Imaging the Alphavirus Exit Pathway - Journal of Virology - American ...

Imaging the Alphavirus Exit Pathway Maria Guadalupe Martinez,a Erik-Lee Snapp,b,c Geoffrey S. Perumal,d Frank P. Macaluso,d Margaret Kieliana Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USAa; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USAb; Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USAc; Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx, New York, USAd

ABSTRACT

Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ⬃2 to 4 ␮m in length and excluded the PM marker, and tubulin-positive extensions that were >10 ␮m long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites. IMPORTANCE

Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.

E

nveloped viruses acquire their membranes from the host cell. While virus assembly and budding are key steps in the production of infectious progeny virions, our understanding of these pathways is incomplete (reviewed in references 1 to 3). Depending on the virus, budding can be targeted to the membrane of a specific host cell compartment, such as the endoplasmic reticulum (ER), Golgi complex, nucleus, or plasma membrane (PM). Viruses also vary in their requirements for viral membrane proteins, capsid protein, and/or matrix proteins during the budding reaction. Cellular factors can play important roles during assembly and budding. For instance, the cellular ESCRT machinery is recruited by some viruses to promote scission of the virus membrane (3, 4), while viruses such as influenza virus are ESCRT independent (5). Alphaviruses are small enveloped plus-sense RNA viruses that bud from the PM (1, 6). They include important human pathogens such as Chikungunya virus and the encephalitic alphaviruses (7). Alphaviruses contain an internal nucleocapsid (NC) core composed of the RNA genome and the capsid protein (Cp) and an outer envelope protein layer composed of closely associated heterodimers of the E2 and E1 transmembrane proteins. Virus particles contain 240 copies each of the Cp, E2, and E1 proteins, which are arranged in the NC and envelope as lattices with T⫽4 icosa-

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hedral quasisymmetry (8, 9). The E2 envelope protein overlays much of E1 in the virus particle and mediates binding of the virus to receptors at the cell surface (10, 11). The E1 protein is the low pH-triggered membrane fusion protein. E2 is initially synthesized in the ER as a precursor, PE2, which is cleaved to the mature E2 protein by cellular furin late in the exocytic pathway (12, 13). Budding of the highly organized alphavirus particle requires both the capsid protein and the envelope proteins (14, 15) and is independent of the ESCRT machinery (16). Budding involves a one-to-one interaction of the cytoplasmic domain of E2 with a hydrophobic pocket on the capsid protein, and mutations in this critical region of E2 block E2-Cp interaction and inhibit budding

Received 26 February 2014 Accepted 30 March 2014 Published ahead of print 2 April 2014 Editor: R. W. Doms Address correspondence to Margaret Kielian, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JVI.00592-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00592-14

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Alphavirus Exit Pathway

(17–20). Alphavirus budding also depends on the correct formation of the E2/E1 heterodimer (21, 22) and on lateral interactions between the envelope proteins that form the lattice (8, 14, 23, 24). Unlike the case for many less-ordered enveloped viruses, structural and biochemical studies indicate that host proteins are strictly excluded from the mature alphavirus envelope (6, 25). Despite the fact that the structures and interactions of the alphavirus capsid and envelope proteins have been extensively characterized, many fundamental questions about alphavirus budding remain. Earlier electron microscopy data suggest that alphavirus budding takes place at localized PM sites (26–28), but it is not clear how these sites are formed or specialized, which viral proteins mediate the exclusion of host membrane proteins from the nascent particles, and what functional roles are played by host and viral proteins during virus assembly and budding. Such questions are amenable to study using live-cell imaging methods (29–31), and published studies indicate that alphaviruses in which E2 is fused to a red fluorescent protein are viable (32–34). In contrast, the E1 and capsid proteins are less permissive to labeling with such fluorescent protein tags (e.g., see reference 35). We constructed and characterized Sindbis virus (SINV) strains containing either a green fluorescent protein (GFP) or mCherry label on the E2 glycoprotein. We then exploited these viruses to study budding using a combination of total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, photobleaching, and correlative light and electron microscopy (CLEM). Our studies demonstrated that alphavirus production occurred at preferential sites on the PM: localized patches and filopodium-like extensions. Two distinct types of E2-positive extensions were induced in virus-infected cells. Tubulin-negative extensions were relatively short and excluded a PM marker protein in a process that required the E2-Cp interaction. In contrast, tubulin-positive extensions were longer, did not exclude the PM marker, and appeared to mediate cell-cell virus particle transfer. The number of tubulin-positive extensions was selectively reduced in cells infected with a nonbudding SINV mutant. MATERIALS AND METHODS Cell lines. BHK-21 cells were maintained at 37°C in Dulbecco’s modified Eagle’s medium containing 5% fetal calf serum, 10% tryptose phosphate broth, 100 U penicillin/ml, and 100 ␮g streptomycin/ml. Vero cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U penicillin/ml, and 100 ␮g streptomycin/ml. Vero cell lines stably expressing plasma membrane-localized GFP were generated using the previously described GFP-22 construct, which produces almost exclusive PM localization of cytoplasmically oriented GFP (36). Briefly, the GFP-22 plasmid contains enhanced GFP (EGFP) fused at its C terminus to a modified tail region of the rat cytochrome b5 and cloned into pcDNA3 (Invitrogen). Vero cells were transfected with GFP-22 using Lipofectamine 2000 according to the manufacturer’s instructions for 6 h, cultured for 48 h in Vero medium, and then selected for 2 weeks in the presence of 1 mg G418/ml. Cells were then sorted to obtain single-cell clones expressing high levels of GFP-22, which are referred to as PM-GFP in this paper. Two independent clones were maintained for this study. Antibodies and reagents. Antibodies used for immunofluorescence against SINV proteins included mouse monoclonal antibodies (MAbs) against E2 (R6) and E1 (R2) (kindly provided by William Klimstra) (37) and the mouse MAb C12-1 against capsid (kindly provided by Irene Greiser-Wilke) (38). The 6G7 anti-␤-tubulin antibody developed by Willi Halfter was obtained from the Developmental Studies Hybridoma Bank (developed under the auspices of NICHD and maintained by the Univer-


