colocalized with E2 ininternal regions of the cell and were small (0.1 to 0.5 �m3) and round.In live cells, these small internal capsid puncta were highly motile(data not shown). As they contain both capsid and E2 protein, wehypothesize that these puncta are vesicles that transport both capsidand envelope proteins. Detailed studies using a virus labeled in boththe capsid and E2 protein are currently in progress to characterize therole of these puncta in virus biogenesis.

A second category contained capsid foci in internal regions ofthe cell (termed “irregular internal capsid structures”) (Fig. 5B)that were larger (0.6 to 3.0 �m3) and more irregularly shaped thanthe first group and showed no detectable E2 protein. Most of thecells that were positive for capsid foci contained both the smallinternal puncta and the irregular internal structures. The fre-quency of small internal capsid puncta was 5.2 per cell (standarddeviations [SD], 5.3; n � 20 cells), which was not significantlydifferent (P � 0.05 by Student’s t test) from the frequency of theirregular internal capsid structures (8.0 per cell; SD, 5.0; n � 20cells). Given their frequencies, these classes of capsid foci were notalways in the same confocal z section. The third group of capsidfoci (Fig. 5C) was observed as many discrete puncta at a focal planethat appeared close to the PM by confocal microscopy. This classof foci (termed “PM-proximal capsid puncta”) also colocalizedwith E2 and was detected in all capsid focus-containing cells.

The irregular internal capsid structures colocalize withG3BP and nsP3. Immunofluorescence analysis demonstratedthat, unlike the other two classes of capsid foci, the irregular in-ternal capsid structures did not colocalize with either the E2 or E1

FIG 3 Electron microscopy of WT- and mutant-infected cells. BHK-21 cells were electroporated with Q94-TC, WT SINV, or GFP-dsTE12Q viral RNA,incubated at 37°C for 12 h, and processed for electron microscopy. Panels A, C, and E show representative examples of the morphology of budding virus particles(indicated by arrows). Panels B and D show representative examples of cytopathic vacuole type II (CPVII) in WT (B)- or Q94-TC (D)-infected cells. Note thatthe CPVII-associated nucleocapsids are larger, denser, and more regular in shape than adjacent ribosomes in the field, differentiating these structures from theendoplasmic reticulum. All images were acquired at a magnification of 20,000�, with the scale bar representing 200 nM.

FIG 4 Specificity of biarsenical dye labeling. Vero cells were infected withQ94-TC or WT SINV at a multiplicity of 0.5 IC/cell, cultured for 7 h at 37°C,and labeled with ReAsH as described in Materials and Methods. Cells werethen fixed, permeabilized, and stained with antibody against the capsid pro-tein. Images are single internal z sections and are representative of two inde-pendent experiments. Bar, 10 �M.

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envelope protein (Fig. 5B and data not shown). We therefore eval-uated cellular markers that might be characteristic of this group ofcapsid foci. The structures did not show colocalization with p115,a marker of the Golgi complex, or with LC3, a marker of autopha-gosomes (data not shown). However, our studies showed thatthese irregular internal capsid foci were strongly positive for theRas-GAP SH3 domain binding protein G3BP (Fig. 6A). G3BP, amultifunctional RNA binding protein, has been reported to be akey factor in the nucleation of cellular stress granules (SGs) and isactivated by dephosphorylation during SG formation (48). To de-termine if the irregular internal capsid structures also containedother SG markers, such as eukaryotic translation initiation factor3 (eIF3), we tested the colocalization of ReAsH-labeled capsidprotein and MAb-stained eIF3. Control cells in which SGs werechemically induced by sodium arsenite treatment (49, 50) dis-played eIF3-positive puncta (data not shown). In contrast, eIF3was diffusely distributed in the cytoplasm of SINV-infected cellsand did not colocalize with any of the three types of capsid struc-tures (Fig. 6A, lower). Consistent with this result, when Q94-TC-or WT-infected cells were stained with the anti-capsid MAb, theirregular internal capsid structures colocalized with G3BP but notwith eIF3 (Fig. 6C and data not shown). Together, our markeranalysis suggested that the capsid-G3BP foci are not typical SGs orautophagosomes. We do observe autophagosome induction andLC3-Q94 capsid colocalization at later times of infection (data notshown), in agreement with the studies of Orvedahl et al. (25).

