IMAGING OF THE ALPHAVIRUS CAPSID PROTEIN DURING ...

IMAGING OF THE ALPHAVIRUS CAPSID PROTEIN DURING VIRUS REPLICATION 1 2 3 4

Yan Zheng and Margaret Kielian* 5 Department of Cell Biology, Albert Einstein College of Medicine 6

Bronx, N.Y. 10461, USA 7 8 9 Running Title: Imaging the alphavirus capsid protein 10 11 12 13 *Address correspondence to Margaret Kielian, Department of Cell Biology, Albert Einstein 14 College of Medicine, 1300 Morris Park Ave., Bronx, NY, 10461. Tel.: (718) 430-3638. Fax: 15 (718) 430-8574. E-mail: [email protected]. 16 17

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.01299-13 JVI Accepts, published online ahead of print on 19 June 2013

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ABSTRACT 18 Alphaviruses are enveloped viruses with highly organized structures. The nucleocapsid 19

(NC) core contains a capsid protein lattice enclosing the plus-sense RNA genome, and is 20 surrounded by a lipid bilayer containing a lattice of the E1 and E2 envelope glycoproteins. 21 Capsid protein is synthesized in the cytoplasm and particle budding occurs at the plasma 22 membrane (PM), but the traffic and assembly of viral components and the exit of virions from 23 host cells are not well understood. To visualize the dynamics of capsid protein during infection, 24 we developed a Sindbis virus infectious clone tagged with a tetracysteine motif. Tagged capsid 25 protein could be fluorescently labeled with biarsenical dyes in living cells without effects on 26 virus growth, morphology, or protein distribution. Live cell imaging and co-localization 27 experiments defined distinct groups of capsid foci in infected cells. We observed highly motile 28 internal puncta that co-localized with E2 protein, which may represent the transport machinery 29 that capsid protein uses to reach the PM. Capsid was also found in larger non-motile internal 30 structures that co-localized with cellular G3BP and viral nsP3. Thus capsid may play an 31 unforeseen role in these previously observed G3BP-positive foci, such as regulation of cellular 32 stress granules. Capsid puncta were also observed at the PM. These puncta co-localized with E2 33 and recruited newly synthesized capsid protein, and thus may be sites of virus assembly and 34 egress. Together our studies provide the first dynamic views of the alphavirus capsid protein in 35 living cells and a system to define detailed mechanisms during alphavirus infection. 36 37 38


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INTRODUCTION 39 Enveloped virus budding reactions can take place at a variety of cellular membranes, and 40

may be dependent on the viral nucleocapsid, envelope proteins, and/or matrix proteins (reviewed 41 in 1, 2). The alphaviruses are small enveloped plus sense RNA viruses with highly organized 42 structures (reviewed in 3, 4, 5). Alphaviruses contain an internal core composed of the ~11kb 43 RNA genome enclosed in an icosahedral capsid protein shell. This nucleocapsid (NC) is 44 enveloped by the virus lipid bilayer containing a lattice of the E1 and E2 membrane 45 glycoproteins. Alphavirus budding takes place at the PM and requires both the NC and the 46 envelope proteins (6). The completed viral particle contains 240 copies of each of these 47 structural proteins, with each capsid protein interacting 1:1 with the cytoplasmic domain of an 48 E2 protein (7-9). 49

During infection, the alphavirus genomic RNA is translated to produce the four 50 nonstructural proteins (nsP1-4) that mediate RNA replication, while the structural proteins are 51 produced as a polyprotein from a subgenomic RNA (reviewed in 3, 4, 10). The N-terminal 52 capsid protein contains a protease domain, and once it is translated, it rapidly folds, auto-cleaves 53 itself from the polypeptide and is released into the cytoplasm. The rest of the polyprotein 54 encodes the viral membrane proteins, which are translocated into the endoplasmic reticulum and 55 transported through the secretory pathway to the PM. 56

Two models have been proposed for alphavirus nucleocapsid assembly (reviewed in 11). 57 One model predicts that the NC is preassembled in the cytoplasm and then drives virus budding 58 by binding to the glycoproteins at the PM. This model is supported by the presence of abundant 59 NC in the cytoplasm of infected cells (12) and by the efficient in vitro assembly of NC in the 60 absence of glycoproteins (13). Microinjection of such preformed NCs into cells expressing the 61


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viral envelope proteins can generate infectious virus-like particles, albeit at a relatively low 62 efficiency (14, 15). 63

An alternative model postulates that a capsid-RNA complex binds the E2 cytoplasmic 64 domain at the PM, where the lateral interactions of the glycoproteins drive formation of the 65 icosahedral NC and subsequent virus budding. In support of this model, particle production for 66 capsid mutants defective in cytoplasmic NC formation is only mildly reduced compared to the 67 WT, indicating that performed NCs are not strictly required for virus budding (9, 16-18). A 68 common feature of both models is that the cytoplasmic NC or the capsid-RNA complex must be 69 transported to the PM. Based on its high protein concentration and extensive cytoskeletal 70 network, the cytoplasmic milieu will greatly restrict the free diffusion of the capsid/NC (19), but 71 potential transport mechanisms are undefined. 72

Early studies of the kinetics of alphavirus particle production indicated that only a 73 fraction of the cellular pool of capsid protein is ultimately released in virus particles (20). 74 Nascent capsid protein can associate at least transiently with ribosomes in infected cells (21-24). 75 Later in infection some capsid proteins associate with the cellular adaptor protein p62, which 76 mediates capsid targeting to autophagosomes (25, 26). It is not clear how or where the remaining 77 capsid proteins might accumulate in the host cell, whether they associate with specific cellular 78 proteins or trigger host cell responses, or what other additional functions the capsid protein may 79 have. 80

Here we set out to characterize the dynamics of the alphavirus capsid protein during virus 81 replication by visualizing the protein in its cellular context in live infected cells. We took 82 advantage of the tetracysteine (TC) motif, a 12 amino acid sequence that can be labeled with 83 high affinity and specificity by biarsenical dyes such as FlAsH and ReAsH (27). Using a Sindbis 84


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virus (SINV) infectious clone, we defined a favorable insertion site for the TC motif on the viral 85 capsid protein, and optimized the system to follow FlAsH- or ReAsH-labeled capsid in real-time. 86 Our studies identified three categories of capsid foci in SINV-infected Vero cells. One group was 87 comprised of small, round internal puncta that were highly motile and co-localized with the E2 88 protein, suggesting a role in transport. A second group consisted of larger more irregular internal 89 structures that co-localized with G3BP and nsP3, two proteins previously shown to interact and 90 regulate stress granule formation. The third group was composed of capsid puncta that co-91 localized with the E2 envelope protein at the PM, suggesting their involvement in the 92 assembly/exit of progeny viruses. 93 94 MATERIALS AND METHODS 95

Cells. BHK-21 cells were cultured at 37°C in complete BHK medium (Dulbecco’s 96 modified Eagle’s medium containing 5% fetal calf serum, 10% tryptose phosphate broth, 100U 97 penicillin/ml, and 100μg streptomycin/ml). Vero cells from ATCC were kindly provided by Dr. 98 Kartik Chandran and cultured in complete Vero medium (Dulbecco’s modified Eagle’s medium 99 containing 10% fetal bovine serum, 100U penicillin/ml, and 100μg streptomycin/ml) at 37°C. 100

Construction of SINV infectious clones. The WT SINV and mCherry SINV used in our 101 studies were derived from the WT dsTE12Q infectious clone (28) and a dsTE12Q clone 102 containing mCherry at the N-terminus of capsid (25), both kindly provided by Dr. Beth Levine. 103 We constructed GFP-dsTE12Q and Toto1101 (29) clones containing superfolder GFP (sfGFP) at 104 the N-terminus of capsid. This insertion was generated using a three-step overlap-extension PCR 105 with sfGFP-C1 (30) (kindly provided by Dr. Erik Snapp) to amplify sfGFP, and dsTE12Q or 106 Toto1101 to amplify the SINV capsid region. The fragment containing sfGFP-capsid was 107


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subcloned into dsTE12Q or Toto1101 infectious clone using HpaI/AatII restriction endonuclease 108 sites. 109

TC tag insertions in capsid protein were generated by overlapping PCR using primers 110 encoding the optimized TC sequence FLNCCPGCCMEP (27). The fragment containing the TC 111 insertion at the N-terminus of the capsid was subcloned into the dsTE12Q infectious clone using 112 the HpaI/AatII restriction sites, and fragments with TC inserted at other sites were subcloned 113 using the HpaI/BclI sites. TC-LL [capsid Q94-TC + capsid L108A/L110A] was constructed 114 using overlapping PCR using Q94-TC dsTE12Q as a template, and primers containing the capsid 115 L108A and L110A mutations (9, 16). The fragment containing Q94-TC insertion and capsid 116 L108A/L110A mutation was subcloned into dsTE12Q using the HpaI/BclI sites. TC Y400K 117 [capsid Q94-TC + E2 Y400K] was constructed by subcloning the E2 Y400K-containing 118 PmlI/BclI fragment from Y400K-dsTE12Q (G. Martinez, unpublished data) into Q94-TC 119 dsTE12Q. 120