sity of Iowa, Department of Biology, Iowa City, IA). The anti-␣-tubulin antibody (ab18251) was purchased from Abcam. Alexa 488-conjugated phalloidin and Alexa 488-, 561-, or 405-conjugated antimouse or antirabbit antibodies were obtained from Molecular Probes. G418, brefeldin A (BFA), and poly-L-lysine were obtained from Sigma-Aldrich. SINV infectious clones. Wild-type SINV was generated from the dsTE12Q infectious clone kindly provided by Beth Levine (39). For fluorescence imaging, SINV E2 was labeled by insertion of either oxGFP or mCherry after the PE2 furin cleavage site. A serine residue was positioned at the ⫹1 position to promote furin cleavage (40). Infectious clones were generated using a two-step overlap extension PCR using the pC1 plasmids encoding oxGFP, a GFP engineered to be cysteine-free (41), or mCherry (42) to amplify oxGFP or mCherry and dsTE12Q to amplify the SINV E3/E2 region. The ⬃2.1-kb restriction fragments encoding oxGFP or mCherry at the N terminus of the E2 protein were then subcloned into the wild-type infectious clone by ligation using the endogenous BclI site on E3 and PmlI site on E2. Sequences of the regions encompassing the mutations were confirmed by automated sequencing (Genewiz Inc., North Brunswick, NJ). Two clones of each construction were tested to confirm the phenotype. The previously described E2 Y400K mutation (17) and capsid L108/A and L110/A mutations (18, 35) were generated by in vitro mutagenesis, subcloned into the wild-type and the mCherry/oxGFP infectious SINV clones, and confirmed by sequencing. These viruses are hereinafter referred to as Y400K-GFP, Y400K-mCherry, CpLL-GFP and CpLLmCherry, respectively. Production of virus stocks. Viral RNAs were in vitro transcribed from the XhoI-linearized SINV infectious clones and electroporated into BHK-21 cells to generate virus stocks (43). All mutant virus stocks were prepared by incubation of electroporated cells for 12 h at 37°C to prevent the possible generation of revertants during extended culture times. Stocks were clarified by low-speed centrifugation, and virus titers were determined by plaque titration on BHK-21 cells. Virus infection. Vero cells were infected with WT virus (WT), WT with GFP-labeled E2 (WT-GFP), or WT with mCherry-labeled E2 (WTmCherry) stocks for 90 min at 37°C at a multiplicity of infection of 5. Cells were then washed twice and incubated in fresh medium for 5 to 6 h at 37°C. Vero cells were infected via transfection of viral RNA using Lipofectamine 2000 in Opti-MEM (Invitrogen) according to the manufacturer’s instructions and incubated for 6 h. Cells were then transferred to fresh culture medium and incubated for the total time described below for each experiment. The time course of protein expression and virus release was similar for the infection and transfection protocols (data not shown). Transfection was used for some experiments in order to study the budding-defective Y400K mutant. Virus assembly assay. The assembly of mutant viruses was evaluated by pulse-chase analysis (44). Confluent BHK cells in six-well plates were electroporated with viral RNA, cultured for 6 h at 37°C, incubated for 15 min in cysteine and methionine-deficient minimal essential medium, pulse-labeled with 100 ␮Ci/ml [35S]-methionine/cysteine (Express Labeling Mix; PerkinElmer Life and Analytical Sciences) for 30 min, and chased for 2 h at 37°C in medium containing a 10-fold excess of unlabeled methionine and cysteine. Medium samples were collected and pelleted through a 20% sucrose cushion, and cells were lysed in buffer containing 1% Triton X-100 and a protease inhibitor cocktail (44) and analyzed by SDS-PAGE. Transmission electron microscopy (TEM). BHK-21 cells were electroporated with viral RNA, plated on 35-mm culture dishes, incubated at 37°C for 12 h, and fixed with 2.5% glutaraldehyde, 2% paraformaldehyde (PFA) in 0.1 M sodium cacodylate buffer for 30 min at room temperature. Samples were then processed by the Albert Einstein College of Medicine Analytical Imaging Facility by postfixation with 1% osmium tetroxide followed by 2% uranyl acetate, dehydration, and embedment in LX112 resin (LADD Research Industries, Burlington, VT). Thin sections were