Previous studies reported that typical SGs are induced at earlytimes (�2 to 4 h) of Semliki Forest virus (SFV) infection and are

disassembled at later times of infection when the cells becomepositive for viral gene expression (49). Studies with SINV, Chi-kungunya virus (CHIKV), and SFV showed that the viral proteinnsP3 interacts with G3BP, sequesters it into cytoplasmic foci, andinhibits the formation of bona fide SGs (38, 50–52). We thereforetested the colocalization of the irregular internal capsid structureswith nsP3 and dsRNA, a marker for the viral replication complex.Approximately 80% of the dsRNA foci in SINV-infected Verocells were associated with the PM at this infection time, with somesmall dsRNA puncta in the cytoplasm (Fig. 6A). However, none ofthe dsRNA foci colocalized with the capsid protein. The irregularinternal capsid structures were also negative for nsP1 (data notshown). Together, these results suggest the structures were notassociated with the viral replication complex. In contrast, the nsP3protein was detected in the irregular internal capsid foci (Fig. 6B),suggesting that these structures represent the previously observednsP3/G3BP foci reported to function in inhibiting formation ofbona fide SG (50, 52).

The role of NC assembly and E2 interactions in the generationof capsid protein-G3BP structures was determined by engineeringadditional mutations into SINV Q94-TC. The capsid substitu-tions L108A/L110A inhibit the formation of the cytoplasmic NCand CPVII but still allow virus budding at the PM (9, 16). Verocells infected with TC-LL showed somewhat more irregular inter-nal capsid structures than Q94-TC (33.0 per cell; SD, 8.2; n � 20cells), but clear colocalization with G3BP was observed (Fig. 6C).Thus, cytoplasmic NC formation is not required for formation ofthese structures. The substitution Y400K in the cytoplasmic tail of

FIG 5 Examples of the three types of capsid foci. Vero cells were infected with Q94-TC and labeled with ReAsH at 7 h postinfection. Cells were then fixed,permeabilized, and stained with a MAb against the SINV E2 protein. The images illustrate three groups of capsid foci. (A) Small internal capsid puncta thatcolocalize with the E2 protein (arrows point to representative examples). (B) Irregular internal capsid structures that did not colocalize with the E2 protein(arrows point to representative examples). (C) PM-proximal capsid puncta that colocalize with E2 protein. The inset shows a zoomed view of the boxed region(3� magnification). All images are single z sections, where panels A and B are internal sections and C is a PM-proximal section. All are representative examplesfrom three experiments. Bar, 10 �M.

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E2 blocks E2-capsid interaction, formation of CPVII, NC localiza-tion at the PM, and virus budding (7). However, cells infected withTC-Y400K showed irregular capsid structures that colocalizedwith G3BP (Fig. 6C), demonstrating that interaction with the E2protein was not necessary for formation of the capsid/G3BP foci.Vero cells infected with the alphaviruses SFV and CHIKV alsoexpress capsid/G3BP foci in the cytoplasm (data not shown), sug-gesting that formation of these structures is conserved among al-phaviruses.

Dynamics of the irregular internal capsid structures. TheTC-tagged virus made it possible to track the movement of theirregular internal capsid structures by ReAsH labeling of Q94-TC-

infected cells and live cell imaging. As shown by a representativetime series (Fig. 7A), this population of capsid foci was relativelyimmobile. Similar results were observed in cells infected with theTC-LL or TC-Y400K mutant (data not shown).

We then wished to address whether these immobile structurescan still actively recruit new capsid proteins. This question couldbe addressed by testing for an increase of fluorescence intensitywith time. However, the ReAsH dye is not very photostable, andthe gradual bleaching of fluorescence, even using low laser powerand short exposure times, made it complicated to directly quantifychanges in fluorescence intensity (data not shown). Alternatively,fluorescence recovery after photobleaching (FRAP) experimentscould be used to monitor delivery of labeled capsid protein. Thissystem requires that the high-power laser photobleach is irrevers-ible, allowing fluorescence recovery exclusively from the un-bleached capsid pool. In our system, bleaching of the ReAsH dyewas reversible and ReAsH signal rapidly reappeared after a com-plete photobleach of the whole cell (data not shown).