Sequence analysis (Genewiz Inc., North Brunswick, NJ) of the entire HpaI/AatII or 121 HpaI/BclI PCR regions confirmed that the TC insertions were in place without additional 122 mutations introduced during PCR amplification. For each insertion mutant, two independent 123 clones were tested to confirm the phenotype. 124

Production and titration of virus stocks. Viral RNAs were in vitro transcribed from the 125 XhoI-linearized SINV infectious clone, and were electroporated into BHK-21 cells to generate 126 virus stocks (31). All mutant virus stocks were prepared by incubation of electroporated cells for 127 12h at 37°C to prevent possible generation of revertants during extended culture time. 128

Virus titers were measured as indicated by plaque titration on BHK-21 cells or by 129 infectious center (IC) assays in BHK-21 cells or Vero cells. For infectious center assays, cells 130


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were infected with serial dilutions of SINV virus for 1.5h at 37°C. Media were replaced with 131 complete growth media containing 20 mM NH4Cl to prevent secondary infection, and incubated 132 at 28°C overnight to allow viral protein expression. Infected cells were quantitated by 133 immunofluorescence using mAbs R2 and R6 to the E1 and E2 proteins, respectively (kindly 134 provided by Dr. William Klimstra, reference 32) 135

Virus assembly assay. The assembly of mutant viruses was evaluated by pulse-chase 136 analysis (33). The viral RNAs were introduced into BHK-21 cells by electroporation. After 6 h 137 incubation at 37°C, cells were pulse-labeled with 50 μCi/ml [35S]-methionine/cysteine (Express 138 Labeling Mix; Perkin Elmer Life and Analytical Sciences) at 37°C for 30 min and chased for 0-3 139 h at 37°C in the absence of radiolabel. The media samples were collected, immunoprecipitated 140 with mAb R6 against E2 (32) in the absence of detergent and analyzed by SDS-PAGE. Cell 141 lysates were collected in the presence of 40μM N-ethylmaleimide to alkylate free cysteine in the 142 TC tag, and aliquots of the lysates were analyzed directly by SDS-PAGE. 143

Electron microscopy of virus-infected cells. BHK-21 cells were electroporated with 144 WT or mutant RNA, plated on 35-mm diameter dishes, and incubated at 37°C for 12 h before 145 fixing with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate buffer for 146 30 min at room temperature. Samples were then processed by the Einstein Analytical Imaging 147 Facility by postfixing with 1% osmium tetroxide followed by 2% uranyl acetate, dehydration, 148 and embedding in LX112 resin (LADD Research Industries, Burlington VT). Thin sections were 149 stained with uranyl acetate followed by lead citrate and examined on a JEOL 1200EX or a JEOL 150 100CXII electron microscope at 80 kv. Images were recorded at a magnification of 20,000X and 151 assembled with the software Adobe Photoshop CS. 152


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Tetracysteine labeling with biarsenical dyes. Vero cells were plated on 8-well Lab-153 TekTM II chambered coverglass (Thermo Scientific), incubated at 37°C overnight, and then 154 infected with WT or TC-tagged SINV at a multiplicity of 0.5 IC/cell. After 2 h at 37°C, the 155 medium was replaced with Vero complete medium and the incubation continued for another 5 h 156 at 37°C to allow the expression of capsid proteins before labeling with biarsenical dyes. For the 157 budding-negative Y400K mutant, Vero cells were transfected with viral RNA and incubated at 158 37°C for 7 h. Labeling protocols were modified from those published (34, 35), with additional 159 helpful suggestions from Drs. Brett Lindenbach and Eric Freed. Cells were incubated in 2.5 μM 160 FlAsH (or ReAsH) for 5 min at 37°C, and washed three times at 37°C with DMEM containing 161 1mM 2,3-dimercapto-1-propanol (British anti-Lewisite [BAL]; Sigma) (34). Live cell imaging 162 was then performed as described below. Alternatively, cells were fixed with 3% 163 paraformaldehyde for immunofluorescence experiments. The time course of infection of the 164 Vero cell culture (and thus the cellular staining patterns) were not fully synchronous, even if the 165 viral innoculum was prebound to the cells in the cold (data not shown). This is likely due to the 166 effects of the local population context including cell density (as described in 36). 167

For FlAsH/ReAsH pulse-chase labeling, cells were labeled with 5 μM FlAsH for 20 min 168 at 37°C. After one wash with DMEM, cells were incubated with Vero growth media for 0-1 h at 169 37°C, then labeled with 2.5 μM ReAsH as described above. 170

Immunofluorescence analysis. To stain for the capsid or cellular marker proteins, Vero 171 cells were infected or transfected as above, incubated at 37°C for 7 h, fixed with 3% 172 formaldehyde, and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. 173 Antibodies used for immunofluorescence included mouse anti-capsid monoclonal antibody 174 (mAb) C12-1 (kindly provided by Dr. Irene Greiser-Wilke, reference 37), rabbit antibodies to 175


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SINV nsP1 and nsP3 (kindly provided by Drs. Margaret MacDonald and Charles Rice, reference 176 38), rabbit anti-LC3 (PM036, MBL International Corporation), rabbit anti-G3BP (anti-G3BP1, 177 G6046, Sigma), mouse anti-dsRNA mAb J2 (English & Science Consulting Kft.), and goat anti-178 eIF3 (sc-16377, Santa Cruz Biotechnology, Inc.). SINV E2 was detected using mouse anti-E2 179 mAb R6 and E1 was detected using mAb R2 (32). Preliminary studies showed that R6 180 recognized the ectodomain of the E2 protein on intact virus but not in cell lysates or detergent-181 solubilized viruses (data not shown), suggesting that the epitope correlates with trimer or lattice 182 assembly. Secondary antibodies were Alexa 488- or 568-conjugated (Molecular Probes). For live 183 cell staining, Vero cells were incubated for 30 min at 37°C with the R6 mAb, placed on ice, 184 washed and incubated with secondary antibody on ice, and fixed with paraformaldehyde on ice 185 for 30 min before imaging. Images were captured using a confocal microscope system (Duoscan; 186 Carl Zeiss MicroImaging, Inc.) with a 63X/1.4 NA oil objective and a 489-nm 100-milliwatt 187 diode laser with a 500-550-nm band pass filter for FlAsH or Alexa 488 and a 40-milliwatt 561-188 nm diode laser with a 580-nm long pass filter for ReAsH or Alexa 568. All the images were 189 prepared with Adobe Photoshop CS4 software (Adobe System, San Jose, CA). 190

Live cell microscopy. Live cells were imaged in imaging media (DMEM w/o phenol red 191 or bicarbonate (D2902 Sigma), 10mM Hepes, pH 7, 10% fetal bovine serum, 100U penicillin/ml, 192 and 100μg streptomycin/ml) using the 37°C environmentally-controlled chamber of the Duoscan 193 confocal microscope system described above. Images were prepared with Volocity 3D Image 194 Analysis Software (PerkinElmer), Adobe Photoshop CS4 and Illustrator CS4 software (Adobe 195 System, San Jose, CA). 196 197 198


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RESULTS 199 Development of functional SINV capsid protein with a fluorescent tag. To visualize 200

the movements of the alphavirus capsid protein during virus replication and assembly, we 201 developed methods to fluorescently label and image capsid protein. SINV was used as a model 202 alphavirus, and our constructs were based on the SINV infectious clone dsTE12Q. 203

We first tested a previously described virus containing mCherry at the N-terminus of the 204 capsid protein (mCherry-dsTE12Q) (25, 26). We also constructed a similar virus in which 205 superfolder GFP was inserted at the capsid N-terminus (GFP-dsTE12Q). BHK cells were 206 electroporated with in vitro transcribed viral RNAs and progeny virus titers measured. The titers 207 of both mCherry-dsTE12Q and GFP-dsTE12Q were about 2 logs lower than those of the 208 unlabeled WT virus at 24 h post electroporation (data not shown). Transmission electron 209 microscopy (EM) of virus-infected BHK cells showed WT SINV budding at the cell surface as 210 spherical particles containing a dense core in the center (as shown below). In contrast, cells 211 infected with GFP-dsTE12Q (as shown below) or mCherry-dsTE12Q (data not shown) produced 212 larger aberrant particles containing multiple patches of dense core material. In addition, 213 immunostaining with an antibody against the capsid protein showed that cells infected for 7 h 214 with GFP- or mCherry-dsTE12Q virus contained unique cytoplasmic patches of fluorescence 215 that were not observed in cells infected with WT SINV (data not shown). Given that the sizes of 216 both GFP and mCherry are similar to those of the capsid protein (247, 250 and 264 amino acids, 217 respectively), it seemed likely that GFP or mCherry interfered with the assembly of viral 218 nucleocapsid and/or caused capsid protein aggregation. 219

