Development of Sindbis Viruses Encoding nsp2/gfp Chimeric Proteins and Their Application for Studying nsp2 Functioning

Transcription

1 JOURNAL OF VIROLOGY, May 2007, p Vol. 81, No X/07/$ doi: /jvi Copyright 2007, American Society for Microbiology. All Rights Reserved. Development of Sindbis Viruses Encoding nsp2/gfp Chimeric Proteins and Their Application for Studying nsp2 Functioning Svetlana Atasheva, 1 Rodion Gorchakov, 1 Robert English, 2 Ilya Frolov, 1 and Elena Frolova 1,3 * Department of Microbiology and Immunology, 1 Mass Spectrometry Laboratory, Biomolecular Resource Facility, 2 and Department of Biochemistry and Molecular Biology, 3 University of Texas Medical Branch, Galveston, Texas Received 13 December 2006/Accepted 20 February 2007 Sindbis virus (SINV) is one of almost 30 currently known alphaviruses. In infected cells, it produces only a few proteins that function in virus replication and interfere with the development of the antiviral response. One of the viral nonstructural proteins, nsp2, not only exhibits protease and RNA helicase activities that are directly involved in viral RNA replication but also plays critical roles in the development of transcriptional and translational shutoffs in the SINV-infected cells. These multiple activities of nsp2 complicate investigations of this protein s functions and further understanding of its structure. Using a transposon-based approach, we generated a cdna library of SINV genomes with a green fluorescent protein (GFP) gene randomly inserted into nsp2 and identified a number of sites that can be used for GFP cloning without a strong effect on virus replication. Recombinant SIN viruses encoding nsp2/gfp chimeric protein were capable of growth in tissue culture and interfering with cellular functions. SINV, expressing GFP in the nsp2, was used to isolate nsp2-specific protein complexes formed in the cytoplasm of the infected cells. These complexes contained viral nsps, all of the cellular proteins that we previously coisolated with SINV nsp3, and some additional protein factors that were not found before in detectable concentrations. The random insertion library-based approach, followed by the selection of the viable variants expressing heterologous proteins, can be applied for mapping the domain structure of the viral nonstructural and structural proteins, cloning of peptide tags for isolation of the protein-specific complexes, and studying their formation by using live-cell imaging. This approach may also be applicable to presentation of additional antigens and retargeting of viruses to new receptors. The alphavirus genus of the Togaviridae family contains almost 30 members that are widely distributed on all continents, except for the Antarctic area. Some of the New World alphaviruses, including the Venezuelan (VEEV), Eastern, and Western equine encephalitis viruses, cause severe encephalitis in humans and animals that can result in death or neurological disorders (9, 20, 22, 29). Others, such as Sindbis virus (SINV), are less pathogenic and rarely cause human-associated disease but rather represent a good model for studying the mechanism of alphavirus replication and some of the aspects of virus interaction with the host cells. Alphavirus pathogenesis and the development of a cytopathic effect (CPE) in cultured cells are likely to be determined by multiple factors, and both structural and nonstructural viral proteins are critically involved in the development of these phenomena. However, our understanding of their molecular mechanisms is very incomplete. The alphavirus genome encodes only a few proteins and consists of a single-stranded RNA molecule of positive polarity that is approximately 11.7 kb in length (24, 46, 48). The genome mimics the structure of cellular messenger RNAs, in that it contains a 5 methylguanylate cap and a 3 polyadenylate tail. Four nonstructural proteins (nsp1 to -4 [nsp1-4]) are translated directly from the viral genome as a polyprotein and form, together with cellular proteins, the replication complex (RC) * Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX Phone: (409) Fax: (409) elfrolov@utmb.edu. Published ahead of print on 28 February required for viral genome replication and transcription of the subgenomic RNA. The latter RNA serves as a template for translation of the structural proteins comprising viral particles. The nsp1-4 polyprotein is differentially processed during the alphavirus replication cycle, and the level of processing determines the ability of RC to synthesize either minus- or plus-strand RNAs from the virus-specific RNA promoters (27, 28, 45). The RC appears to have a complex structure, with all of the viral nsps involved in its functioning (47). In previous studies, nsp1 demonstrated both guanine-7-methyltransferase and guanylyl-transferase activities required for capping of newly synthesized viral genomic and subgenomic RNAs. This protein was also predicted to bind to cellular membranes (2, 3). The exact functions of nsp3 have not been determined yet. However, both our studies and results from other research groups demonstrated that, in infected cells, this protein forms highorder complexes, which contain other viral nsps and virusspecific RNAs (8, 14, 16, 35). nsp4, which expresses RNAdependent RNA polymerase activity, is present at a very low concentration in the infected cells and is a key component of viral replicative complexes (47). Another nonstructural protein, nsp2, is widely distributed in the cytoplasm. This protein was detected in the cytoplasm both in the large, nsp3-containing complexes (14, 19) and outside of these structures and in the nuclei of the infected cells (19, 36). nsp2 is critically involved in the development of transcriptional and translational shutoffs occurring in mammalian cells during SINV and Semliki Forest virus (SFV) replication (13, 15, 19, 37), and the expression of this protein alone was shown to be highly cyto- 5046

2 VOL. 81, 2007 SINDBIS VIRUS ENCODING nsp2/gfp 5047 toxic and inhibited the transcription of cellular ribosomal and poly(a)-containing RNAs (17). Some of the mutations outside of the protease and helicase domains, in the carboxy-terminal fragment of nsp2, make viruses and replicons less cytopathic and highly attenuated (13, 17, 37, 38). Moreover, the carboxyterminal fragment of VEEV nsp2 has been recently crystallized and was found to have not only a protease domain but also an additional distinct domain with a structural similarity to S-adenosyl-L-methionine-dependent RNA methyltransferases (42). All of the adaptive mutations that make viral RNA replication less efficient and reduce the cytopathogenicity of the viruses (10, 13, 37, 38) are concentrated in a short peptide of the latter carboxy-terminal domain whose function remains to be defined. These data strongly indicated that alphavirus nsp2 is involved not only in the replication of alphavirus genome and transcription of the subgenomic RNA but has additional activities in the interaction of these viruses with the host cells. The multiple functions of alphavirus nsp2 suggest that this protein is one of the most important targets in alphavirus research; on the other hand, the presence of such a large number of activities strongly complicates structure-functional investigation of this protein. In reverse genetics experiments, the mutations in nsp2 might have multiple effects on virus replication and generate data that are difficult to interpret. An additional problem in studying the mechanism(s) of nsp2 s