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stained with uranyl acetate followed by lead citrate and examined on a JEOL 1200EX or a JEOL 100CXII electron microscope at 80 kV at a magnification of ⫻20,000 to ⫻30,000. Images were assembled with Adobe Photoshop CS software (Adobe Systems, San Jose, CA). Immunofluorescence microscopy. Vero cells were cultured on MatTek glass-bottom culture dishes (number 1.5; P35G-1.5-14-C; MatTek Corporation), transfected with viral RNAs, incubated at 37°C for 6 to 8 h, and fixed with 4% paraformaldehyde (Electron Microscopy Science) for 20 min at room temperature. Cells were then stained with MAbs to E1 or E2 proteins and Alexa 561-conjugated secondary antibody. To visualize internal proteins, fixed cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature and then stained to detect capsid protein or cellular proteins. Images were acquired using TIRFM or confocal microscopy, as indicated below, for individual experiments. For quantitation of extensions, sparsely plated Vero cells stably expressing PM-GFP were mock infected, infected with WT-mCherry, or transfected with Y400K-mCherry RNA. After 8 h of infection, cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100 for 5 min at room temperature, and stained with anti-␣-tubulin antibody. Images were acquired with a confocal microscope, and a total of 30 cells were counted in 3 different experiments. To determine the total number of extensions, PM-GFP was used as a marker in mock-infected cells. Extensions in infected cells were defined by the presence of E2, since extensions could potentially exclude the PM-GFP marker. Measurements of the length of the tubulin-positive or -negative extensions were performed using Volocity software (version 6.2.1; PerkinElmer). The Microsoft Excel program was used to calculate statistical significance by a two-tailed unpaired Student’s t test, as indicated in the appropriate figures. Live-cell microscopy, data acquisition, and image analysis. Vero cells were grown on MatTek glass-bottom culture dishes and transfected with viral RNAs. At 6 h after transfection, the medium was replaced with imaging medium (Dulbecco’s modified Eagle medium without phenol red or NaHCO3 [D-2902; Sigma] supplemented with 10 mM HEPES, pH 8.0, 10% fetal bovine serum, 100 U penicillin/ml, and 100 ␮g streptomycin/ml). Imaging chambers were sealed with Parafilm, and cells were maintained on a heated microscope stage at 37°C. Total internal reflection fluorescence (TIRF) microscopy used a modified commercial, objective-based TIRF Olympus IX71 microscope (45) in the Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine. The light sources of the Olympus IX71 microscope were replaced by five lasers, two of which were used in this work: 488-nm (IMA series; Melles Griot) and 561-nm (Jive 10; Cobolt) lasers. Rapid switching and shuttering of the lasers was accomplished with an acousto-optic tunable filter. A high-speed direct current servo motor (M-235.2DD; Physics Instrumente) was used to repeat the setting of different TIRF angles for each laser and rapidly switch between the TIRF position and epifluorescence illumination for each laser line. A ⫻60 oil objective (numerical aperture [NA], 1.45) was used, and sequential images were acquired every 10 s with a 50-ms exposure using a back-illuminated electron-multiplying chargecoupled-device camera (DU-897; Andor). Images were collected using Metamorph software (Molecular Devices, Sunnyvale, CA). Confocal images were acquired using an LSM5 Live DuoScan confocal microscope system (DuoScan microscope; Carl Zeiss MicroImaging, Inc.) with a ⫻63 oil objective (NA, 1.4). Imaging of GFP or mCherry was achieved by excitation with a 100-mW 488-nm diode laser with a 500- to 550-nm band-pass filter or with a 40-mW 561-nm diode laser and a 580-nm long-pass filter. Simultaneous dual-color imaging was achieved with a dual-emission splitter. Images were analyzed using Volocity three-dimensional image analysis software. Time series were generated using ImageJ software (National Institutes of Health, Bethesda, MD), and images for figures were processed with Adobe Photoshop software. Microscopy of viral particles. Virus stocks were incubated on poly-Llysine-coated MatTek glass-bottom culture dishes for 2 h at 37°C, and samples were examined by TIRFM in imaging medium. Where indicated,

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samples were fixed, permeabilized with 0.2% Triton X-100, and immunostained with antibodies against the E1 or capsid protein. CLEM. Vero cells were cultured on glass coverslips imprinted with three fiduciary markers. Pilot experiments demonstrated better postfixation retention of fluorescence by mCherry-labeled E2 than GFP-labeled E2. Cells were therefore transfected with WT-mCherry or Y400KmCherry RNA, transferred to imaging medium after 6 h, and cultured for 4 h. Cells were then fixed for 20 min at room temperature with 4% paraformaldehyde, 2% glutaraldehyde in phosphate-buffered saline (PBS), and quenched with 0.2% sodium borohydride. Samples were imaged using a Zeiss AxioObserver microscope equipped with AxioVision software with a shuffle-and-find feature to mark cell locations. Cells were then fixed with 2.5% glutaraldehyde, postfixed in 2% osmium tetroxide, dehydrated in ethanol, critical-point dried (Tousimis Samdri 790 critical point dryer), and coated with chromium (EMS 150T-ES sputter coater). Selected cells were automatically located in a Zeiss Supra 40 field emission scanning electron microscope (SEM) and imaged with a secondary electron detector. Fluorescence and SEM images were correlated with AxioVision software. Fluorescence recovery after photobleaching (FRAP). Vero cells stably expressing the PM-GFP marker were transfected for 6 h with RNA for the indicated mCherry-labeled viruses, transferred to imaging medium, and imaged for 50 s in the DuoScan confocal microscope. Selected areas were photobleached using three iterations of the 488- or 561-nm diode laser at scan speed 6 and observed for an additional 200 s, with images being acquired at 10-s time intervals. The laser intensity remained unchanged during the course of the experiment. Fluorescence recovery was calculated as a percentage of the prebleach intensity. Vero cells were also labeled with the lipid label CellMask orange plasma membrane stain (Life Technologies) according to the manufacturer’s recommendation. Briefly, cells were incubated with dye for 5 min at 37°C, washed 5 times in PBS to remove free dye, transferred to imaging medium, and imaged for approximately 5 s in the DuoScan confocal microscope. To control for free dye that could interfere with FRAP analysis, we evaluated the recovery of CellMask stain in a large FRAP area of uninfected cells. Analysis showed that the borders of the FRAP area recovered faster than its center, compatible with lateral diffusion of the dye rather than insertion of free dye from the medium (data not shown). The same CellMask stain labeling conditions were then used in Vero cells infected for 6 h with wild-type SINV in which the E2 protein was labeled with oxGFP (WT-GFP). Selected areas were photobleached using three iterations of the 561-nm and 488-nm diode lasers and observed for an additional 20 s, with images being acquired continuously. The laser intensity remained unchanged during the course of the experiment. Fluorescence recovery was calculated as a percentage of the prebleach intensity.