As an alternative strategy, we tested insertion of the TC motif and its labeling with 220 biarsenical dyes (27, 39). This system has been successfully used to study the trafficking of the 221


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human immunodeficiency virus (HIV) GAG protein, the hepatitis C virus core protein, and the 222 vesicular stomatitis virus M protein (34, 35, 40-42). A TC motif introduced at the N-terminus of 223 the SINV capsid protein appeared to be rapidly lost during virus infection as detected by 224 migration on SDS-PAGE (data not shown). Insertion of a flexible linker between the TC tag and 225 the N-terminus of the capsid protein stabilized the insertion, but the TC-tagged capsid protein 226 was not successfully labeled by biarsenical dye treatment of infected cells (data not shown). This 227 presumably reflects limited accessibility of the TC motif in the folded/assembled capsid protein. 228 The alphavirus capsid protein assembles into NC with its N-terminal region located internally in 229 association with the viral RNA (43) and its C-terminal protease domain arrayed on the NC 230 surface (44). We therefore narrowed our target site selection to the C-terminal domain and the 231 region close to it in the hope of obtaining better dye accessibility. Within those regions of the 232 capsid protein, we selected specific sites (Table 1) that appeared more likely to tolerate insertions 233 based on the results of a whole genome transposon-mediated insertion screen of the alphavirus 234 Venezuelan equine encephalitis virus (45). Based on the structure of the SINV capsid protease 235 domain (46), our selected sites were all localized at the tips of flexible loops, in keeping with 236 their potential to tolerate a small insertion (data not shown). A series of TC-tagged SINV strains 237 was constructed and the viral growth kinetics were compared to those of WT SINV (Table 1). 238 Most viruses with a TC tag inserted within the C-terminal capsid protease domain produced viral 239 titers at least 2 logs lower than that of WT SINV. The single exception was D113-TC, which 240 grew to a titer only 1 log lower than that of WT virus. D113 is located at the junction between 241 the N-terminal and the C-terminal regions of the capsid protein. Insertion sites just N-terminal to 242 this junction (E89, Q94, K97 and G101) proved more amenable to the TC tag, with all of the 243 resultant viruses showing comparable growth to WT SINV (Table 1). All of the TC-tagged 244


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viruses that grew to within 1 log of the WT titer (D113-TC, E89-TC, Q94-TC, K97-TC, and 245 G101-TC) were then tested for their labeling intensity with the biarsenical dyes, FlAsH or 246 ReAsH. Among these, K97- and Q94-TC showed the brightest signal. Q94-TC was selected for 247 further studies. 248

Characterization of Q94-TC virus. BHK-21 cells were electroporated with WT or Q94-249 TC viral RNA and the kinetics of progeny virus production determined (Fig. 1A). Efficient 250 production of both viruses was observed by 6 h post-electroporation, and titers reached maximal 251 levels by ~14 h. Similar results were observed when the progeny viruses were titered using an 252 infectious center assay on Vero cells, the cell line used for our imaging studies (data not shown). 253 The growth kinetics of Q94-TC were therefore comparable to those of WT SINV. 254

The assembly of virus particles was evaluated by pulse-chase analysis of BHK-21 cells 6 255 h after RNA electroporation. Cells infected with Q94-TC or WT virus produced comparable 256 amounts of viral proteins (Fig. 2, lysate samples). The E2 precursor pE2 was clearly detected at 257 the 0 h chase time, and comparable maturation to E2 was observed in WT vs. Q94-TC-infected 258 cells during a 3 h chase. Analysis of media samples indicated that the budding efficiencies of 259 Q94-TC and WT SINV were comparable. The capsid protein in both Q94-TC media and lysate 260 samples migrated as a single band that ran slightly above the position of WT SINV capsid (Fig. 261 2), consistent with the stable incorporation of the 12-amino acid TC motif in Q94-TC. 262

The morphology of Q94-TC virus particles was evaluated by transmission EM of infected 263 BHK cells at 12 h post-electroporation (Fig. 3). Abundant Q94-TC virus particles were observed 264 budding at the plasma membrane. Their morphology was comparable to that of WT virus, with a 265 particle diameter of about 70 nm and a dense NC core in the center (Fig. 3A vs. 3C). In contrast, 266 the GFP- or mCherry-capsid viruses both produced aberrant particles (e.g., Fig. 3E, and data not 267


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shown). Typical membranous replication structures termed CPVI (3, 47) were detected within 268 WT and Q94-TC-infected cells (data not shown). In addition, both WT and Q94-TC-infected 269 cells contained CPVII with associated NC (Fig. 3B and 3D). Together all the evidence indicated 270 that the introduction of a TC tag at the capsid Q94 position did not cause detectable effects on 271 virus replication or progeny virus production. 272

ReAsH labeling specificity and effects of ReAsH labeling on virus assembly. We next 273 tested the specificity of ReAsH labeling and its effects on virus production. Vero cells were 274 infected with Q94-TC or WT viruses for 7 h and stained with ReAsH as described in the 275 methods. Vero cells were chosen for imaging given their efficient infection by SINV and flat 276 morphology. After labeling the cells were fixed, permeabilized, and stained with a mAb against 277 the capsid protein (Fig. 4). No ReAsH signal was detected in WT SINV-infected cells while in 278 Q94-TC-infected cells clear intracellular staining was observed. The distribution pattern of the 279 ReAsH staining in Q94-infected cells co-localized with the signal from the mAb against capsid 280 protein. Thus, the Q94-TC motif was specifically labeled by the ReAsH dye with minimal 281 background, and accurately reflected the distribution of capsid protein in infected cells. Similar 282 results were obtained with FlAsH dye labeling (data not shown). 283

We then measured the titers of Q94-TC progeny viruses collected at different time points 284 following ReAsH labeling or mock-treatment. Similar titers were detected in the ReAsH vs. 285 mock-ReAsH-treated samples (Fig. 1B), indicating that the ReAsH treatment and the binding of 286 ReAsH dye to the capsid protein had minimal effect on the production of infectious virus 287 particles. 288

Distribution of the capsid protein in infected cells. We then used dye labeling to 289 follow the distribution of the capsid protein after various times of infection. No significant 290


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staining of Q94-TC cells was detectable during the first ~5 h of infection (data not shown). After 291 5.5-6 h of infection, Q94-TC-infected cells showed relatively weak ReAsH staining that was 292 diffusely distributed in the cytoplasm (data not shown). By 7-8 h post-infection, some cells still 293 showed weak ReAsH staining but the majority of the cells contained a much brighter ReAsH 294 capsid signal. This staining was distributed diffusely in the cytoplasm as before, but also in 295 distinct foci as discussed in detail below and documented in Fig. 5. The absence of clear capsid 296 foci at earlier times of infection could reflect their formation at a certain point in the infection 297 cycle, or simply be due to their intensities lying below the detection limit compared to the 298 surrounding cytoplasmic capsid signal. 299

We performed a detailed analysis of the intracellular capsid foci by confocal microscopy 300 of Vero cells infected for 7-8 h, approximately at the start of exponential virus production (as 301 shown in Fig. 1). Q94-TC-infected cells were labeled with ReAsH at 7 h post-infection, fixed, 302 permeabilized and stained with mAb R6 against the SINV E2 protein (32). The cytoplasmic 303 capsid foci were separated into three groups based on their size, shape, approximate cellular 304 location and co-localization with E2. The first category of capsid foci (termed “small internal 305 capsid puncta”) (Fig. 5A) co-localized with E2 in internal regions of the cell, and were small 306 (0.1-0.5 µm3) and round in shape. In live cells, the small internal capsid puncta were highly 307 motile (data not shown). As they contain both capsid and E2 protein, we hypothesize that these 308 puncta are vesicles that transport both capsid and envelope proteins. Detailed studies using a 309 virus labeled in both the capsid and E2 protein are currently in progress to characterize the role 310 of these puncta in virus biogenesis. 311

A second category of capsid foci in internal regions of the cell (termed “ irregular internal 312 capsid structures”) (Fig. 5B) were larger (0.6-3.0 µm3) and more irregularly shaped than the first 313


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group, and showed no detectable E2 protein. Most of the cells that were positive for capsid foci 314 contained both the small internal puncta and the irregular internal structures. The frequency of 315 small internal capsid puncta was 5.2 per cell (SD 5.3, n=20 cells), which was not significantly 316 different (p>0.05, student’s t-test) from the frequency of the irregular internal capsid structures 317 (8.0 per cell, SD 5.0, n=20 cells). Given the frequencies, these classes of capsid foci were not 318 always in the same confocal z-section. The third group of capsid foci (Fig. 5C) was observed as 319 many discrete puncta at a focal plane that appeared close to the PM by confocal microscopy. 320 This class of foci (termed “PM-proximal capsid puncta”) also co-localized with E2, and was 321 detected in all capsid foci-containing cells. 322