Therefore, we used FlAsH/ReAsH pulse-chase experiments tomonitor the newly synthesized capsid protein. Cells were infectedfor 6.5 h with Q94-TC and pulse labeled with FlAsH to saturate theavailable TC sites. After incubation in growth media at 37°C for 30min (chase), the cells were treated with ReAsH to label the newlysynthesized capsid protein. No ReAsH labeling was detected in theabsence of chase, confirming that the FlAsH labeling is complete(Fig. 7B, upper). In contrast, ReAsH labeling was readily detectedin the cells following a 30-min chase (Fig. 7B, lower). In the chase

FIG 6 Irregular internal capsid structures colocalize with G3BP and nsP3. (A)Vero cells were infected with Q94-TC and labeled with ReAsH (left) at 7 hpostinfection. Cells were then fixed, permeabilized, and stained with antibod-ies against G3BP, dsRNA, or eIF3 (middle panels). (B) Vero cells were infectedwith Q94-TC virus, incubated for 7 h, fixed, and costained with MAb recog-nizing the capsid protein and rabbit antibody against nsP3. (C) Vero cells wereinfected with either Q94-TC or Q94-TC plus L108A/L110A (indicated as TC-LL). Alternatively, cells were transfected with RNA for the budding-defectivemutant Q94-TC plus E2Y400K (indicated as TC-Y400K). Cells were incubatedfor 7 h, fixed, and costained with MAb to capsid and rabbit antibody againstG3BP. Images are representative of two independent experiments. Scale bar,10 �M.

FIG 7 Dynamics of irregular internal capsid structures. (A) Time series ofinternal capsid structures. Vero cells were infected with Q94-TC and labeledwith ReAsH at 7 h postinfection. The positions of specific foci (indicated byarrows) were tracked by images acquired every second. Images collected at 0,20, and 40 s are shown, documenting the relative immobility of these struc-tures. (B) Recruitment of newly synthesized capsid protein to preexisting ir-regular internal capsid foci. Vero cells were infected with Q94-TC and labeledwith FlAsH at 7 h postinfection (right column). At the indicated chase time,the cells were labeled with ReAsH (left column). Images all were acquired at thesame gain and are representative of two independent experiments. Bar, 10 �M.

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samples, the irregular internal capsid structures were labeled withboth FlAsH and ReAsH, suggesting that newly synthesized capsidwas being delivered into preexisting capsid/G3BP/nsP3 struc-tures. Thus, although these structures were relatively immobile,they were dynamic in acting as delivery sites for newly synthesizedcapsid protein.

Characterization of capsid puncta at the plasma membrane.As shown in Fig. 5C, distinct capsid puncta that colocalized withE2 were detected close to the PM and were particularly clear whenfocusing at the basolateral region of the cells. To address whetherthese PM-proximal capsid puncta are actually localized to the PM,we labeled Q94-TC-infected Vero cells with ReAsH and then per-formed immunostaining on ice to specifically detect the PM-lo-calized pool of E2. Under these conditions, E2 was not detectedinside the cells (Fig. 8A), confirming that only the PM pool wasvisualized. Strong colocalization of capsid ReAsH signal and E2protein was observed at the surface of infected cells. We will referto this group of capsid structures as “PM capsid puncta” from thispoint on in the text. Live cell imaging showed that most of the PMcapsid puncta were relatively immobile (Fig. 8B).

Tests of TC-LL-infected cells (Fig. 9A) showed that the forma-tion of the PM capsid puncta did not require cytoplasmic NCs. Incontrast, PM capsid puncta were not detected in TC-Y400K-in-fected cells, indicating that the E2-capsid interaction was critical(data not shown). Our earlier data showed that at this time ofinfection in Vero cells, the majority of dsRNA-positive punctawere associated with the PM (Fig. 6A). While we readily detectedboth dsRNA puncta and PM capsid puncta at the basolateralmembrane of infected cells, no colocalization was observed(Fig. 9B). Thus, the capsid puncta at the PM were not associatedwith the viral RNA replication complex.