RESULTS

Construction and characterization of E2-tagged SINV. We constructed SINV infectious clones in which the E2 protein was labeled with either oxGFP or mCherry, both of which lack cysteines. Both fluorescent proteins are thus amenable to expression in the oxidizing environment of the ER lumen (46) and were positioned after the PE2 furin cleavage site to create an N-terminal extension of E2. These viruses are referred to as WT-GFP and WT-mCherry, respectively. Both labeled viruses grew efficiently (maximal titers, ⬃107 PFU/ml), although their final titers were ⬃1 log unit lower than the final titer of wild-type SINV (data not shown). Serial passage of the labeled viruses confirmed that the tags were stable to multiple cycles of infection (data not shown). The two types of labeled viruses displayed equivalent properties during characterization, and representative results with WT-GFP are summarized below. Pulse-chase assays of virus assembly demonstrated efficient production of WT and WT-GFP particles (Fig. 1A, right), suggest-

Journal of Virology


FIG 1 Labeled virus characterization. (A) BHK-21 cells were mock electroporated or electroporated with WT or WT-GFP RNA, incubated at 37°C for 6 h, pulse-labeled with [35S]methionine/cysteine for 30 min, and chased for 2 h at 37°C. Cells were lysed, chase media were pelleted through a sucrose cushion to recover intact virus particles, and aliquots of the samples were analyzed by SDS-PAGE. Lysate samples were visualized by Western blotting using a MAb against GFP (left), which identifies both GFP-E2 and GFP-PE2 (multiple bands are due to the heterogeneity of glycosylation). Medium samples were detected by fluorography (right). Uninf., uninfected. (B) Transmission electron microscopy of wild-type SINV- and WT-GFP-infected cells. BHK-21 cells were electroporated with viral RNA, incubated at 37°C for 12 h, and processed for transmission electron microscopy. All images were acquired at a magnification of ⫻20,000. Bar ⫽ 200 nm. (C) Fluorescence intensity distribution of WT-GFP. BHK-21 cells were electroporated with WT-GFP RNA and incubated at 37°C for 12 h. The virus-containing culture medium was adsorbed to poly-L-lysine-treated coverslips at 37°C for 2 h. Samples were then fixed, permeabilized, stained with MAb against the E1 or the capsid protein, and visualized by TIRFM (top). TIRFM images in the GFP channel were acquired, and the fluorescence intensity of each spot was quantified using Volocity software (bottom). Data are representative of those from 2 to 3 independent experiments. A.U., arbitrary units.

ing that the somewhat decreased final titer of the labeled virus was largely due to the effect of the tag on E2 receptor binding. The released WT-GFP contained mature, GFP-tagged E2 protein, and no untagged E2 was detectable (Fig. 1A, right). Western blot analysis of the lysates of WT-GFP-infected cells confirmed the presence of GFP-tagged E2 and PE2 and the absence of free GFP (Fig. 1A, left). Transmission electron microscopy (TEM) demonstrated the budding of spherical, dense core-containing virus particles of comparable morphology at the PM of WT- and WT-GFP-infected cells (Fig. 1B). WT-GFP stocks were adsorbed to poly-L-lysinecoated slides and stained with antibodies to E1 and Cp. As predicted, GFP particle fluorescence exhibited a high degree of colocalization with both E1 (96%, n ⫽ 512 particles) and Cp (95%, n ⫽ 490 particles) (Fig. 1C, top). The amount of GFP fluorescence per particle was quantitated (29, 47). A single peak of distribution of fluorescence intensity was observed, consistent with homogeneous virus labeling and the absence of significant multimeric or aggregated particles (Fig. 1C, bottom). Together, our data indicate that the GFP- and mCherry-tagged viruses are functional for both assembly and infection and that the fluorescence of the tag accurately reflects the distribution of the viral E2 protein. Characterization of E2 distribution at the PM. Cells infected with WT or WT-GFP were fixed, permeabilized, stained with


MAb to the E2 protein, and analyzed by TIRFM to restrict imaging to the PM and the region within ⬃200 nm of the culture glass surface. The unlabeled wild-type E2 and GFP-tagged E2 proteins were similarly distributed in a patchy pattern in the PM region (data not shown). Either GFP fluorescence or E2 immunostaining of the WT-GFP-infected cells produced a comparable patchy pattern (data not shown). Together, these data confirm that the fluorescent tag did not affect the E2 distribution at the cell surface. We then used TIRFM and live-cell imaging to follow the E2 protein in cells transfected with WT-GFP RNA. The E2 protein was detected in motile puncta close to the PM focal plane at 5 to 6 h posttransfection (Fig. 2A). These puncta exhibited a fluorescence intensity at least 10 times higher than that of individual viral particles imaged under the same microscope settings. Initially, the individual puncta displayed lateral motility in the TIRF field and a gradual increase in fluorescence intensity, consistent with vesicles transporting E2 to the focal plane of the PM. At later time points, these motile E2 foci became stationary, while their fluorescence intensity continued to increase. These E2 PM patches also contained the major viral structural proteins E1 and Cp (data not shown). To address the role of the E2-Cp interaction in E2 dynamics, we performed similar imaging of cells transfected with Y400K-

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FIG 2 Formation and growth of E2 PM patches. (A and B) Vero cells were transfected with WT-GFP (A) or Y400K-GFP (B) RNA. After 6 h the cells were transferred to imaging medium, and images were acquired every 10 s for 75 min using TIRFM. Most of the surface of a single representative cell at 50 min is shown. Bar ⫽ 10 ␮m. (Bottom) The region demarcated by the white dashed box is shown as a zoom image (⫻1.5), where images acquired every 25 min from 0 to 75 min are presented. Arrowheads, the tracking of one E2 vesicle to the PM. Images are representative of the results in three independent experiments. (C and D) Growth of E2 patches. Vero cells were infected with WT-GFP. After incubation for 7 h at 37°C, the culture medium was replaced by control imaging medium (C) or imaging medium plus 3 ␮g BFA/ml (D). Images were acquired every 10 s for 90 min using TIRFM. The fluorescence intensity of individual E2 patches was quantitated using Volocity software, setting the fluorescence at T⫽0 equal to 100%. Each graph shows the mean and standard error of the mean of 60 E2 patches visualized in 6 different cells in three independent experiments.