The irregular internal capsid structures co-localize with G3BP and nsP3. 323 Immunofluorescence analysis demonstrated that, unlike the other two classes of capsid foci, the 324 irregular internal capsid structures did not colocalize with either the E2 or E1 envelope proteins 325 (Fig. 5B and data not shown). We therefore evaluated cellular markers that might be 326 characteristic of this group of capsid foci. The structures did not show co-localization with p115, 327 a marker of the Golgi complex, or with LC3, a marker of autophagosomes (data not shown). 328 However, our studies showed that all of the irregular internal capsid foci were strongly positive 329 for the Ras-GAP SH3 domain binding protein G3BP (Fig. 6A). G3BP, a multifunctional RNA 330 binding protein, has been reported to be a key factor in the nucleation of cellular stress granules 331 (SGs) and is activated by dephosphorylation during SG formation (48). To determine if the 332 irregular internal capsid structures also contained other SG markers such as eukaryotic 333 translation initiation factor 3 (eIF3), we tested the co-localization of ReAsH-labeled capsid 334 protein and mAb-stained eIF3. Control cells in which SGs were chemically induced by sodium 335 arsenite treatment (49, 50) displayed eIF3-positive puncta (data not shown). In contrast, eIF3 was 336


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diffusely distributed in the cytoplasm of SINV-infected cells and did not co-localize with any of 337 the three types of capsid structures (Fig. 6A, lower panel). Consistent with this result, when Q94-338 TC- or WT-infected cells were stained with the anti-capsid mAb, the irregular internal capsid 339 structures co-localized with G3BP but not with eIF3 (Fig. 6C and data not shown). Together our 340 marker analysis suggested that the capsid-G3BP foci are not typical SGs or autophagosomes. We 341 do observe autophagosome induction and LC3-Q94 capsid co-localization at later times of 342 infection (data not shown), in agreement with the studies of Orvedahl et al. (25). 343

Earlier studies reported that typical SGs are induced at early times (~2-4 h) of Semliki 344 Forest virus (SFV) infection and are disassembled at later times of infection when the cells 345 become positive for viral gene expression (49). Studies with SINV, Chikungunga virus (CHIKV) 346 and SFV showed that the viral protein nsP3 interacts with G3BP, sequesters it into cytoplasmic 347 foci and inhibits the formation of bona fide SGs (38, 50-52). We therefore tested the co-348 localization of the irregular internal capsid structures with nsP3 and dsRNA, a marker for the 349 viral replication complex. Approximately 80% of the dsRNA foci in SINV-infected Vero cells 350 were associated with the PM at this infection time, with some small dsRNA puncta in the 351 cytoplasm (Fig. 6A). However, none of the dsRNA foci co-localized with the capsid protein. The 352 irregular internal capsid structures were also negative for nsP1 (data not shown) and thus 353 together these results suggest the structures were not associated with the viral replication 354 complex. In contrast, the nsP3 protein was detected in the irregular internal capsid foci (Fig.6B), 355 suggesting that these structures represent the previously observed nsP3/G3BP foci reported to 356 function in inhibiting formation of bona fide SG (50, 52). 357

The role of NC assembly and E2 interactions in the generation of capsid protein-G3BP 358 structures was determined by engineering additional mutations into SINV Q94-TC. The capsid 359


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substitutions L108A/L110A inhibit the formation of the cytoplasmic NC and CPVII but still 360 allow virus budding at the PM (9, 16). Vero cells infected with TC-LL showed somewhat more 361 irregular internal capsid structures than Q94-TC (33.0 per cell, SD 8.2, n=20 cells), but clear co-362 localization with G3BP was observed (Fig. 6C). Thus, cytoplasmic NC formation is not required 363 for formation of these structures. The substitution Y400K in the cytoplasmic tail of E2 blocks 364 E2-capsid interaction, formation of CPVII, NC localization at the PM, and virus budding (7). 365 However, cells infected with TC-Y400K showed irregular capsid structures that colocalized with 366 G3BP (Fig. 6C), demonstrating that interaction with the E2 protein was not necessary for 367 formation of the capsid/G3BP foci. Vero cells infected with the alphaviruses SFV and CHIKV 368 also express capsid/G3BP foci in the cytoplasm (data not shown), suggesting that formation of 369 these structures is conserved among alphaviruses. 370

Dynamics of the irregular internal capsid structures. The TC-tagged virus made it 371 possible to track the movement of the irregular internal capsid structures by ReAsH labeling of 372 Q94-TC-infected cells and live cell imaging. As shown by a representative time series (Fig. 7A), 373 this population of capsid foci was relatively immobile. Similar results were observed in cells 374 infected with the TC-LL or TC-Y400K mutants (data not shown). 375

We then wished to address whether these immobile structures can still actively recruit 376 new capsid proteins. This question could be addressed by testing for an increase of fluorescence 377 intensity with time. However, the ReAsH dye is not very photo-stable, and the gradual bleaching 378 of fluorescence even using low laser power and short exposure times made it complicated to 379 directly quantify changes in fluorescence intensity (data not shown). Alternatively, fluorescence 380 recovery after photobleaching (FRAP) experiments could be used to follow delivery of labeled 381 capsid protein. This system requires that the high-power laser photobleach is irreversible, thus 382


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allowing fluorescence recovery exclusively from the unbleached capsid pool. In our system, 383 bleaching of the ReAsH dye was reversible and ReAsH signal rapidly re-appeared after a 384 complete photobleach of the whole cell (data not shown). 385

We therefore used FlAsH/ReAsH pulse-chase experiments to follow the newly 386 synthesized capsid protein. Cells were infected for 6.5 h with Q94-TC and pulse-labeled with 387 FlAsH to saturate the available TC sites. After incubation in growth media at 37 °C for 30 min 388 (“chase”), the cells were treated with ReAsH to label the newly synthesized capsid protein. No 389 ReAsH labeling was detected in the absence of chase, confirming that the FlAsH label is 390 complete (Fig. 7B upper panel). In contrast, ReAsH-labeling was readily detected in the cells 391 following a 30 min chase (Fig. 7B lower panel). In the chase samples the irregular internal capsid 392 structures were labeled with both FlAsH and ReAsH, suggesting that newly synthesized capsid 393 was being delivered into pre-existing capsid/G3BP/nsP3 structures. Thus, although these 394 structures were relatively immobile they were dynamic in acting as delivery sites for newly 395 synthesized capsid protein. 396

Characterization of capsid puncta at the plasma membrane. As shown in Fig. 5C, 397 distinct capsid puncta that colocalized with E2 were detected close to the PM, and were 398 particularly clear when focusing at the baso-lateral region of the cells. To address whether these 399 PM-proximal capsid puncta are actually localized to the PM, we labeled Q94-TC-infected Vero 400 cells with ReAsH and then performed immunostaining on ice to specifically detect the PM-401 localized pool of E2. Under these conditions, E2 was not detected inside the cells (Fig. 8A), 402 confirming that only the PM pool was visualized. Strong co-localization of capsid ReAsH signal 403 and E2 protein was observed at the surface of infected cells. We will refer to this group of capsid 404


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structures as “ PM capsid puncta” from this point on in the text. Live cell imaging showed that 405 most of the PM capsid puncta were relatively immobile (Fig. 8B). 406

Tests of TC-LL-infected cells (Fig. 9A) showed that the formation of the PM capsid 407 puncta did not require cytoplasmic NCs. In contrast, PM capsid puncta were not detected in TC-408 Y400K infected cells, indicating that the E2-capsid interaction was critical (data not shown). Our 409 earlier data showed that at this time of infection in Vero cells, the majority of dsRNA-positive 410 puncta were associated with the PM (Fig. 6A). While we readily detected both dsRNA puncta 411 and PM capsid puncta at the baso-lateral membrane of infected cells, no co-localization was 412 observed (Fig. 9B). Thus, the capsid puncta at the PM were not associated with the viral RNA 413 replication complex. 414

We then used FlAsH/ReAsH pulse-chase experiments as described above to address 415 whether the PM capsid puncta can actively recruit newly synthesized capsid protein. As early as 416 1 h after chase, newly synthesized capsid proteins were detected in PM puncta that were also 417 labeled with the FlAsH signal (Fig. 9C). Based on their enrichment for viral structural proteins 418 and recruitment of newly synthesized capsid protein, our results suggest that the PM capsid 419 puncta are sites of virus assembly and budding. 420 421 DISCUSSION 422

In summary, we developed a TC-based system to image the movements of the SINV 423 capsid protein in live infected cells without affecting its biological activity. At early times of 424 infection, labeling of the TC-capsid produced diffuse cytoplasmic staining that may include 425 ribosome-bound capsid proteins. By the beginning of exponential virus production, three distinct 426 types of intracellular capsid foci were detected. Based on properties including motility and co-427