function lies in its presence in different cellular compartments and its likely formation of a variety of function-specific complexes with cellular proteins. Studying the structure and functioning of these complexes requires their affinity purification using antibodies specific to nsp2 or to specific tags. However, such tag insertions should not interfere with the protein s activities in virus replication; otherwise, the isolation and analysis makes little sense. In the present study, we were interested in further understanding the role of nsp2 in SINV-host cell interactions. Using a transposon-based approach, we generated a cdna library of SINV genomes with GFP randomly inserted into the nsp2 gene and, after selecting viable, green fluorescent protein (GFP)-expressing variants, we identified a number of sites in nsp2 suitable for the insertion of large peptides and proteins without a strong effect on virus replication. The insertions generated important information about functional domains of the nsp2 and were used for isolation of the nsp2-specific protein complexes from the cytoplasm of the SINV-infected cells. MATERIALS AND METHODS Cell cultures. BHK-21 cells were kindly provided by Sondra Schlesinger (Washington University, St. Louis, MO). Cells were maintained at 37 C in alpha minimum essential medium ( MEM) supplemented with 10% fetal bovine serum (FBS) and vitamins. Construction of cdna library of SINV genomes with GFP gene randomly inserted into nsp2. The protocol was designed using the library construction strategies described by London et al. (31) and Moradpour et al. (33). (i) The 2,876-nucleotide (nt) fragment of SINV genome that contains 248 nt of nsp1, the entire nsp2 gene, and 189 nt of nsp3 was cloned into the prs2 plasmid (analog of puc-19). Then, the transprimer sequence encoding a kanamycin resistance gene was cloned into this plasmid using the GPS-LS scanning system (New England Biolabs). The library of random insertions was represented by 57,000 ampicillin-resistant, kanamycin-resistant Escherichia coli clones. The clones were harvested from the plates and, after 3 h of additional growth at 37 C in liquid medium, plasmid DNA was isolated. (ii) Next, 10 g of transprimer-containing plasmid library was digested by PmeI to remove the kanamycin resistance gene, and the GFP sequence was cloned into the same site for further manipulations. This secondary library was also represented by more than 54,000 clones, and they were also harvested from the plates to isolate the plasmid DNA. (iii) The isolated plasmid pool was digested by AfeI and AvrII, and a fragment, containing the SINV nsp2 sequence with random GFP insertions, was isolated and cloned into the plasmids encoding the infectious cdna of SINV Toto1101 genome (40) to replace the original sequence between the AfeI and AvrII restriction sites. These final libraries were also represented by more than 45,000 E. coli clones that were harvested from the agar plates. Isolated plasmids were used for in vitro transcription, followed by manipulations with RNA, as described below. Plasmids. Plasmids encoding the infectious clone of the wild-type (wt) SINV Toto1101 and SIN/2V/389 viral genomes were described elsewhere (14, 40). They were used without additional modifications. psinrep/nsp2gfp/472 and psin/ nsp2gfp/472 encoded the SINV replicon (having a Pac gene under control of the subgenomic promoter) (13) and the SINV genome, respectively, with the insertion of the entire GFP-coding sequence after amino acids (aa) 472 of the nsp2. psin/nsp2gfp/8 encoded the SINV genome with the GFP insertion after aa 8 of the nsp2. psin/nsp2gfp/nsp3cherry encoded the SINV genome with GFP insertion after aa 472 of the nsp2 and Cherry (44) after aa 389 of the nsp3. The schematic representation of the constructs is shown in the corresponding figures. Sequences can be provided upon request. RNA transcriptions. Plasmids were purified by centrifugation in CsCl gradients. Before the transcription reaction, plasmids were linearized by using the XhoI restriction site located downstream of the poly(a) sequence. RNAs were synthesized by SP6 RNA polymerase in the presence of cap analog under the previously described conditions (40). The yield and integrity of transcripts were analyzed by gel electrophoresis under nondenaturing conditions. Transcription reactions were used for electroporation without additional purification. RNA transfections. BHK-21 cells were electroporated by using previously described conditions (30). For the individual plasmids, the infectious center assay was performed in parallel as previously described (19). For the GFP insertion library, 5 g of the in vitro-synthesized viral genome RNA was transfected into BHK-21 cells, and different numbers of these cells were seeded into 100-mm dishes containing subconfluent monolayers of naive BHK-21 cells. After 1 h of incubation at 37 C, the monolayers were covered with 0.5% agarose, supplemented with MEM and 2% FBS. Individual, GFP-positive plaques were isolated after 1 day of incubation at 37 C. These plaque-purified viruses were further analyzed. Identification of the insertion sites. Viruses eluted from the individual plaques were used for infection of naive BHK-21 cells in 35-mm dishes. Stocks were harvested at 24 h postinfection, and cells were used for RNA isolation. The insertion sites were identified by synthesizing DNA fragments using reverse transcription-pcr, followed by their direct sequencing. One of the PCR primers used was specific to GFP sequence; the second primer depended on the synthesized fragment and was specific either to the nsp1 or the nsp3 gene. Viral replication analysis. BHK-21 cells were seeded at a concentration of cells/35-mm dish. After 4hofincubation at 37 C, the formed subconfluent monolayers were infected with different viruses at a multiplicity of infection (MOI) of 10 PFU/cell for 1 h, washed three times with prewarmed medium, and overlaid with 1 ml of complete medium. At the indicated times postinfection, the media were replaced, and virus titers in the harvested samples were determined via a plaque assay on BHK-21 cells as previously described (26). Analysis of SINV P123 processing by Western blotting and pulse-chase experiments. BHK-21 cells were seeded at a concentration of cells per 35-mm dish and infected with different viruses at the MOIs indicated in the figure legends. At 9 h postinfection the cells were harvested, and equal amounts of proteins were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis. After protein transfer, the nitrocellulose membranes were processed by rabbit anti-sinv nsp2 antibodies or anti-nsp3 antibodies. Horseradish peroxidase-conjugated secondary donkey anti-rabbit antibodies were purchased from Santa Cruz Biotechnology. Horseradish peroxidase was detected by using the Western blotting Luminol reagent according to the manufacturer s recommendations (Santa Cruz Biotechnology). In the pulse-chase experiments, proteins were metabolically labeled at 3 h postinfection with [ 35 S]methionine (20 Ci/ml) in DMEM lacking methionine supplemented with 0.1% FBS. After 30 min incubation at 37 C, media were replaced by 2 ml of complete MEM supplemented with 10% FBS, and incubation was continued for 15, 30, or 60 min at 37 C. The cells were lysed, and proteins were immunoprecipitated by rabbit anti-sinv nsp2 antibodies and Omnisorb (Calbiochem) as described elsewhere (1) and then analyzed by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis, followed by autoradiography.