GFP RNA. Y400K-GFP contains the GFP tag on E2 and a tyrosineto-lysine substitution at residue 400 in the E2 cytosolic tail. Cells infected with an E2 Y400K mutant virus efficiently transport E1 and E2 to the PM and form a cytoplasmic NC but are blocked in E2-Cp interaction and virus particle production (17). Cells transfected with Y400K GFP RNA showed motile puncta close to the PM focal plane. However, these puncta had a relatively short life span and disappeared rapidly rather than forming immobile E2 patches at the PM. Instead, a gradual increase of diffusely distributed E2 intensity was observed at the PM of Y400K-GFP-infected cells (Fig. 2B). As expected, no Cp was observed at the PM of these cells, and E1 showed the same diffuse distribution as E2 (data not shown). We also performed live-cell imaging of cells infected with Cp LL-GFP, which contains the GFP tag fused to E2 plus L108A and L110A substitutions in the Cp. These Cp mutations were previously shown to inhibit the formation of cytosolic NC, while they allowed NC assembly at the PM and budding of morphologically normal virus (48). Cells infected with Cp LLGFP showed E2 traffic similar to that of WT-GFP, including the motile/nonmotile transition and formation of E2 patches at the PM (data not shown). Together, these results suggest that E2 was initially visualized en route to the cell surface in motile

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vesicles that fuse with the PM. Interaction with the Cp/NC acted as a local scaffold that inhibited the lateral diffusion of E2 in the PM and led to the accumulation of patches of E2 protein. While cytoplasmic NCs were not required for the formation of E2 patches, stable patches did not form in the absence of the E2-Cp interaction. We then characterized the gradual increase of fluorescence intensity in the E2 PM patches that we observed in Fig. 2A. Vero cells were infected with WT-GFP, and individual E2 patches were imaged in the TIRF microscope. A steady increase of the fluorescence intensity in E2 patches was detected during the ⬃90-min time course of the experiment (Fig. 2C). This increase could require vesicular delivery of new E2 to the PM or could occur though lateral diffusion of the E2 already present on the PM. To determine the mechanism of E2 recruitment, patch formation was allowed to occur during 7 h of incubation of Vero cells transfected with WT-GFP RNA, as shown in Fig. 2C. The cells were then treated with brefeldin A (BFA), a well-characterized inhibitor of the secretory pathway (49), and imaged by TIRFM starting approximately 30 min after BFA addition (Fig. 2D). Addition of BFA rapidly inhibited the increase in fluorescence of E2 PM patches. Similar results were obtained with monensin, an ionophore that inhibits transport from the medial cisternae to the trans-Golgi

Journal of Virology


FIG 3 Virus-induced plasma membrane extensions. (A) (Three left panels) Vero cells stably expressing the plasma membrane marker PM-GFP were mock infected (uninfected [Uninf.]) or infected with WT or WT-mCherry, incubated at 37°C for 8 h, and fixed. Cells infected with WT were permeabilized and stained with antibody against E2. Images were acquired with the DuoScan confocal microscope and are representative of the images from three independent experiments. Bar ⫽ 10 ␮m. IF, immunofluorescent. (Right panel) The number of extensions per cell was determined by counting the total number of extensions in 10 cells. (B) (Three left panels) Vero cells were infected with WT or WT-mCherry, incubated at 37°C for 8 h, fixed, permeabilized, and stained with antibody against tubulin. Images were acquired with the DuoScan confocal microscope and are representative of the images from three independent experiments. Bar ⫽ 5 ␮m. Representative images for WT-mCherry are shown in the leftmost panel. Arrows, short extensions that are positive for E2 but do not colocalize with tubulin; arrowheads, longer E2-positive extensions that are also positive for tubulin. (Right panel) The length of extensions in cells infected with WT or WT-mCherry was quantitated as described for panel A, and the extensions were separated on the basis of their tubulin contents. (C) Vero cells stably expressing PM-GFP were infected with WT-mCherry or transfected with Y400K-mCherry RNA and incubated, stained, and quantitated as described for panel B. The graphs in panels A to C represent the means and standard error of the means of three independent experiments. **, P ⬍ 0.01. All other differences were not significant. (D) Quantitation of the extensions in noninfected cells or in cells infected as described for panel C. The bars show the total number of tubulin-positive or -negative extensions in 30 cells (10 cells from 3 different experiments).