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localization with viral and cellular proteins, we hypothesize that these foci are involved in capsid 428 protein delivery to the PM, in the regulation of cellular SGs, and in virus assembly/exit from the 429 PM. 430

Fluorescent labeling of the alphavirus capsid protein. The alphavirus NC has a 431 diameter of ~400Å and contains 240 copies of the ~30kDa capsid protein arranged in a T=4 432 icosahedral lattice (53, 54). Given this organized NC structure, introducing the ~27kDa, 42Å 433 long β-barrel structure of GFP or mCherry (55) into the complete capsid protein is spatially 434 challenging. Insertion of GFP or mCherry at the N-terminus of the capsid protein did produce 435 infectious virus, but its growth was reduced by ~2 logs, and both constructs produced aberrant 436 particle morphology and capsid protein distribution. Aberrant budding of HIV GPF-Gag is 437 rescued by co-expression of WT-Gag (56, 57). We tested an analogous complementation strategy 438 for rescue of the budding defects in SINV GFP-capsid. We co-expressed the GFP- or mCherry-439 SINV infectious clone with the WT capsid protein, reasoning that the incorporation of WT 440 capsid might reduce the spatial hindrance in NC formation. However, even though WT capsid 441 protein was well expressed and displayed a normal cellular distribution pattern, it was not 442 efficiently recruited into the GFP- or mCherry-labeled virus (data not shown). The highly 443 symmetric structure of the alphavirus NC and/or the possible importance of cis-expression of 444 capsid and envelope proteins may limit such complementation approaches. We conclude that the 445 GFP- or mCherry-capsid virus does not accurately reflect the complete WT capsid pathway and 446 is not optimal for imaging studies of capsid dynamics. However, such GFP or mCherry-capsid 447 viruses have clearly been very useful tools to identify specific alphavirus-host protein 448 interactions that confirm using WT SINV (25, 26). 449


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We chose the small TC motif as an alternative strategy for live cell imaging of the 450 alphavirus capsid protein. Virus particles in the medium of ReAsH-labeled WT or Q94-TC-451 infected cells could be captured on poly-L-lysine-coated culture slides (data not shown). Both 452 WT and Q94-TC produced particles that labeled with mixtures of mAbs against the E1 and E2 453 proteins. ReAsH-labeled particles were only detected in the Q94-TC sample, and the number of 454 particles was dependent on the multiplicity of infection and time of incubation after labeling. 455 This result indicates that the Q94-TC virus supports imaging of both intracellular and particle-456 assembled capsid protein. Q94-TC virus particles did not show labeling when stained with 457 ReAsH after absorption to coverslips (data not shown), presumably reflecting inhibition of free 458 diffusion of the biarsenical dye into the Q94-TC site in assembled virus. 459

Capsid protein traffic and NC production. While the alphavirus envelope proteins use 460 the endogenous cellular secretory pathway for delivery to the PM, the traffic of the capsid 461 protein is not clear. We observed a diffuse distribution of capsid in the cytoplasm, which could 462 provide a local source during virus budding. Our labeling studies also identified small highly 463 motile capsid puncta, which we hypothesize are capsid transport vehicles. Immunofluorescence 464 experiments showed that this class of capsid foci co-localized with the E2 protein, suggesting 465 their vesicular nature. Further characterization is necessary to determine if these puncta deliver 466 the capsid/NC to the PM and what mediates their rapid movement. We are currently analyzing 467 their properties using a virus containing both the TC-94 capsid and a GFP-E2 protein, which 468 permits live cell imaging of both proteins. If the capsid/NC does travel with the E2 protein, it 469 will be interesting to explore the cellular location where this E2-capsid interaction initiates and 470 the specific mechanisms of transport. 471


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Labeling of Q94-TC virus-infected cells also identified a group of capsid puncta at the 472 PM. Based on the co-enrichment of the viral envelope proteins at these sites and the absence of 473 such PM capsid puncta in the budding-defective Y400K mutant (data not shown), we propose 474 that they represent assembly/budding sites for alphavirus particles. Further experiments will 475 address whether the capsid protein is delivered to these sites as pre-assembled NCs or as capsid-476 RNA complexes. FlAsH-based super-resolution microscopy (e.g., 58) and/or correlative electron 477 microscopy of ReAsH-labeled capsid (27, 59) may provide the spatial resolution to differentiate 478 between these two states of the capsid protein. 479

G3BP/nsP3-positive capsid structures. Labeling of Q94-TC virus-infected cells 480 revealed a population of large internal capsid structures that co-localize with G3BP and nsP3. 481 Immunofluorescence analysis of WT-infected cells demonstrated similar capsid foci that were 482 positive for G3BP and nsP3. To our knowledge, while the G3BP/nsP3 foci have been quite 483 extensively studied, this is the first report that these foci can contain the alphavirus capsid 484 protein. Further co-localization studies showed that this group of intracellular capsid foci was 485 negative for viral envelope proteins, nsP1, dsRNA, and eIF3. 486

DsRNA is a hallmark of plus-strand RNA virus replication complexes (60) and can be 487 detected by staining with the J2 mAb. In SFV-infected BHK cells, replication complexes are first 488 localized on regions of the PM (1-3 h post-infection), then in small and scattered cytoplasmic 489 vesicles, and finally (~8 h post-infection) in large perinuclear CPVI vacuolar structures (52, 61). 490 We observed cell line differences in this immunofluorescence pattern, detecting large perinuclear 491 dsRNA structures at 8h post-infection in SFV-infected BHK cells but not in SFV-infected Vero 492 cells (data not shown). Similarly, at ~6-8 h post-infection SINV-infected Vero cells showed mAb 493 J2 staining on specific regions of the PM or on small vesicles scattered in the cytoplasm, but no 494


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large perinuclear dsRNA structures then or at later times of infection. The dsRNA structures in 495 WT or Q94-TC-infected Vero cells did not co-localize with capsid mAb or capsid ReAsH 496 staining (data not shown). Thus, it appears that the internal capsid/G3BP foci are not viral 497 replication complexes. 498

As part of the host defense against virus infection, cells can trigger the formation of 499 canonical SGs that sequester translation factors and inhibit protein synthesis (reviewed in 62, 63, 500 64). Some viruses have evolved strategies to inhibit or reverse the formation of cellular SGs at 501 later times of infection. For example, the poliovirus 3C protease cleaves G3BP (65), while West 502 Nile virus sequesters the SG protein TIAR (66). Alphaviruses such as SFV and CHIKV have 503 been reported to reduce the SG response late in infection by sequestering the G3BP protein into 504 cytoplasmic foci (49, 50, 52). These G3BP foci do not contain other SG markers such as eIF3 or 505 TIAR, and thus are not bona fide cellular SGs (50, 52). G3BP sequestration into these 506 cytoplasmic structures is mediated by the alphavirus nsP3 protein, which contains a short 507 sequence at its C-terminus that binds G3BP (50, 52, 67). 508

At late times of infection a number of RNA viruses including hepatitis C virus, SFV and 509 dengue virus are observed to induce the dynamic assembly and disassembly of cytoplasmic SGs 510 over the time span of hours (68). Such oscillations allow cells to cycle between translational 511 activity and arrest, potentially promoting cell survival and chronic infection (68). Alphaviruses 512 inhibit the formation of bona fide SGs at about 6-8 h post infection, and the G3BP/nsP3-513 mediated SG inhibition might reflect the disassembly phase of SG oscillation. As the time-lapse 514 experiments we performed to track the movement of the GBP3/capsid foci were relatively short, 515 it is not clear if there are oscillations in these capsid structures that might influence the 516


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oscillations in cellular SGs. The overall process of SG disassembly has been shown to occur in 517 cells infected with recombinant alphaviruses lacking the capsid protein (50, 52). 518

Although G3BP depletion by siRNA produces a small enhancement of SINV virus 519 production (69), the effect is relatively modest. This might be explained by the important roles 520 G3BP plays in mediating both initial SG assembly and subsequent SG disassembly. In contrast, a 521 SINV nsP3 mutant that fails to form nsP3/G3BP cytoplasmic foci or to dissociate SG displays a 522 relatively strong (2 log) decrease in virus titer (67). In this case, the robust inhibition of virus 523 growth could be due to initial SG formation in the absence of subsequent SG disassembly. We 524 speculate that the capsid protein in the nsP3/G3BP foci may play a role in the complex biology 525 controlling viral replication. Clearly, future studies will be needed to determine the mechanism 526 of capsid protein recruitment to G3BP/nsP3 foci, the potential interactions of capsid protein with 527 the components of these foci, and the role(s) of capsid in SG regulation and oscillation. 528 529 530 Acknowledgments. 531 We thank all the members of our lab for their helpful discussions and experimental 532 suggestions and Drs. Mathieu Dube, Guadalupe Martinez, and Claudia Sánchez-San Martín for 533 critical reading of the manuscript. We thank Drs. Erik Snapp and Guadalupe Martinez for many 534 helpful discussions and input on imaging, Youqing Xiang for her excellent technical assistance, 535 and the staff of the Einstein Analytical Imaging Facility for their support. The data in this paper 536 are from a thesis submitted by YZ in partial fulfillment of the requirements for the Degree of 537 Doctor of Philosophy in the Graduate Division of Medical Sciences, Albert Einstein College of 538 Medicine, Yeshiva University. 539