We then used FlAsH/ReAsH pulse-chase experiments as de-scribed above to address whether the PM capsid puncta can ac-tively recruit newly synthesized capsid protein. As early as 1 h afterchase, newly synthesized capsid proteins were detected in PMpuncta that were also labeled with the FlAsH signal (Fig. 9C).Based on their enrichment for viral structural proteins and re-cruitment of newly synthesized capsid protein, our results suggestthat the PM capsid puncta are sites of virus assembly and budding.

DISCUSSION

In summary, we developed a TC-based system to image the move-ments of the SINV capsid protein in live infected cells withoutaffecting its biological activity. At early times of infection, labelingof the TC-capsid produced diffuse cytoplasmic staining that mayinclude ribosome-bound capsid proteins. By the beginning of ex-ponential virus production, three distinct types of intracellularcapsid foci were detected. Based on properties such as motility andcolocalization with viral and cellular proteins, we hypothesize thatthese foci are involved in capsid protein delivery to the PM, in theregulation of cellular SGs, and in virus assembly/exit from the PM.

Fluorescent labeling of the alphavirus capsid protein. Thealphavirus NC has a diameter of �400 Å and contains 240 copiesof the �30-kDa capsid protein arranged in a TA4 icosahedrallattice (53, 54). Given this organized NC structure, introducingthe �27-kDa, 42-Å-long �-barrel structure of GFP or mCherry(55) into the complete capsid protein is spatially challenging. In-sertion of GFP or mCherry at the N terminus of the capsid proteindid produce infectious virus, but its growth was reduced by �2logs, and both constructs produced aberrant particle morphologyand capsid protein distribution. Aberrant budding of HIV GFP-Gag is rescued by coexpression of WT-Gag (56, 57). We tested ananalogous complementation strategy for rescue of the buddingdefects in SINV GFP-capsid. We coexpressed the GFP- ormCherry-SINV infectious clone with the WT capsid protein, rea-soning that the incorporation of WT capsid might reduce the spa-tial hindrance in NC formation. However, even though WT capsidprotein was well expressed and displayed a normal cellular distri-bution pattern, it was not efficiently recruited into the GFP- ormCherry-labeled virus (data not shown). The highly symmetricstructure of the alphavirus NC and/or the possible importance ofcis expression of capsid and envelope proteins may limit suchcomplementation approaches. We conclude that the GFP- ormCherry-capsid virus does not accurately reflect the completeWT capsid pathway and is not optimal for imaging studies ofcapsid dynamics. However, such GFP- or mCherry-capsid viruseshave clearly been very useful tools to identify specific alphavirus-host protein interactions that confirm using WT SINV (25, 26).

We chose the small TC motif as an alternative strategy for livecell imaging of the alphavirus capsid protein. Virus particles in themedium of ReAsH-labeled WT- or Q94-TC-infected cells couldbe captured on poly-L-lysine-coated culture slides (data notshown). Both the WT and Q94-TC produced particles that labeledwith mixtures of MAbs against the E1 and E2 proteins. ReAsH-labeled particles were detected only in the Q94-TC sample, andthe number of particles was dependent on the multiplicity of in-fection. This result indicates that the Q94-TC virus supports im-aging of both intracellular and particle-assembled capsid protein.Q94-TC virus particles did not show labeling when stained withReAsH after adsorption to coverslips (data not shown), presum-

FIG 8 Localization and dynamics of the PM-proximal capsid puncta. Verocells were infected with Q94-TC and labeled with ReAsH at 7 h postinfection.(A) Localization of the PM-proximal capsid puncta. Following ReAsH label-ing, cells were immunolabeled on ice to detect the cell surface E2 protein asdescribed in Materials and Methods. A middle section in the z direction isshown. (B) Time series of PM capsid puncta. The positions of specific puncta(indicated by arrows) were tracked by images acquired every second during a37°C incubation. Images of a z section at the PM collected at 0, 20, and 40 s areshown, with a zoomed view of the boxed region on the upper-right side, doc-umenting the relative immobility of this puncta type. Images are representa-tive examples from two independent experiments. Bar, 10 �M.