3 5048 ATASHEVA ET AL. J. VIROL. RNA analysis. Cells were infected with viruses at an MOI of 20 PFU/cell. At the times indicated in the figure legends, virus-specific RNAs were labeled with [ 3 H]uridine in the presence of 1 g of dactinomycin (ActD)/ml. RNAs were isolated from the cells by TRIzol reagent, as recommended by the manufacturer (Invitrogen), and then they were denatured with glyoxal in dimethyl sulfoxide and analyzed by agarose gel electrophoresis using the previously described conditions (5). In some of the experiments, the RNA bands were excised from the 2,5-diphenyloxazole-impregnated gels, and the radioactivity was measured by liquid scintillation counting. Analysis of protein synthesis. BHK-21 cells were seeded into six-well Costar plates at a concentration of cells/well. After 4hofincubation at 37 C in 5% CO 2, the cells were infected at an MOI of 20 PFU/cell. At 4, 8, and 20 h postinfection, the cells were washed three times with phosphate-buffered saline (PBS) and then incubated for 30 min at 37 C in 0.8 ml of Dulbecco modified Eagle medium lacking methionine, supplemented with 0.1% FBS and 20 Ci of [ 35 S]methionine/ml. After this incubation, cells were scraped into the media, pelleted by centrifugation, and dissolved in 400 l of standard protein gel loading buffer. Equal amounts of proteins were loaded onto sodium dodecyl sulfate 10% polyacrylamide gels. After electrophoresis, gels were dried, autoradiographed, and analyzed on a Storm 860 PhosphorImager (Molecular Dynamics). The amount of radioactivity detected in the protein band corresponding to actin was used to evaluate the residual host cell protein synthesis. The results were normalized on the amount of radioactivity detected in the actin-containing fragment of the lane representing the uninfected cells. Isolation of nsp2-specific protein complexes. Subconfluent BHK-21 cells (10 7 cells per 150-mm-diameter dish) were infected either with SIN/nsP2GFP/472 virus or SINrep/nsP2GFP/472 replicon (or control SINrep/GFP replicon) at an MOI of 20 PFU/cell or infectious units/cell, respectively. Cell lysis and purification of the proteins were performed as previously described (4). Briefly, at 8 h postinfection, cells were washed with PBS and then scraped and pelleted by centrifugation at 1,000 g. Next, they were suspended in hypotonic buffer (10 mm Tris-HCl [ph 7.5], 10 mm NaCl, 5 mm MgCl 2, and 1 protease inhibitor cocktail [Roche]) and, after 15 min of incubation on ice, broken in a tight glass Dounce homogenizer. Nuclei were pelleted by centrifugation at 900 g for 5 min at 5 C. The postnuclear supernatant was used for coimmunoprecipitation of cellular proteins. The NaCl concentration was adjusted to 150 mm, and NP-40 was slowly added to 1%, after which incubation on ice continued for 30 min. Next, the lysate was centrifuged at 15,000 g for 10 min at 5 C, and the supernatant was mixed with 50 l of MACS beads (Miltenyi Biotec) with covalently linked, affinity-purified anti-gfp antibodies. After 1 h of incubation at 5 C, the suspension was loaded on the Columns (Miltenyi Biotec), and then the columns were washed with NP buffer (10 mm Tris-HCl [ph 7.5], 150 mm NaCl, 5 mm MgCl 2, 1% NP-40, and 1 protease inhibitor cocktail), and bound proteins were eluted in 40 l of protein gel sample buffer. They were separated on sodium dodecyl sulfate 10% polyacrylamide gels (25) and stained with Coomassie brilliant blue R-250. In parallel, the same affinity purification procedure was applied to the cells infected with control virus or replicon, encoding native GFP under control of the subgenomic promoter. The Coomassie blue-stained bands were excised from the gels, and the proteins were identified by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS). The data were acquired with an Applied Biosystems 4700 MALDI-TOF/ TOF Proteomics Analyzer. The Applied Biosystems software package included a 4000 Series Explorer (v.3.0 RC1) with an Oracle Database Schema Version (v ), Data Version (3.80.0), to acquire both MS and MS/MS spectral data. Applied Biosystems GPS Explorer (v.3.0) software was used in conjunction with MASCOT to search the NCBI database using both MS and MS/MS spectral data for protein identification. Protein match probabilities were determined by using expectation values and/or MASCOT protein scores. For protein identification, both rodent and viral taxonomies were searched in the National Center for Biotechnology Information and/or Swiss-Prot database(s). Microscopy. For confocal microscopy, BHK-21 cells were seeded on glass chamber slides and infected at an MOI of 20 PFU/cell with recombinant viruses and incubated at 37 C. At 8 h postinfection, they were fixed in 1% formaldehyde in PBS and analyzed on a Zeiss LSM510 META confocal microscope with a NA oil immersion planapochromal lens. For three-dimensional analysis, infected cells were fixed with 1% formaldehyde for less than 10 min at different times postinfection, and images were acquired by using a NA oil immersion planapochromal lens. The image stacks were further processed using Huygens Essential v2.7 deconvolution software (Scientific Volume Imaging) and 3D rendering software Imaris v4.2 (Bitplane AG). FIG. 1. Selection of replicating SIN viruses encoding nsp2/gfp protein. A library of viral genomes having random GFP insertions in the nsp2 gene was generated as described in Materials and Methods. After transfection of the RNA pools, the genomes of the GFP-expressing viruses from the individual plaques were sequenced to identify the sites of the insertions. The numbers of amino acids, after which the insertions occurred, are indicated. RESULTS Recombinant, nsp2/gfp-expressing viruses. The SINV nsp2 appears to have a wide spectrum of functions in alphavirus replication, and analysis of its amino acid sequence suggested the existence in this protein of several structural and functional domains. There was not enough information in the literature about the sites in the SINV nsp2 that could be potentially used for insertion of the tag polypeptides without altering the activities of this protein in virus replication. Our previous experience suggested that the GFP tag is large but appears to be convenient for affinity purification of the protein complexes, formed during virus replication, and for studies of the complexes formation and functioning in live cells (14). The nspbound proteins can be isolated by using GFP-specific antibodies linked to magnetic beads, and the recombinant, GFP-containing viral nsps can be easily monitored in live cells at different times postinfection, using either confocal or inverted UV microscopes. To identify site(s) that can be used for GFP insertions, we randomly cloned GFP-encoding DNA fragment into the nsp2 gene by using a transposon-based approach (see Fig. 1 and Materials and Methods for details). The entire library of the recombinant nsp2/gfp genes was then transferred into wt SINV Toto1101 genome. Almost clones containing SINV cdna with random GFP insertions between nt 1404 and 4285 were generated. This fragment covers the entire nsp2 and short sequences of nsp1 and nsp3. In vitro-synthesized, recombinant SINV RNA pool was transfected into BHK-21 cells, and different numbers of these cells were seeded on the monolayer of naive cells and covered by the agarose overlay to prevent mixing of the viruses (see Materials and Methods for details).