network (50) (data not shown). Thus, the E2 PM patches acquired new E2 via vesicular delivery. Virus-induced formation of plasma membrane extensions. Earlier studies (27, 28, 51) and our observations of infected cells suggested that alphavirus infection causes the formation of filopodium-like extensions. As a tool to follow this, we generated a Vero cell line stably expressing a fluorescent PM marker protein, here referred to as PM-GFP. PM-GFP contains cytoplasmically oriented EGFP fused to a membrane anchor and diffuses freely within the PM (36). Preliminary studies showed that the level of PM-GFP in the cell line was relatively unchanged during 10 h of culture in the presence of cycloheximide (data not shown), indicating the relative resistance of the marker to virus inhibition of host protein synthesis. We used this cell line to compare the number of extensions in uninfected cells with that in cells infected with wild-type SINV or WT-mCherry (Fig. 3A). Uninfected cells showed relatively homogeneous PM-GFP staining and smooth cell borders with few extensions. Virus infection significantly increased the number of filopodium-like extensions, with compara-


ble induction by WT and WT-mCherry. Similar levels of extensions were also induced by WT-GFP or by infection of the parental (unlabeled) Vero cells or in BHK, U-2 OS, C6/36, HeLa, or CHO cells (data not shown). We characterized these extensions for tubulin, a frequent component of such elongated structures in noninfected cells (52, 53). Two morphologically distinct types of extensions were detected: tubulin-negative extensions that were relatively short (⬃2 to 4 ␮m; Fig. 3B, arrows and graph) and tubulin-positive extensions that were significantly longer (⬎10 ␮m; Fig. 3B, arrowheads and graph). The proportion of each type of extension and the length of each type were not affected by the E2 fluorescent tag (Fig. 3B, graph; data not shown). The tubulin-positive extensions also stained with phalloidin, indicating the presence of polymerized actin along the length of the extension (data not shown). Actin was also frequently detected at the base of the tubulin-negative extensions (data not shown). Similar studies were then performed in cells infected with the nonbudding Y400K-mCherry. Tubulin-negative extensions were

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FIG 4 Exclusion of a PM marker during biogenesis. Vero cells stably expressing the plasma membrane marker PM-GFP were transfected with WT-mCherry RNA (top) or Y400K-mCherry RNA (bottom). Cells were incubated at 37°C for 8 h, fixed, permeabilized, stained with antibody against tubulin, and imaged by the DuoScan confocal microscope. Images from an optical section close to the coverslip surface are shown. Arrows, tubulin-negative extensions; arrowheads, tubulin-positive extensions. Images are representative examples of the images from three independent experiments. Bar ⫽ 10 ␮m.

of similar length in the absence of the E2-Cp interaction, but the tubulin-positive extensions were significantly shorter (Fig. 3C). Strikingly, in Y400K-mCherry-infected cells, the number of tubulin-positive extensions was reduced almost to the level found in uninfected cells (Fig. 3D, left). In contrast, Y400K-mCherry-infected cells showed a large increase (⬃4-fold) in the number of tubulin-negative extensions compared to that for WT-infected cells (Fig. 3D, right). Thus, alphavirus infection induced two distinct types of extensions: shorter, tubulin-negative extensions that occurred with or without virus budding and longer, tubulin-positive extensions that correlated with active virus budding. We henceforth define short extensions as those from 2 to 4 ␮m in length and long extensions as those ⱖ10 ␮m in length. Exclusion of PM proteins from extensions. Alphavirus particles have highly organized structures and do not incorporate cellular PM proteins into their envelope. Thus, at some point during virus biogenesis, cellular proteins are excluded from virus assembly/budding sites. To monitor exclusion in live cells, we used our Vero cell line stably expressing PM-GFP. As expected, mocktransfected cells showed few extensions (data not shown). Transfection with WT-mCherry RNA produced a significant increase in PM extensions, with a high proportion of the short, tubulin-negative E2-labeled extensions showing exclusion of the PM-GFP marker (Fig. 4, top, arrows). Some short extensions contained distinct, nonoverlapping areas of E2 and PM-GFP fluorescence, while others completely excluded PM-GFP throughout their length. Live-cell imaging demonstrated that the short extensions tended to progress from partial to complete exclusion with continued time of infection (see Movie S1 in the supplemental material). In contrast, the long, tubulin-positive extensions did not show a significant exclusion of the PM-GFP marker (Fig. 4, top, arrowhead). The overall patterns of exclusion were comparable when the endogenous PM protein Na⫹/K⫹ ATPase was used as a marker (data not shown). In addition, exclusion of PM-GFP occurred in the short extensions of cells infected with unlabeled WT and immunostained for E2 or those of cells infected with Cp LLmCherry (data not shown). However, while cells infected with the Y400K-mCherry had large numbers of short tubulin-negative extensions, all showed colocalization of the PM-GFP marker with E2-mCherry (Fig. 4, bottom). Dynamics of viral envelope proteins in extensions. Although earlier studies showed that E1/E2 heterodimers become relatively immobile at late times of SINV infection, the meaning of this

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finding remained unclear (54). We used fluorescence recovery after photobleaching (FRAP) to study the mobility of PM-GFP, the E2 protein from WT-mCherry, and the E2 protein from Y400K-mCherry. As predicted, the fluorescence intensity of the PM-GFP marker displayed rapid and almost complete recovery from photobleaching, consistent with its free diffusion in the PM (Fig. 5A). FRAP analysis was performed on the short extensions in WT-mCherry-infected Vero cells (Fig. 5B). The E2 protein present in these structures was immobile, showing essentially no recovery of fluorescence intensity during the time course of the experiment. In contrast, FRAP analysis of Y400K-mCherry-infected cells showed significant recovery of the fluorescence intensity in the short extensions (Fig. 5B). Recovery was more efficient than that in WT-mCherry-infected cells, although it was less complete than that for the small PM marker. We then compared the dynamics of a lipid probe, CellMask orange, with those of the E2 protein in WT-GFP-infected Vero cells (Fig. 5C). While the E2 protein in short extensions was essentially immobile, the fluorescence of the lipophilic dye in the same extensions showed rapid recovery. Together, our results indicate that formation of the glycoprotein lattice in Y400K mutant-infected cells was not sufficient to immobilize the envelope proteins or to exclude PM proteins. Instead, the interaction of the E2 cytoplasmic domain with Cp/NC mediated the immobilization of E2 and the exclusion of PM proteins, while it still allowed the rapid diffusion of a lipophilic dye. Sites of virus budding at the PM. We used CLEM methods to characterize the E2 PM patches and extensions and to determine if they were sites of virus particle production. Vero cells were transfected with WT-mCherry or Y400K-mCherry RNA. The cells were fixed at 9 h posttransfection, and mCherry-labeled E2 was imaged using wide-field fluorescence microscopy. Since our CLEM system requires the use of wide-field microscopy, we focused on the cell borders to ensure visualization of the fluorescence at the PM. The samples were then processed for scanning electron microscopy (SEM), and the cells imaged in the fluorescence microscope were located by the use of fiduciary markers on the coverslips. Representative SEM images of cells transfected with WTmCherry or Y400K-mCherry RNA or mock-transfected cells are shown in Fig. 6. The WT-mCherry-infected cells showed abundant particles across the cell surface, along the cell borders, and on plasma membrane extensions (Fig. 6D, E, and H). Correlation between the fluorescence and SEM images of WT-mCherry-in-