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ably reflecting inhibition of free diffusion of the biarsenical dyeinto the Q94-TC site in assembled virus.

Capsid protein traffic and NC production. While the alpha-virus envelope proteins use the endogenous cellular secretorypathway for delivery to the PM, the traffic of the capsid protein isnot clear. We observed a diffuse distribution of capsid in the cy-toplasm that could provide a local source during virus budding.Our labeling studies also identified small, highly motile capsidpuncta, which we hypothesize are capsid transport vehicles. Im-munofluorescence experiments showed that this class of capsidfoci colocalized with the E2 protein, suggesting their vesicular na-ture. Further characterization is necessary to determine if thesepuncta deliver the capsid/NC to the PM and what mediates theirrapid movement. We are currently analyzing their properties us-ing a virus containing both the TC-94 capsid and a GFP-E2 pro-tein, which permits live cell imaging of both proteins. If the cap-sid/NC does travel with the E2 protein, it will be interesting toexplore the cellular location where this E2-capsid interaction ini-tiates and the specific mechanisms of transport.

Labeling of Q94-TC virus-infected cells also identified a groupof capsid puncta at the PM. Based on the coenrichment of the viralenvelope proteins at these sites and the absence of such PM capsidpuncta in the budding-defective Y400K mutant (data not shown),we propose that they represent assembly/budding sites for alpha-virus particles. Further experiments will address whether the cap-

sid protein is delivered to these sites as preassembled NCs or ascapsid-RNA complexes. FlAsH-based superresolution micros-copy (58) and/or correlative electron microscopy of ReAsH-la-beled capsid (27, 59) may provide the spatial resolution to differ-entiate between these two states of the capsid protein.

G3BP/nsP3-positive capsid structures. Labeling of Q94-TCvirus-infected cells revealed a population of large internal capsidstructures that colocalize with G3BP and nsP3. Immunofluores-cence analysis of WT-infected cells demonstrated similar capsidfoci that were positive for G3BP and nsP3. To our knowledge,while the G3BP/nsP3 foci have been quite extensively studied, thisis the first report that these foci can contain the alphavirus capsidprotein. Further colocalization studies showed that this group ofintracellular capsid foci was negative for viral envelope proteins,nsP1, dsRNA, and eIF3.

dsRNA is a hallmark of plus-strand RNA virus replicationcomplexes (60) and can be detected by staining with the J2 MAb.In SFV-infected BHK cells, replication complexes are first local-ized on regions of the PM (1 to 3 h postinfection), then in smalland scattered cytoplasmic vesicles, and finally (�8 h postinfec-tion) in large perinuclear CPVI vacuolar structures (52, 61). Weobserved cell line differences in this immunofluorescence pattern,detecting large perinuclear dsRNA structures at 8 h postinfectionin SFV-infected BHK cells but not in SFV-infected Vero cells (datanot shown). Similarly, at �6 to 8 h postinfection, SINV-infected

FIG 9 Properties of PM capsid puncta. (A) Generation of PM capsid puncta does not require cytoplasmic NC formation. Vero cells infected with TC-LL for 7 h werelabeled with ReAsH and then fixed, permeabilized, and stained with E2 MAb R6. (B) PM capsid puncta do not colocalize with dsRNA. Vero cells were infected withQ94-TC for 7 h, labeled with ReAsH, and then fixed, permeabilized, and stained with MAb against dsRNA. (C) Newly synthesized capsid protein was delivered topreexisting PM capsid puncta. Vero cells were infected with Q94-TC for 7 h and stained with FlAsH to label the existing capsid protein pool (second column). At theindicated chase time, the cells were labeled with ReAsH (first column). After 1 h of chase, ReAsH-labeled capsid protein was detected in PM capsid puncta containing theFlAsH signal. The inset is a zoomed view of the boxed region (2.5� magnification). Images are representative of two independent experiments. Bar, 10 �M.