4 VOL. 81, 2007 SINDBIS VIRUS ENCODING nsp2/gfp 5049 FIG. 2. Recombinant viruses encoding nsp2/gfp and analysis of their replication in BHK-21 cells. (A) Schematic representation of wt SINV Toto1101 genome and genomes of SIN/nsP2GFP/8 and SIN/ nsp2gfp/472, containing in-frame insertions of the entire GFP gene after aa 8 and 472 of nsp2, respectively. Arrows indicate the positions of the subgenomic promoter. (B) Infectivity of in vitro-synthesized viral RNAs and comparative plaque sizes determined in the infectious center assay (see Materials and Methods for details). Plaques were stained after 48 h of incubation under agarose cover. (C) BHK-21 cells were infected with the indicated viruses at an MOI of 10 PFU/cell. At the times indicated, media were replaced, and virus titers were determined by a plaque assay as described in Materials and Methods. The results of one of two reproducible experiments are presented. The insertions dramatically reduced the RNA s infectivity, i.e., the characteristic that is usually used to evaluate the RNAs ability to initiate replication. Instead of the usual 10 6 PFU/ g of transfected RNA, determined for the wt SINV Toto1101 genome, the infectivity of the library-derived RNA was close to 10 2 PFU/ g (Fig. 1). The majority of the developed plaques were either GFP negative right after transfection or rapidly lost the ability to express GFP during subsequent rounds of virus propagation. Some of the plaques were very small, indicating strong alterations in virus replication caused by the GFP insertion, and were not used for further experiments. However, a number of plaques were large and contained viruses that continued to express GFP during propagation in BHK-21 cells. These selected variants formed plaques of a size comparable to that of wt SINV Toto1101 (Fig. 1 and 2) and were used in further experiments to identify the insertion sites by reverse transcription-pcr. The sequencing data indicated that nsp2 could tolerate GFP insertion into a very few sites. The GFP sequence was mainly found either in the amino terminus of the nsp2 (after aa 8 to 11), between the helicase and protease domains (after aa 469 to 472), or after aa 627. Some of the insertions were detected in the beginning of SINV nsp3 (Fig. 1). The latter mutants were not further investigated. It should be noted that most of the insertions were usually present in more than one plaquepurified variant, an indication that the constructed library was representative. Two of the virus genomes with a GFP sequence after aa 8 and 472 were assembled in a cdna form, rescued from the in vitro-synthesized RNA, and used in the experiments described below. The cdna construct with insertion after aa 627 produced virus in which the insertion was unstable and, therefore, no further experiments with this insertion mutant were performed. Replication of the GFP insertion mutants. It was not reasonable to expect that the insertion of a 240-aa-long GFPcontaining sequence could have no effect on the nsp2 functioning. The SIN/nsP2GFP/8 and SIN/nsP2/472 viruses, with GFP insertions after aa 8 and 472, respectively (Fig. 2A), were rescued from the in vitro-synthesized RNA and evaluated for their growth, RNA replication, and processing of the nonstructural proteins. The RNA infectivity was above 10 6 PFU/ g (Fig. 2B) and was very similar to the infectivity determined for the RNA of wt SINV Toto1101. This was an indication that no additional, adaptive mutations were required for viral replication in BHK-21 cells. Stocks of the recombinant viruses generated by electroporation approached titers that were 10 8 PFU/ml. However, plaques developed by SIN/nsP2GFP/8 and SIN/nsP2GFP/472 in BHK-21 cells were smaller (Fig. 2B) than those formed by SINV Toto1101, and both recombinants demonstrated slower growth rates (Fig. 2C), suggesting that, to some extent, the insertions had a negative effect on virus replication. This was most likely a result of a decrease in RNA replication at the early times postinfection (Fig. 3A and B). The insertions also affected transcription of the subgenomic RNA, which was detected by a lower ratio of genomic versus subgenomic RNA synthesis (Fig. 3C). However, taken together, the data indicated that RNA replication (but not transcription of the subgenomic RNA) was not strongly affected by the GFP insertions. nsp2/gfp was capable of efficient functioning in SINV replication complexes that could synthesize RNA and, ultimately, produce virus at a level comparable to that of wt SINV. Effects of GFP insertions on SINV nonstructural polyprotein processing. Changes in the genomic and/or subgenomic RNA synthesis ratio suggested that the GFP insertions affected either the processing of the nonstructural polyprotein or the nsp2 functioning (or both processes). To distinguish between these possibilities, we analyzed protein processing in more detail by Western blotting and by pulse-chase experiments. In addition to the wt SINV Toto1101, SIN/nsP2GFP/8 and SIN/