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This work was supported by grants to M.K. from the National Institute of General 540 Medicine (R01-GM057454) and by Cancer Center Core Support Grant NIH/NCI P30-CA13330. 541 The content is solely the responsibility of the authors and does not necessarily represent the 542 official views of the National Institute of General Medicine or the National Institutes of Health. 543 544 545 REFERENCES 546 1. Garoff, H., R. Hewson, and D.-J. E. Opstelten. 1998. Virus maturation by budding. 547

Microbiol.and Mol.Biol.Rev. 62:1171-1190. 548 2. Weissenhorn, W., E. Poudevigne, G. Effantin, and P. Bassereau. 2013. How to get 549

out: ssRNA enveloped viruses and membrane fission. Curr Opin Virol 3:159-67. 550 3. Kuhn, R. J. 2007. Togaviridae: The Viruses and Their Replication, p. 1001-1022. In D. 551

M. Knipe and P. M. Howley (ed.), Fields Virology, Fifth ed, vol. 1. Lippincott, Williams 552 and Wilkins, Philadelphia, PA. 553

4. Jose, J., J. E. Snyder, and R. J. Kuhn. 2009. A structural and functional perspective of 554 alphavirus replication and assembly. Future Microbiol 4:837-56. 555

5. Vaney, M. C., S. Duquerroy, and F. A. Rey. 2013. Alphavirus structure: activation for 556 entry at the target cell surface. Curr Opin Virol 3:151-8. 557

6. Suomalainen, M., P. Liljeström, and H. Garoff. 1992. Spike protein-nucleocapsid 558 interactions drive the budding of alphaviruses. J.Virol. 66:4737-4747. 559

7. Zhao, H., B. Lindqvist, H. Garoff, C.-H. von Bonsdorff, and P. Liljeström. 1994. A 560 tyrosine-based motif in the cytoplasmic domain of the alphavirus envelope protein is 561 essential for budding. EMBO J. 13:4204-4211. 562


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

26

8. Skoging, U., M. Vihinen, L. Nilsson, and P. Liljeström. 1996. Aromatic interactions 563 define the binding of the alphavirus spike to its nucleocapsid. Structure 4:519-529. 564

9. Lee, S., K. E. Owen, H.-K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. 565 Rossmann, and R. J. Kuhn. 1996. Identification of a protein binding site on the surface 566 of the alphavirus nucleocapsid and its implication in virus assembly. Structure 4:531-541. 567

10. Strauss, J. H., and E. G. Strauss. 1994. The alphaviruses: gene expression, replication, 568 and evolution. Microbiol.Rev. 58:491-562. 569

11. Garoff, H., M. Sjoberg, and R. H. Cheng. 2004. Budding of alphaviruses. Virus Res. 570 106:103-16. 571

12. Acheson, N. H., and I. Tamm. 1967. Replication of Semliki Forest virus: an electron 572 microscopic study. Virol. 32:128-43. 573

13. Tellinghuisen, T. L., A. E. Hamburger, B. R. Fisher, R. Ostendorp, and R. J. Kuhn. 574 1999. In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in 575 Escherichia coli. J.Virol. 73:5309-5319. 576

14. Snyder, J. E., O. Azizgolshani, B. Wu, Y. He, A. C. Lee, J. Jose, D. M. Suter, C. M. 577 Knobler, W. M. Gelbart, and R. J. Kuhn. 2011. Rescue of infectious particles from 578 preassembled alphavirus nucleocapsid cores. J.Virol. 85:5773-81. 579

15. Cheng, F., and S. Mukhopadhyay. 2011. Generating enveloped virus-like particles with 580 in vitro assembled cores. Virol. 413:153-60. 581

16. Skoging-Nyberg, U., and P. Liljestrom. 2001. M-X-I motif of semliki forest virus 582 capsid protein affects nucleocapsid assembly. J.Virol. 75:4625-32. 583

17. Forsell, K., G. Griffiths, and H. Garoff. 1996. Preformed cytoplasmic nucleocapsids 584 are not necessary for alphavirus budding. EMBO J. 15:6495-6505. 585


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

27

18. Forsell, K., L. Xing, T. Kozlovska, R. H. Cheng, and H. Garoff. 2000. Membrane 586 proteins organize a symmetrical virus. EMBO J. 19:5081-5091. 587

19. Sodeik, B. 2000. Mechanisms of viral transport in the cytoplasm. Trends in Microbiol. 588 8:465-72. 589

20. Scheele, C. M., and E. R. Pfefferkorn. 1969. Kinetics of incorporation of structural 590 proteins into Sindbis Virions. J.Virol. 3:369-375. 591

21. Soderlund, H., and I. Ulmanen. 1977. Transient association of Semliki Forest virus 592 capsid protein with ribosomes. J.Virol. 24:907-9. 593

22. Ulmanen, I., H. Soderlund, and L. Kaariainen. 1976. Semliki Forest virus capsid 594 protein associates with the 60S ribosomal subunit in infected cells. J.Virol. 20:203-10. 595

23. Ulmanen, I., H. Soderlund, and L. Kaariainen. 1979. Role of protein synthesis in the 596 assembly of Semliki forest virus nucleocapsid. Virol. 99:265-76. 597

24. Wengler, G., and C. Gros. 1996. Analyses of the role of structural changes in the 598 regulation of uncoating and assembly of alphavirus cores. Virol. 222:123-132. 599

25. Orvedahl, A., S. MacPherson, R. Sumpter, Jr., Z. Talloczy, Z. Zou, and B. Levine. 600 2010. Autophagy protects against Sindbis virus infection of the central nervous system. 601 Cell Host Microbe 7:115-27. 602

26. Orvedahl, A., R. Sumpter, Jr., G. Xiao, A. Ng, Z. Zou, Y. Tang, M. Narimatsu, C. 603 Gilpin, Q. Sun, M. Roth, C. V. Forst, J. L. Wrana, Y. E. Zhang, K. Luby-Phelps, R. 604 J. Xavier, Y. Xie, and B. Levine. 2011. Image-based genome-wide siRNA screen 605 identifies selective autophagy factors. Nature 480:113-7. 606

27. Adams, S. R., R. E. Campbell, L. A. Gross, B. R. Martin, G. K. Walkup, Y. Yao, J. 607 Llopis, and R. Y. Tsien. 2002. New biarsenical ligands and tetracysteine motifs for 608


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

28

protein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem 609 Soc 124:6063-76. 610

28. Hardwick, J. M., and B. Levine. 2000. Sindbis virus vector system for functional 611 analysis of apoptosis regulators. Meth.Enzym. 322:492-508. 612

29. Rice, C. M., R. Levis, J. H. Strauss, and H. V. Huang. 1987. Production of infectious 613 RNA transcripts from Sindbis virus cDNA clones: Mapping of lethal mutations, rescue of 614 a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. 615 J.Virol. 61:3809-38l9. 616

30. Pedelacq, J. D., S. Cabantous, T. Tran, T. C. Terwilliger, and G. S. Waldo. 2006. 617 Engineering and characterization of a superfolder green fluorescent protein. Nat 618 Biotechnol 24:79-88. 619

31. Liljeström, P., S. Lusa, D. Huylebroeck, and H. Garoff. 1991. In vitro mutagenesis of 620 a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight 621 membrane protein modulates virus release. J.Virol. 65:4107-4113. 622

32. Meyer, W. J., and R. E. Johnston. 1993. Structural rearrangement of infecting Sindbis 623 virions at the cell surface: Mapping of newly accessible epitopes. J.Virol. 67:5117-5125. 624

33. Chanel-Vos, C., and M. Kielian. 2004. A conserved histidine in the ij loop of the 625 Semliki Forest virus E1 protein plays an important role in membrane fusion. J.Virol. 626 78:13543-13552. 627

34. Counihan, N. A., S. M. Rawlinson, and B. D. Lindenbach. 2011. Trafficking of 628 hepatitis C virus core protein during virus particle assembly. PLoS Pathog. 7:e1002302. 629


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

29

35. Gousset, K., S. D. Ablan, L. V. Coren, A. Ono, F. Soheilian, K. Nagashima, D. E. 630 Ott, and E. O. Freed. 2008. Real-time visualization of HIV-1 GAG trafficking in 631 infected macrophages. PLoS Pathog. 4:e1000015. 632