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FIG 5 Dynamics of E2 in virus-infected cells. (A and B) (Left) Vero cells stably expressing the PM-GFP marker were mock transfected (A) or transfected with WT-mCherry or Y400K-mCherry RNA (B), incubated at 37°C for 6 h, and transferred to imaging medium. FRAP analysis was performed using the DuoScan confocal microscope. Images were acquired at 10-s intervals for 250 s. White dotted boxes, bleached areas. Bars ⫽ 5 ␮m. (Right) Mean and standard error of the mean of the percentage of fluorescence intensity at time zero of the bleached areas from 14 cells in 2 independent experiments. (C) (Left) Vero cells were infected with WT-GFP and incubated at 37°C for 6 h. Cells were stained with CellMask orange lipid marker and transferred to imaging medium. FRAP analysis was performed, and the results were analyzed as described for panels A and B, with images acquired continuously in both channels over 25 s. (Top) The dynamics of E2-GFP; (bottom) the dynamics of the lipid marker in the same FRAP region in the same cell.

fected cells showed that the previously observed fluorescent patches and extensions corresponded to sites containing nascent virus particles (Fig. 6E to J). The size of the particles observed by SEM was consistent with that of alphaviruses (diameter, ⬃70 nm). These virus-sized particles were not detected in cells infected with the nonbudding Y400K-mCherry, although numerous short PM extensions containing diffusely distributed E2 fluorescence were observed (Fig. 6K to N). In keeping with our earlier results, SEM of mock-transfected cells revealed a relatively smooth surface with few extensions (Fig. 6A to C). Long extensions were also visualized by CLEM. In agreement with the prior fluorescence analysis (Fig. 3D), these structures were rare in the Y400K-mCherry-infected cells (data not shown). The long extensions in the WT-mCherry-infected cells showed nascent virus particles concentrated at the tip (Fig. 6O to Q). Interestingly, these extensions were frequently in contact with cells that did not show detectable virus particles or E2 fluorescence. Long extensions and virus transmission. To characterize the potential role of the long extensions in virus transmission, we cocultured WT-mCherry-infected Vero cells with noninfected Vero cells expressing the PM-GFP marker. During coculture we


observed long extensions with a single bulky tip originating from infected cells and contacting neighboring uninfected cells (Fig. 7), consistent with the CLEM results in Fig. 6O to Q. Live-cell imaging revealed that the tip of these extensions contained fluorescent foci corresponding to the amount of E2 fluorescence in individual virus particles imaged under the same conditions. These foci were frequently released close to or in contact with the noninfected cell and were subsequently internalized. The extension then retracted back to the infected cell, as shown in the time series in Fig. 7 and in Movies S2 and S3 in the supplemental material. Such foci were observed emanating from WT-mCherry-infected cells but not from Y400K-mCherry-infected cells (data not shown). Together, our CLEM and live-cell imaging results indicate that the E2 PM patches and short extensions corresponded to sites of nascent virus particle production. In contrast, long extensions preferentially showed virus particles at the tip, where they could be transferred to noninfected cells. DISCUSSION

We describe the use of SINVs engineered with fluorescent protein tags to follow the transit and dynamics of E2 during alphavirus

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FIG 7 Long extensions are involved in cell-to-cell particle transfer. Vero cells were infected with WT-mCherry and incubated for 4.5 h at 37°C. Uninfected Vero cells stably expressing PM-GFP were then plated onto the infected cells, and the cocultures were incubated for 3 h at 37°C. The cells were then imaged using the TIRFM in the wide-field mode and both the 561-nm and 488-nm lasers, and images were acquired every 10 s for 90 min. The time series in panels A and B show the same fields, with only the E2-mCherry signal being shown in panel A and both E2-mCherry and the PM-GFP marker being shown in panel B. Arrowheads, a released virus-sized particle. Images are representative of those from three independent experiments. Bar ⫽ 5 ␮m.

infection. Previous morphological studies of alphavirus assembly and budding were based primarily on electron microscopy methods, such as transmission, scanning, freeze-etch, and freeze fracture electron microscopy (26–28), and/or on surface labeling of cells with antibodies to the envelope proteins (54, 55). These early studies demonstrated local enrichment of envelope proteins in patches and extensions and exclusion of host membrane proteins from nascent virus particles but by necessity were based on static measurements. The intrinsic nature of a fluorescent protein fusion enables live-cell imaging of E2 in real time and provides uniform labeling without concerns of antibody accessibility. Our characterization indicated that GFP- or mCherry-tagged E2 proteins were accurate reporters for the virus exit pathway, displaying an authentic cellular distribution, producing morphologically normal virus particles by budding from the PM, and showing excellent stability during virus replication (see also references 32 to 34). We used this system to image the delivery of E2 to the PM by exocytic vesicular traffic. Our studies demonstrated that vesicles containing GFP-labeled E2 (GFP-E2) had ⬃10 times the fluorescence intensity of one viral particle. TIRFM showed that after the initial arrival of a WT E2-GFP vesicle within the PM focal plane, the vesicle displayed brief movements underneath the cell surface, followed by rapid immobilization in localized patches at the PM. Once they were docked at the PM, these patches showed a gradual and sustained increase in fluorescence, a process mediated by the continued exocytic delivery of E2 to the PM. In contrast, when the Cp-E2 interaction was inhibited by the E2 Y400K mutation, E2 was delivered to the PM but rapidly diffused to form a homogeneous distribution on the PM. This result suggests that the Cp may serve as scaffolding on the E2-transporting vesicle, impairing the diffusion of E2 once the vesicle fuses with the PM.