5 5050 ATASHEVA ET AL. J. VIROL. FIG. 3. Synthesis of virus-specific RNAs in the cells infected with recombinant viruses. (A) BHK-21 cells in 6-well Costar plates were infected with SINV Toto1101, SIN/nsP2GFP/8, and SIN/ nsp2gfp/472 at an MOI of 20 PFU/cell. At the indicated times, the medium in the wells was replaced by 0.8 ml of MEM supplemented with 10% FBS, ActD (1 g/ml), and [ 3 H]uridine (20 Ci/ml). After 4 h of incubation at 37 C, RNAs were isolated and analyzed by agarose gel electrophoresis as described in Materials and Methods. (B) The synthesis of viral genome RNA was analyzed by excising the bands corresponding to 49S viral genome RNA from the gel shown in panel A, followed by measurement of the radioactivity by scintillation counting. (C) Bands corresponding to 26S subgenome RNA (at 6 h postinfection) were also excised from the gel, the radioactivity was measured by scintillation counting, and the molar ratio of subgenome to genome RNA synthesis was evaluated. G and SG indicate the positions of viral genomic and subgenomic RNAs. The results for one of two reproducible experiments are presented. nsp2gfp/472, we used, in the present study, the previously described SIN/2V/389 virus that contained GFP insertion after aa 389 of nsp3 and a mutation in the cleavage site between nsp2 and nsp3 that blocked P23 processing (45). The GFP insertion after the eighth amino acid of nsp2 in the SIN/nsP2GFP/8 blocked the processing of one of the cleavage sites (Fig. 4B and C) and, surprisingly, the affected cleavage was not between nsp1 and nsp2, but between nsp2 and nsp3, on the carboxy terminus of the nsp2. The effect was very similar to that detected in the processing of the P123 polyprotein of SIN/2V/389 virus (see Fig. 4B). The pulse-chase experiments revealed that processing proceeds but at a very low rate. The barely detectable nsp2 band could be found on the gel in the lines corresponding to longer chase time points (Fig. 4C) or by Western blotting (Fig. 4B). The insertion after aa 472 also had a negative effect on the rate of polyprotein processing (Fig. 4C). P23/GFP was clearly detectable in the pulse-chase experiments even after a long chase; however, both nsp2/gfp and the phosphorylated forms of nsp3 were found by Western blotting in the cells (Fig. 4B). It should be noted that GFP insertion in SIN/nsP2GFP/8 had some negative effect on the stability of either P123 or P23 because multiple degradation products were present in the gel in the pulse-chase experiments. In the SIN/nsP2GFP/472, the GFP insertion caused the formation process for nsp2/gfp to pass through an additional step of processing, in which the intermediate product of a higher molecular weight was initially formed (Fig. 4C) and then processed to a final form of nsp2/gfp. In addition to the standard forms of phosphorylated and nonphosphorylated nsp3 found in both SINV Toto1101- and SIN/nsP2GFP/472- infected cells, we also detected an additional band of the protein recognized by nsp3-, but not by the nsp2-specific antibodies (Fig. 4B, upper panel). Taken together, the alterations in polyprotein processing provide a plausible explanation for the less-efficient synthesis of viral subgenomic RNA, produced only by the RC that contains completely processed nsps (28). In accordance with the cleavage alterations, nsp2/gfp produced by SIN/nsP2GFP/8 and SIN/nsP2GFP/472 were differently distributed in the infected cells. The SIN/nsP2GFP/8- specific fusion protein was present in the cytoplasm in large complexes, and very little was found in the nuclei; nsp2/gfp of SIN/nsP2GFP/472 was distributed both in the cytoplasm and in the nucleus (Fig. 4D). It formed granulated structures, indicating its association with complexes whose nature is not yet defined. One of the most important questions was whether the recombinant viruses expressing nsp2 with GFP insertion are capable of modifying cellular metabolism to the same level as does wt SINV. BHK-21 cells infected with SINV Toto1101 demonstrated an inhibition of cellular protein synthesis that developed within 8 h postinfection (Fig. 5). Both SIN/ nsp2gfp/472 and SIN/nsP2GFP/8 also induced translational shutoff; however, it developed slower and in good agreement with the delayed processing of the nonstructural proteins and synthesis of virus-specific RNAs (Fig. 3 and 5). Taken together, the data indicated that the GFP insertion affected P123 and P23 cleavage and the distribution of nsp2 expressed by SIN/nsP2GFP/8 but had only a moderate effect on the SIN/nsP2GFP/472 nonstructural polyprotein processing

6 VOL. 81, 2007 SINDBIS VIRUS ENCODING nsp2/gfp 5051 Downloaded from FIG. 4. Processing and distribution of SINV nsps in the cells infected with different recombinant viruses. (A) Schematic representation of the genomes of the viruses used in these experiments. SIN/2V/389 was previously described (14). It had GFP insertion after aa 389 of nsp3 and a single-point mutation leading to replacement of glycine by valine in the P2 position of the cleavage site between nsp2 and nsp3 (45). This nsp2 G 806 3V mutation blocked P2/3 cleavage during P123 processing. It also contained an additional mutation leading to the replacement of a stop codon between nsp3 and nsp4 by the cysteine-coding one. Other recombinant viruses are described in the legend to Fig. 2. (B) At 9 h postinfection, cells were harvested, and equal amounts of protein were used for gel electrophoresis. After transfer, the nitrocellulose membranes were processed by rabbit anti-nsp3 (upper panel) or anti-nsp2 (lower panel) antibodies and horseradish peroxidase-conjugated, secondary donkey anti-rabbit immunoglobulin G antibodies. (C) Analysis of the nonstructural polyprotein processing. At 3 h postinfection, proteins were labeled with [ 35 S]methionine as described in Materials and Methods and chased for 