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Vero cells showed MAb J2 staining on specific regions of the PMor on small vesicles scattered in the cytoplasm but no large peri-nuclear dsRNA structures then or at later times of infection. ThedsRNA structures in WT- or Q94-TC-infected Vero cells did notcolocalize with capsid MAb or capsid ReAsH staining (data notshown). Thus, it appears that the internal capsid/G3BP foci arenot viral replication complexes.

As part of the host defense against virus infection, cells cantrigger the formation of canonical SGs that sequester translationfactors and inhibit protein synthesis (reviewed in references 62–64). Some viruses have evolved strategies to inhibit or reverse theformation of cellular SGs at later times of infection. For example,the poliovirus 3C protease cleaves G3BP (65), while West Nilevirus sequesters the SG protein TIAR (66). Alphaviruses such asSFV and CHIKV have been reported to reduce the SG responselate in infection by sequestering the G3BP protein into cytoplas-mic foci (49, 50, 52). These G3BP foci do not contain other SGmarkers, such as eIF3 or TIAR; thus, they are not bona fide cellularSGs (50, 52). G3BP sequestration into these cytoplasmic struc-tures is mediated by the alphavirus nsP3 protein, which contains ashort sequence at its C terminus that binds G3BP (50, 52, 67).

At late times of infection a number of RNA viruses, includinghepatitis C virus, SFV, and dengue virus, are observed to induce thedynamic assembly and disassembly of cytoplasmic SGs over a timespan of hours (68). Such oscillations allow cells to cycle betweentranslational activity and arrest, potentially promoting cell survivaland chronic infection (68). Alphaviruses inhibit the formation ofbona fide SGs at about 6 to 8 h postinfection, and the G3BP/nsP3-mediated SG inhibition might reflect the disassembly phase of SGoscillation. As the time-lapse experiments we performed to track themovement of the GBP3/capsid foci were relatively short, it is not clearif there are oscillations in these capsid structures that influence theoscillations in cellular SGs. The overall process of SG disassembly hasbeen shown to occur in cells infected with recombinant alphaviruseslacking the capsid protein (50, 52).

Although G3BP depletion by short interfering RNA produces asmall enhancement of SINV virus production (69), the effect isrelatively modest. This might be explained by the important rolesG3BP plays in mediating both initial SG assembly and subsequentSG disassembly. In contrast, an SINV nsP3 mutant that fails toform nsP3/G3BP cytoplasmic foci or to dissociate SG displays arelatively strong (2 log) decrease in virus titer (67). In this case, therobust inhibition of virus growth could be due to initial SG for-mation in the absence of subsequent SG disassembly. We specu-late that the capsid protein in the nsP3/G3BP foci plays a role inthe complex biology controlling viral replication. Clearly, furtherstudies will be needed to determine the mechanism of capsid pro-tein recruitment to G3BP/nsP3 foci, the potential interactions ofcapsid protein with the components of these foci, and the role(s)of capsid in SG regulation and oscillation.

ACKNOWLEDGMENTS

We thank all members of our laboratory for their helpful discussions andexperimental suggestions and Mathieu Dube, Guadalupe Martinez, andClaudia Sánchez-San Martín for critical readings of the manuscript. Wethank Erik Snapp and Guadalupe Martinez for many helpful discussionsand input on imaging, Youqing Xiang for her excellent technical assis-tance, and the staff of the Einstein Analytical Imaging Facility for theirsupport.

The data in this paper are from a thesis submitted by Y.Z. in partial

fulfillment of the requirements for the Degree of Doctor of Philosophy inthe Graduate Division of Medical Sciences, Albert Einstein College ofMedicine, Yeshiva University.

This work was supported by a grant to M.K. from the National Insti-tute of General Medicine (R01-GM057454) and by Cancer Center CoreSupport Grant NIH/NCI P30-CA13330.

The content of this work is solely the responsibility of the authors anddoes not necessarily represent the official views of the National Institute ofGeneral Medicine or the National Institutes of Health.

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