Previous FRAP studies with labeled antibodies showed that SINV envelope proteins on the PM are immobilized early in infection and suggested that this effect is mediated primarily by interactions between the envelope proteins, rather than E2-Cp interactions (54). Our FRAP experiments confirmed that the GFP-E2 protein was strikingly immobile on the PM of WT virusinfected cells. However, inhibition of the E2-Cp interaction significantly increased E2 mobility, in keeping with the diffuse distribution of Y400K E2 that we observed on the PM. Together, our results suggest that important interactions between E2 and Cp may already have occurred before delivery of the envelope proteins to the PM. These interactions may prevent the escape of the protein by limiting its diffusion into the bulk PM protein pool. The patchy distribution of E2 in the Cp LL mutant suggests that these key interactions do not require the formation of cytoplasmic nucleocapsids, and thus, they may occur with Cp monomers and/or Cp oligomers that may already be binding the viral RNA genome (as discussed in references 14 and 56). By infecting a stable cell line expressing the PM-GFP marker with mCherry E2-labeled viruses, we were able to image the process of exclusion of host membrane proteins during SINV infection. The PM marker protein was excluded from both the E2 PM patches and the short filopodial extensions. Exclusion required the E2-Cp interaction but not the presence of cytoplasmic NC. Live-cell imaging of the extensions suggested a gradual process of exclusion, and FRAP studies using E2-GFP and a lipophilic dye showed that an extension could simultaneously show rapid lipid diffusion but drastic immobilization of E2. Further studies will be required to determine if host PM proteins are selectively cleared from the extensions by endocytosis or if other mechanisms prevent their continued delivery into the forming extension. Our imaging studies provide the first evidence that alphavirus

FIG 6 CLEM analysis of nascent virus particles. Vero cells were mock transfected (A to C) or transfected with WT-mCherry (D to J, O to Q) or Y400K-mCherry (K to N) RNA, incubated at 37°C for 9 h, fixed, and imaged using a Zeiss AxioObserver microscope with software with a shuffle-and-find feature. Cells were then processed for SEM and imaged using a Zeiss Supra 40 field emission SEM. (A, D, and K) Representative SEM images of cells infected with the indicated viruses. Magnification, ⫻6,000. Bar ⫽ 1 ␮m. (B, C, E, H, L) SEM images of the regions indicated by the dashed white boxes in panels A, D, and K. Magnification, ⫻10,000. Bar ⫽ 1 ␮m. (F, I, and M) Fluorescence of the indicated E2-mCherry proteins. Magnification, ⫻10,000. (G, J, and N) Overlay of the SEM and fluorescence images. (O to Q) SEM (O) and fluorescence (P) images of an infected cell strongly labeled by E2-mCherry showing many virus-sized particles (arrowheads) interacting with an E2/particle-negative cell (arrow). Magnification, ⫻10,000. The region demarcated by the dashed white box is shown in panel Q as the overlay of the SEM and fluorescence images. Magnification, ⫻25,000. Bars ⫽ 1 ␮m.


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infection induces the formation of two distinct types of PM extensions. The short extensions contained all of the viral structural proteins, strongly excluded the PM marker protein, and were negative for tubulin and actin. CLEM analysis of WT-infected cells showed that these extensions contained numerous virus budding structures distributed all along their length. In contrast, long extensions contained tubulin and actin filaments and did not exclude the PM marker. While the long extensions showed generalized staining for E2, CLEM analysis showed nascent virus particles only at the tip of the extension. While Y400K mutant-infected cells produced abundant short extensions, long extensions were selectively decreased, suggesting that formation of these extensions required the process of active virus budding. Coculture experiments demonstrated that such long extensions interacted with adjacent noninfected cells and released particles with the size and fluorescence level observed in our imaging analyses of isolated virus particles. These data suggest that the long extensions mediate cell-tocell virus particle transfer. Many questions about this process remain to be addressed, including the frequency of these cell-cell contacts, their selectivity for infected versus noninfected target cells, and their biological importance in virus transmission. For both types of extensions, the proteins and mechanisms that mediate their formation, interactions, and retraction remain to be determined. Our current work defines aspects of the complex process by which alphaviruses assemble and bud from host cells and suggests important mechanistic questions for future study. ACKNOWLEDGMENTS We thank Youqing Xiang for technical assistance, all of the members of the Kielian lab for their experimental advice and discussion, and Mathieu Dube, Katie Stiles, and Yan Zheng for their helpful comments on the manuscript. We thank Claudia Sánchez-San Martín for the electron microscopy images in Fig. 1B and Nicolas Fourel for computer programming and software assistance. We thank the staff of the Albert Einstein College of Medicine Analytical Imaging Facility for their helpful assistance with light and electron microscopy and David Entenberg and the Gruss Lipper Biophotonics Center for assistance with and use of the TIRF microscope. This work was supported by a grant to M.K. from the National Institute of General Medical Sciences (GM-057454) and by Cancer Center Core support grant NIH/NCI P30-CA13330. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

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