15, 30, or 60 min. The positions of the processed and partially processed proteins are indicated. (D) BHK-21 cells were infected with SIN/nsP2GFP/8 and SIN/nsP2GFP/472 as described in Materials and Methods. At 8 h postinfection, the cells were fixed with 1% formaldehyde, and images were acquired on a Zeiss LSM510 META confocal microscope using a NA oil immersion planapochromal lens. and the nsp2 compartmentalization. Both insertions did not make SINV incapable of interfering with cellular translation. Recombinant SINV expressing two fluorescent markers. Formation of virus-specific protein complexes is a sophisticated process and, in the case of alphaviruses, protein complexes that are involved in viral RNA replication and modification of the intracellular environment appear to change their composition and distribution at different times postinfection. Further investigation of these complexes includes live-cell imaging studies that are easier to perform with viruses expressing fluorescent markers in more than one nsp. To test the possibility of making such viruses, we designed SINV, encoding GFP after aa 472 of nsp2 and one of the red fluorescent proteins, Cherry (44), after aa 389 of nsp3. The position for the latter insertion was defined based on our previous study (14). After we cloned two genes into the ns polyprotein (Fig. 6A), we found that the in vitro-synthesized RNA was as infectious as the RNA of wt SINV Toto1101. Moreover, the virus remained viable, stable, and cytopathic and approached titers of 10 8 PFU/ml. Viral RNA replicated at a level comparable to that of the wt SINV Toto1101; however, transcription of the subgenomic RNA proceeded at a noticeably lower rate (Fig. 6B). Therefore, virus growth rates were similar to those described above for SIN/nsP2GFP/472, but not wt SINV (data not shown). Protein complexes were easily detectable in the infected cells (Fig. 6C): nsp2/gfp was found both in the nuclei and in the cytoplasm, where it was either present in the highorder, nsp3/cherry-containing structures or distributed in a granular fashion outside of these complexes. nsp3/cherry formed only large complexes, which were present exclusively in on September 17, 2018 by guest

7 5052 ATASHEVA ET AL. J. VIROL. FIG. 5. Analysis of protein synthesis in the cells infected with recombinant viruses BHK-21 cells were infected with SINV Toto1101, SIN/nsP2GFP/8, and SIN/nsP2GFP/472 at an MOI of 20 PFU/cell. At the indicated times, proteins were pulse-labeled with [ 35 S]methionine for 30 min and analyzed on a sodium dodecyl sulfate 10% polyacrylamide gel. The gels were dried and autoradiographed (A) or analyzed on a Storm 860 PhosphorImager (B) as described in Materials and Methods. The levels of the synthesis of cellular proteins were determined by measuring radioactivity in the protein band corresponding to actin and normalized to radioactivity in the actin band in the lane representing the uninfected cells. The results from one of two reproducible experiments are presented. the cytoplasm of the infected cells. It was possible even to distinguish the substructures in these complexes: nsp3/cherry was always colocalized with nsp2/gfp, but nsp2/gfp could be readily detected in the non-nsp3-associated form (Fig. 6C, panel c and d). Thus, two fluorescent markers can be used to study the genesis of the virus-specific protein complexes, but some changes in virus replication, caused by insertions, certainly must be considered. Isolation of the cellular proteins binding to SINV nsp2. During SINV replication, nsp2 is detected both in the nuclei and in the cytoplasm of the infected cells. Moreover, in the cytoplasm, it is associated with large protein complexes with a high concentration of nsp3, and at least a high percentage of these complexes contain SINV-specific RNAs (14). In addition, nsp2 is also distributed in the cytoplasm outside of these complexes, suggesting its functioning in processes other than only viral RNA replication. In our previous work (14), we isolated the nsp3-specific protein complexes and identified the components of both cellular and viral origin that were present FIG. 6. Replication of the SINV/nsP2GFP/nsP3Cherry virus expressing two fluorescent proteins fused with different nsps and distribution of nsp2/gfp and nsp3/cherry in the infected cells. (A) Schematic representation of wt SINV Toto1101 and SINV/nsP2GFP/ nsp3cherry genomes. (B) A total of BHK-21 cells in six-well Costar plates were infected with SINV Toto1101 and SINV/nsP2GFP/ nsp3cherry at an MOI of 20 PFU/cell. At the indicated times, the medium in the wells was replaced by 0.8 ml of MEM supplemented with 10% FBS, ActD (1 g/ml), and [ 3 H]uridine (20 Ci/ml). After 4 h of incubation at 37 C, the RNAs were isolated and analyzed by agarose gel electrophoresis as described in Materials and Methods. (C) BHK-21 cells were infected with SINV/nsP2GFP/nsP3Cherry at an MOI of 20 PFU/cell. At 12 h postinfection, they were fixed with 1% formaldehyde, and the three-dimensional images were acquired on a Zeiss LSM510 META confocal microscope using a NA oil immersion planapochromal lens. The enlarged cell fragment presented in panel d is indicated in panel c. at detectable concentrations. These complexes certainly contained nsp2, but the question remained as to whether the nsp2 fraction distributed outside the nsp3-containing structures was associated with other host protein factors.