36. Snijder, B., R. Sacher, P. Ramo, E. M. Damm, P. Liberali, and L. Pelkmans. 2009. 633 Population context determines cell-to-cell variability in endocytosis and virus infection. 634 Nature 461:520-3. 635

37. Greiser-Wilke, I., V. Moennig, O.-R. Kaaden, and L. T. M. Figueiredo. 1989. Most 636 alphaviruses share a conserved epitopic region on their nucleocapsid protein. J.Gen.Virol. 637 70:743-748. 638

38. Cristea, I. M., J. W. Carroll, M. P. Rout, C. M. Rice, B. T. Chait, and M. R. 639 MacDonald. 2006. Tracking and elucidating alphavirus-host protein interactions. 640 J.Biol.Chem. 281:30269-78. 641

39. Griffin, B. A., S. R. Adams, and R. Y. Tsien. 1998. Specific covalent labeling of 642 recombinant protein molecules inside live cells. Science 281:269-72. 643

40. Rudner, L., S. Nydegger, L. V. Coren, K. Nagashima, M. Thali, and D. E. Ott. 2005. 644 Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells 645 by biarsenical labeling. J.Virol. 79:4055-65. 646

41. Coller, K. E., N. S. Heaton, K. L. Berger, J. D. Cooper, J. L. Saunders, and G. 647 Randall. 2012. Molecular determinants and dynamics of hepatitis C virus secretion. 648 PLoS Pathog. 8:e1002466. 649

42. Das, S. C., D. Panda, D. Nayak, and A. K. Pattnaik. 2009. Biarsenical labeling of 650 vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic 651 imaging of m protein and virus uncoating in infected cells. J.Virol. 83:2611-22. 652


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

30

43. Coombs, K. M., and D. T. Brown. 1989. Form-determining functions in Sindbis virus 653 nucleocapsids: nucleo somelike organization of the nucleocapsid. J.Virol. 63:883-891. 654

44. Mukhopadhyay, S., W. Zhang, S. Gabler, P. R. Chipman, E. G. Strauss, J. H. 655 Strauss, T. S. Baker, R. J. Kuhn, and M. G. Rossmann. 2006. Mapping the structure 656 and function of the E1 and E2 glycoproteins in alphaviruses. Structure 14:63-73. 657

45. Beitzel, B. F., R. R. Bakken, J. M. Smith, and C. S. Schmaljohn. 2010. High-658 resolution functional mapping of the venezuelan equine encephalitis virus genome by 659 insertional mutagenesis and massively parallel sequencing. PLoS Pathog. 6:e1001146. 660

46. Choi, H.-K., L. Tong, W. Minor, P. Dumas, U. Boege, M. G. Rossman, and G. 661 Wengler. 1991. Structure of Sindbis virus core protein reveals a chymotrypsin-like serine 662 proteinase and the organization of the virion. Nature 354:37-43. 663

47. Salonen, A., T. Ahola, and L. Kaariainen. 2005. Viral RNA replication in association 664 with cellular membranes. Curr.Topics Microbiol.Immunol. 285:139-73. 665

48. Tourriere, H., K. Chebli, L. Zekri, B. Courselaud, J. M. Blanchard, E. Bertrand, 666 and J. Tazi. 2003. The RasGAP-associated endoribonuclease G3BP assembles stress 667 granules. J.Cell Biol. 160:823-31. 668

49. McInerney, G. M., N. L. Kedersha, R. J. Kaufman, P. Anderson, and P. Liljestrom. 669 2005. Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus 670 translation regulation. Mol.Biol.Cell 16:3753-63. 671

50. Fros, J. J., N. E. Domeradzka, J. Baggen, C. Geertsema, J. Flipse, J. M. Vlak, and 672 G. P. Pijlman. 2012. Chikungunya Virus nsP3 Blocks Stress Granule Assembly by 673 Recruitment of G3BP into Cytoplasmic Foci. J.Virol. 86:10873-9. 674


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

31

51. Frolova, E., R. Gorchakov, N. Garmashova, S. Atasheva, L. A. Vergara, and I. 675 Frolov. 2006. Formation of nsP3-specific protein complexes during Sindbis virus 676 replication. J.Virol. 80:4122-34. 677

52. Panas, M. D., M. Varjak, A. Lulla, K. Er Eng, A. Merits, G. B. Karlsson Hedestam, 678 and G. M. McInerney. 2012. Sequestration of G3BP coupled with efficient translation 679 inhibits stress granules in Semliki Forest virus infection. Mol.Biol.Cell 23:4701-12. 680

53. Cheng, R. H., R. J. Kuhn, N. H. Olson, M. G. Rossman, H.-K. Choi, T. J. Smith, and 681 T. S. Baker. 1995. Nucleocapsid and glycoprotein organization in an enveloped virus. 682 Cell 80:621-630. 683

54. Paredes, A. M., D. T. Brown, R. Rothnagel, W. Chiu, R. J. Schoepp, R. E. Johnston, 684 and B. V. V. Prasad. 1993. Three-dimensional structure of a membrane-containing 685 virus. Proc.Natl.Acad.Sci.USA 90:9095-9099. 686

55. Ormo, M., A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington. 687 1996. Crystal structure of the Aequorea victoria green fluorescent protein. Science 688 273:1392-5. 689

56. Larson, D. R., M. C. Johnson, W. W. Webb, and V. M. Vogt. 2005. Visualization of 690 retrovirus budding with correlated light and electron microscopy. 691 Proc.Natl.Acad.Sci.USA 102:15453-8. 692

57. Jouvenet, N., P. D. Bieniasz, and S. M. Simon. 2008. Imaging the biogenesis of 693 individual HIV-1 virions in live cells. Nature 454:236-40. 694

58. Lelek, M., F. Di Nunzio, R. Henriques, P. Charneau, N. Arhel, and C. Zimmer. 695 2012. Superresolution imaging of HIV in infected cells with FlAsH-PALM. 696 Proc.Natl.Acad.Sci.USA 109:8564-9. 697


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

32

59. Gaietta, G., T. J. Deerinck, S. R. Adams, J. Bouwer, O. Tour, D. W. Laird, G. E. 698 Sosinsky, R. Y. Tsien, and M. H. Ellisman. 2002. Multicolor and electron microscopic 699 imaging of connexin trafficking. Science 296:503-7. 700

60. Weber, F., V. Wagner, S. B. Rasmussen, R. Hartmann, and S. R. Paludan. 2006. 701 Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but 702 not in detectable amounts by negative-strand RNA viruses. J.Virol. 80:5059-64. 703

61. Spuul, P., G. Balistreri, K. Hellstrom, A. V. Golubtsov, E. Jokitalo, and T. Ahola. 704 2011. Assembly of alphavirus replication complexes from RNA and protein components 705 in a novel trans-replication system in mammalian cells. J.Virol. 85:4739-51. 706

62. Reineke, L. C., and R. E. Lloyd. 2013. Diversion of stress granules and P-bodies during 707 viral infection. Virol. 436:255-67. 708

63. Valiente-Echeverria, F., L. Melnychuk, and A. J. Mouland. 2012. Viral modulation of 709 stress granules. Virus Res. 169:430-7. 710

64. Beckham, C. J., and R. Parker. 2008. P bodies, stress granules, and viral life cycles. 711 Cell Host Microbe 3:206-12. 712

65. White, J. P., A. M. Cardenas, W. E. Marissen, and R. E. Lloyd. 2007. Inhibition of 713 cytoplasmic mRNA stress granule formation by a viral proteinase. Cell Host Microbe 714 2:295-305. 715

66. Emara, M. M., and M. A. Brinton. 2007. Interaction of TIA-1/TIAR with West Nile 716 and dengue virus products in infected cells interferes with stress granule formation and 717 processing body assembly. Proc.Natl.Acad.Sci.USA 104:9041-6. 718

67. Foy, N. J., M. Akhrymuk, I. Akhrymuk, S. Atasheva, A. Bopda-Waffo, I. Frolov, 719 and E. I. Frolova. 2013. Hypervariable Domains of nsP3 Proteins of New World and 720


http://jvi.asm.org/

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http://jvi.asm.org/

33

Old World Alphaviruses Mediate Formation of Distinct, Virus-Specific Protein 721 Complexes. J.Virol. 87:1997-2010. 722

68. Ruggieri, A., E. Dazert, P. Metz, S. Hofmann, J. P. Bergeest, J. Mazur, P. Bankhead, 723 M. S. Hiet, S. Kallis, G. Alvisi, C. E. Samuel, V. Lohmann, L. Kaderali, K. Rohr, M. 724 Frese, G. Stoecklin, and R. Bartenschlager. 2012. Dynamic oscillation of translation 725 and stress granule formation mark the cellular response to virus infection. Cell Host 726 Microbe 12:71-85. 727