8 VOL. 81, 2007 SINDBIS VIRUS ENCODING nsp2/gfp 5053 To isolate all of the nsp2-specific protein complexes present in the cytoplasm of infected cells, we applied a SINV replicon encoding GFP after aa 472 of the nsp2 (SINrep/nsP2GFP/ 472), and a SIN virus with a GFP insertion in the same position of nsp2 (SIN/nsP2GFP/472). In our previous studies, we demonstrated that replication of viral and replicon genomes proceeds in a very similar fashion and leads to highly comparable changes in cellular machinery; however, our knowledge of the alphavirus replication mechanism is far from complete and, to date, the possibility of capsid functioning in the replication processes has not been ruled out. Therefore, in the following experiments, we used both SINV replicon and SIN virus. Based on the data presented above, replication of the virus-specific RNAs encoding this chimeric nsp2 was similar to that of the wt SINV genome and caused comparable changes in cell biology; thus, the results were expected to be biologically relevant. The SINrep/nsP2GFP/472 and the control replicon SINrep/ GFP, encoding an unmodified nsp2 and GFP, cloned under the control of the subgenomic promoter, were packaged into viral structural proteins by using a two-helper system (12). BHK-21 cells were infected at the same MOI with both packaged replicons and SIN/nsP2GFP/472 virus and, at 8 h postinfection, lysed as described in Materials and Methods. The nsp2-bound proteins were isolated using GFP-specific antibodies, covalently linked to magnetic beads. In repeated experiments, we reproducibly isolated and identified, by MS, the spectra of the same proteins that were copurified with nsp2/ GFP (Fig. 7). The sets isolated from the replicon- and virusinfected cells differed only by the presence of capsid. In the additional experiments, we demonstrated that the coisolation of capsid was not a result of its nonspecific binding to GFPspecific antibodies but was, more likely, because of the isolation of large complexes containing SINV nucleocapsids (data not shown). Therefore, the possibility that capsid functions in RNA replication is very doubtful but cannot be completely ruled out. Protein complexes purified from the infected cells contained other viral nsps, nsp1 and nsp3; nsp4 was not definitely detected because of its presence at a very low concentration. The isolated complexes contained RNAbinding proteins [different hnrnps, myb-1b, poly(a)-binding protein (PABP), and G3bp], proteins forming different cellular filaments (vimentin and -actin) and heat shock protein Hsc70. As we expected, the spectrum of the proteins isolated by this procedure was similar to what we previously isolated from cells infected with SINV replicons encoding a GFP insertion-containing nsp3. However, in the nsp2/gfp-specific complexes, some of the proteins, namely, Ddx5, PABP, and hnrnp C, were present at higher concentrations and definitively identified. The ribosomal proteins were also found in the isolated samples at higher concentrations, and almost the entire sets composing both the 60S and 40S subunits were unambiguously recognized. Thus, the data indicated that GFP inserted into nsp2 was accessible to specific antibodies and capable of functioning as an efficient tag for the isolation of protein complexes formed during SINV replication. FIG. 7. Analysis of proteins coisolated with nsp2/gfp from the replicon- or virus-infected cells. BHK-21 cells were infected with packaged SINrep/nsP2GFP/472/Pac virus and SINrep/GFP or SIN/ nsp2gfp/472 virus at an MOI of 20 infectious units or PFU per cell. Proteins were isolated by using GFP-specific antibodies as described in Materials and Methods, and after elution from the affinity columns were separated on sodium dodecyl sulfate 10% polyacrylamide gels and stained with Coomassie brilliant blue R-250. Coomassie bluestained bands were excised from the gels, and the proteins were identified by MALDI-TOF analysis. The names of the identified proteins are indicated. An open box indicates a GFP-containing band present in the lysate of SINrep/GFP replicon-infected cells. The results for one of five reproducible experiments are presented. DISCUSSION Alphaviruses express very few proteins during their replication in the infected cells. These virus-specific proteins efficiently function in the synthesis of virus-specific RNA and the formation of infectious viral particles required for the subsequent rounds of infection. In vertebrate cells, alphavirusspecific proteins also modify the intracellular environment to promote efficient virus replication and to downregulate a virusinduced cell response and cell signaling. This interference is a key element of virus pathogenesis on the molecular and cellular levels and is a multicomponent, poorly understood process. SINV nsp2 is an important factor in viral RNA replication and modification of cell biology. Specific mutations in this protein differentially affect the inhibition of cellular transcription or translation during virus replication and strongly influence the replication of the viral genome and/or transcription of the subgenomic RNA. Based on accumulated data, nsp2 is

9 5054 ATASHEVA ET AL. J. VIROL. directly involved not only in nsps processing, because it has protease activity, but also in RNA replication, because of its RNA helicase, NTPase and RNA triphosphatase activities (18, 41, 50). Moreover, this protein accumulates compensatory mutations in response to modification of the 5 cis-acting RNA elements, indicating that it might mediate binding of the RC to viral RNA (11). In addition, the presence of SINV nsp2 in the cell in a completely processed form causes transcriptional shutoff, required for downregulation of cell signaling (19). It should also be noted that point mutations in the carboxy-terminal domain of nsp2, which is similar structurally to the S-adenosyl- L-methionine-dependent RNA methyltransferases (42), make this protein nontoxic for the cells and incapable of interfering with cellular transcription (13, 37). Multiple functions of SINV nsp2 strongly complicate investigation of its activities in virus replication and suggest a possibility of its interaction with different cellular protein factors. Intracellular distribution of this protein correlates with its multiple functions. It was found both in the cytoplasm and in the nuclei of the infected cells (19, 36). Moreover, in the cytoplasm, SINV nsp2 is present in large complexes that can be isolated with the P15 fraction that contains the RNA-dependent RNA polymerase activity and from the so-called S15 fraction, which has almost no cellular organelles, but, nevertheless, contains high levels of nsp2 comparable to that found in P15 (14). Traditional methods of isolation of protein-specific complexes are usually based on their expression as fusions with different tags that are cloned into the carboxy or amino-terminal ends of the protein being studied. However, this strategy is not applicable to the SINV nsp2. The most biologically relevant way to express this protein is to produce it in the context of replicating virus, but in this case one faces the problem of tag sequence insertion. It cannot be cloned into the amino or carboxy terminus because they are required for polyprotein processing. Moreover, the short tags cloned into other sites might make the protein nonfunctional, or they can be hidden inside the folded domains and unavailable to antibodies. Therefore, we have chosen to pursue a strategy in which the tag is supposed to be large and exposed on the protein surface. The GFP tag met this requirement, and the magnetic beads covalently linked to affinity-purified, GFP-specific antibodies were commercially available. However, there was no information about potential sites in SINV nsp2 that could be used for GFP cloning. To identify places in nsp2 that can be used for GFP insertion, we modified the transposon-based insertion protocol that was previously applied for developing HCV replicons with GFP insertions in an NS5A sequence (33). We were aware of the possibility that SINV nsp2 