69. Cristea, I. M., H. Rozjabek, K. R. Molloy, S. Karki, L. L. White, C. M. Rice, M. P. 728 Rout, B. T. Chait, and M. R. MacDonald. 2010. Host factors associated with the 729 Sindbis virus RNA-dependent RNA polymerase: role for G3BP1 and G3BP2 in virus 730 replication. J.Virol. 84:6720-32. 731

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735 Table 1. Summary of TC motif insertion sites tested in SINV capsid protein. 736

737 a. The TC motif plus an N-terminal ATG codon was introduced just before the first amino acid 738 (Methionine) of the capsid protein. 739 b The TC motif plus an N-terminal ATG codon and a C-terminal GSSGGSSGGSSG flexible 740 linker region was introduced before the first amino acid of the capsid protein. 741 c The TC motif was introduced directly after the indicated residue. 742

Insert region on capsid protein 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 N/A

S182-TC 5 logs lower N/A

E186-TC 6 logs lower ++++

S199-TC >7 logs lower N/A

E259-TC >7 logs lower N/A

N-terminal to protease domainc E89-TC WT ++

Q94-TC WT +++

K97-TC WT +++

G101-TC WT ++


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d The growth of all TC insertion mutants were compared to WT SINV at 24 h post 743 electroporation (methods). The log of the WT SINV titer was ~8.6 as measured by plaque assay 744 on BHK-21 cells. Titer of all mutants designated (WT) was > 8. 745 e The labeling efficiency of virus-infected Vero cells with biarsenical dyes. “N/A” indicates not 746 applicable, “+” indicates barely detectable fluorescence, “++” indicates low fluorescence 747 intensity, “+++” indicates strong fluorescence intensity, “++++” indicates very bright 748 fluorescence intensity. 749 750 751


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FIGURE LEGENDS 752 Figure 1. Growth properties of Q94-TC SINV. A. Growth kinetics of Q94-TC vs. WT 753

virus. BHK-21 cells were electroporated with WT or mutant virus RNA and incubated at 37° C 754 for the indicated time points. Media were collected and progeny viruses titered by plaque assay. 755 B. The effect of ReAsH labeling on Q94-TC virus production. Vero cells were infected with 756 Q94-TC at a multiplicity of 0.5 IC/cell, cultured for 7 h at 37°C, and treated with ReAsH or 757 mock-treated using the conditions described in the methods. The incubation was continued at 758 37°C for the indicated time points and progeny virus production was quantitated by infectious 759 center assays on Vero cells. Data are the average of two independent experiments with the range 760 indicated. 761 762

Figure 2. Assembly properties of Q94-TC SINV. BHK-21 cells were electroporated 763 with Q94-TC (TC) or WT virus RNA, incubated at 37°C for 6 h, pulse-labeled with [35S]-764 methionine/cysteine, and chased for 0 or 3 h at 37°C. Left panel: Cell lysates were collected and 765 an aliquot analyzed directly by SDS-PAGE. The positions of TC-capsid (TC-C) and WT capsid 766 (C) proteins are indicated, with TC-C migrating slightly slower than WT C. The pE2 protein is 767 visible in the 0 h lysate sample but E1 and E2 are obscured by the host cell proteins. Right panel: 768 Samples of the chase media were immunoprecipitated with an antibody against SINV E2 in the 769 absence of detergent to recover intact virus particles, and analyzed by SDS-PAGE. The E1 and 770 E2 proteins migrate as a doublet. 771 772

Figure 3. Electron microscopy of WT and mutant-infected cells. BHK-21 cells were 773 electroporated with Q94-TC, WT SINV, or GFP-dsTE12Q viral RNA, incubated at 37°C for 12 774


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h, and processed for electron microscopy. Panels A, C, and E show representative examples of 775 the morphology of budding virus particles (indicated by arrows). Panels B and D show 776 representative examples of cytopathic vacuole type II (CPVII) in WT (B) or Q94-TC (D) 777 infected cells. Note that the CPVII-associated nucleocapsids are larger, denser, and more regular 778 in shape than adjacent ribosomes in the field, differentiating these structures from the 779 endoplasmic reticulum. All images were acquired at a magnification of 20,000 X, with the scale 780 bar representing 200 nM. 781

782 Figure 4. Specificity of biarsenical dye labeling. Vero cells were infected with Q94-TC 783

or WT SINV at a multiplicity of 0.5 IC/cell, cultured for 7 h at 37°C, and labeled with ReAsH as 784 described in the methods. Cells were then fixed, permeabilized and stained with antibody against 785 the capsid protein. Images are a single internal z-section and are representative of two 786 independent experiments. Bar = 10 µM. 787 788

Figure 5. Examples of the three types of capsid foci. Vero cells were infected with 789 Q94-TC and labeled with ReAsH at 7 h post infection. Cells were then fixed, permeabilized, and 790 stained with a mAb against the SINV E2 protein. The images illustrate three groups of capsid 791 foci: A. Small internal capsid puncta that co-localize with the E2 protein (arrows point to 792 representative examples). B. Irregular internal capsid structures that did not co-localize with the 793 E2 protein (arrows point to representative examples). C. PM-proximal capsid puncta that co-794 localize with E2 protein. The inset shows a zoomed view of the boxed region (3X 795 magnification). All images are single z-sections, where A and B are internal sections and C is a 796 PM-proximal section. All are representative examples of three experiments. Bar = 10 µM. 797


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798 Figure 6. Irregular internal capsid structures co-localize with G3BP and nsP3. A. 799

Vero cells were infected with Q94-TC and labeled with ReAsH (left panels) at 7 h post infection. 800 Cells were then fixed, permeabilized, and stained with antibodies against G3BP, dsRNA, or eIF3 801 (middle panels). B. Vero cells were infected with Q94-TC virus, incubated for 7 h, fixed and co-802 stained with mAb recognizing the capsid protein and rabbit antibody against nsP3. C. Vero cells 803 were infected with either Q94-TC or [Q94-TC + L108A/L110A] (indicated as TC-LL). 804 Alternatively, cells were transfected with RNA for the budding-defective mutant [Q94-TC + 805 E2Y400K] (indicated as TC-Y400K). Cells were incubated for 7 h, fixed, and co-stained with 806 mAb to capsid and rabbit antibody against G3BP. Images are representative of two independent 807 experiments, with scale bar = 10 μM. 808 809

Figure 7. Dynamics of irregular internal capsid structures. A. Time series of internal 810 capsid structures. Vero cells were infected with Q94-TC and labeled with ReAsH at 7 h post 811 infection. The positions of specific foci (indicated by arrow) were tracked by images acquired 812 every second. Images collected at 0, 20 and 40 seconds are shown, documenting the relative 813 immobility of these structures. B. Recruitment of newly synthesized capsid protein to pre-814 existing irregular internal capsid foci. Vero cells were infected with Q94-TC and labeled with 815 FlAsH at 7 h post-infection (right column). At the indicated chase time, the cells were labeled 816 with ReAsH (left column). Images were all acquired at the same gain, and are representative of 817 two independent experiments. Bar = 10 µM. 818 819


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Figure 8. Localization and dynamics of the PM-proximal capsid puncta. Vero cells 820 were infected with Q94-TC and labeled with ReAsH at 7 h post infection. A. Localization of the 821 PM-proximal capsid puncta. Following ReAsH labeling cells were immunolabeled on ice to 822 detect the cell surface E2 protein as described in the methods. A middle section in the z direction 823 is shown. B. Time series of PM capsid puncta. The positions of specific puncta (indicated by 824 arrow) were tracked by images acquired every second during a 37°C incubation. Images of a z 825 section at the PM collected at 0, 20 and 40 seconds are shown, with a zoomed view of the boxed 826 region on the upper right-hand side, documenting the relative immobility of this puncta type. 827 Images are representative examples of two independent experiments. Bar = 10 µM. 828 829

Figure. 9. Properties of PM capsid puncta. A. Generation of PM capsid puncta does 830 not require cytoplasmic NC formation. Vero cells infected with TC-LL for 7 h were labeled with 831 ReAsH and then fixed, permeabilized, and stained with E2 mAb R6. B. PM capsid puncta do not 832 co-localize with dsRNA. Vero cells were infected with Q94-TC for 7 h, labeled with ReAsH, and 833 then fixed, permeabilized, and stained with mAb against dsRNA. C. Newly synthesized capsid 834 protein was delivered to pre-existing PM capsid puncta. Vero cells were infected with Q94-TC 835 for 7 h and stained with FlAsH to label the existing capsid protein pool (second column). At the 836 indicated chase time, the cells were labeled with ReAsH (first column). After 1 h chase, ReAsH-837 labeled capsid protein was detected in PM capsid puncta containing the FlAsH signal. Inset is a 838 zoomed view of the boxed region (2.5X magnification). Images are representative of two 839 independent experiments. Bar = 10 µM. 840 841


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