would have very few sites that tolerate insertions or no sites at all. Therefore, the library was represented by more than 10 4 clones and expected to contain random GFP insertions after each nucleotide. The restriction digestion of a plasmid pool with recombinant SINV genomes indicated that the insertions were indeed distributed all over the nsp2, and no preferential sites were noticed (data not shown). After the transfection of the in vitro-synthesized RNA, followed by selection of the GFP-expressing, plaque-forming viruses, stable insertions were identified only at two sites. In these cytopathic and stable viruses, GFP sequences were found either in the amino terminus (after aa 8, 9, and 11) or in the short peptide between the two known, RNA helicase and proteinase, domains. The strategy used for making the insertion mutants is efficient, but, most likely, not the one that can be universally applied. Attempts to use the same protocol for making the insertions into the SINV nsp1 were unsuccessful, in spite of the very large libraries of recombinant clones used (data not shown). The lack of viable viruses indicated that the latter protein is, most likely, incapable of tolerating the insertions of long peptides. It should also be noted that finding the sites for GFP insertions in SINV nsp2 does not necessarily indicate that the same insertions can be made into the nsp2 of other alphaviruses. For example, cloning of GFP between the protease and helicase domains of nsp2 in VEEV TC-83 genome to make an analog of SIN/nsP2GFP/472 resulted in a virus that was not viable, and the transposon-based GFP insertion mutagenesis had to be repeated (data not shown). In spite of only minor effects on viral RNA replication, the insertions into the amino-terminal portion of nsp2 affected the activity of this protein in the proteolytic cleavage of the nsp2/ nsp3 site that is located on the opposite, carboxy terminus of the protein. This fact supports the previously suggested hypothesis that the amino-terminal fragment of the alphavirus nsp2 might be a protease cofactor (51) and function in a mode similar to those previously described for HCV NS4A (21) or flavivirus NS2B (6, 52, 53). This finding points to an interesting structure of the nsp2 that should be further investigated. Similar to what we previously described for a SINV variant with a mutated cleavage site between nsp2 and nsp3, the alteration of P23 processing made nsp2/gfp incapable of translocating to the nucleus (19) and could alter the structure of the nsp2- specific protein complexes in the cytoplasm. Therefore, this mutant was not used in the protein isolation experiments. The GFP insertion into the interdomain region (after aa 472) also affected the rate and pattern of polyprotein processing, with an additional cleavage product being formed (see Fig. 4B and C for details). Nevertheless, the RNA replication was efficient, and the nsp2/gfp synthesized by SIN/nsP2GFP/472 mutant was distributed in the cytoplasm and nuclei of the infected cells as previously described for the natural nsp2 (Fig. 4D) (19). Moreover, this virus was capable of inducing translational shutoff in the infected cells, which would suggest that the cytoplasm-specific functions of nsp2 in both viral RNA replication and modification of translational machinery were not strongly affected. However, in additional studies, we found that, in spite of the nsp2/gfp s presence in the nuclei, the insertion significantly affected this protein s functions in inhibiting cellular transcription (data not shown). As reported previously for P23 cleavage mutant (19), SIN/nsP2GFP/8 was incapable of causing transcriptional shutoff, and SIN/nsP2GFP/472 downregulated cellular RNA synthesis at a significantly lower rate. Therefore, the latter insertion mutant was further used only to isolate cellular and viral proteins that interact with SINV nsp2 in the cytoplasm of infected cells. Thus, the nuclear function of SINV nsp2 is now being studied by other approaches (data not shown). The nsp2-specific protein complexes, formed in the cytoplasm of cells infected with nsp2gfp/472-encoding viruses and replicons, contained nsp1, nsp3, and a combination of cellular proteins similar to ones we (14) and another group (8) previously reported in nsp3-related studies. However, Ddx5, PABP,

10 VOL. 81, 2007 SINDBIS VIRUS ENCODING nsp2/gfp 5055 FIG. 8. Summary of the data about the structure and functional domains in alphavirus nsp2. (A) Positions of RNA helicase, protease, and putative S-adenosyl-L-methionine-dependent RNA methyltransferase domains. (B) Positions of GFP insertions that do not cause deleterious effect on virus replication. (C) Positions of adaptive point mutations in the putative methyltransferase domains of SINV, SFV, and VEEV that have a strong effect on both RNA replication and the ability of replicating alphaviruses or replicons to cause CPE (13, 37, 38). (D) Positions of the compensatory mutations found in the SINV and VEEV genomes with a mutated replication enhancer, the 51-nt CSE (11). (E) Positions of deletion in SFV nsp2 (51) and GFP insertions in SINV nsp2 that make these proteins incapable of P23 polyprotein processing. and hnrnp C were coisolated with SINV nsp2/gfp at a higher concentration and were identified by MS. The distinguishing feature of the newly isolated complexes was in the fact that they contained almost all of the ribosomal proteins at concentrations higher than those of complexes isolated through nsp3/gfp (14). Coisolation of the ribosomes correlates with previously published data that the nsp2 of another alphavirus, VEEV, binds to the ribosomal S6 protein and can be detected in the polyribosomes fractionated on the sucrose gradient (32). SFV nsp2 was also detected as being bound to ribosomes isolated from the infected cells (39). Another interesting finding in the protein isolation experiments was that capsid protein efficiently and reproducibly coprecipitates with nsp2/gfp from the SIN/nsP2GFP/472 virus-infected cells. No capsid was detected in the samples derived from the cells infected with SINV that expressed GFP encoded by additional subgenomic RNA (data not shown). The biological significance of coisolation of capsid and nsp2/gfp is not clear yet and will be further investigated. This efficient capsid isolation may be due to its previously demonstrated binding to ribosomes (49) or because of an additional function in RNA replication that we do not yet understand. An alternative explanation is that it is the result of the colocalization of newly formed nucleocapsids with replicative complexes, as readily detected by electron microscopy (data not shown). In addition, the recently designed variants of SINV with altered processing of the nsps demonstrated an efficient RNA replication and synthesis of viral structural proteins, but formed infectious viral particles very inefficiently (19). These data also suggest a possibility that viral replicative machinery and the RNA encapsidation are cocompartmentalized. The experiments described here and in our previous publication (14) allowed us to identify a number of cellular proteins that might be involved in alphavirus replication due to their direct functioning in RNA replication or an ability to modify the intracellular environment. Identification of the exact mechanism of their functioning is certainly a major direction of our future work. In particular, we are interested in understanding the roles of heat shock proteins, because a growing number of publications suggests their importance for replication of a variety of RNA viruses (7, 23, 34, 43). The results of the present study suggest a method of designing chimeric viral proteins that express the heterologous sequence and remain efficiently functioning in viral replication. The random insertion library-based approach, followed by the selection of the viable variants expressing heterologous peptide can be applied to mapping of the domain structure of the viral nonstructural and structural proteins, for cloning of peptide tags required for isolation of the protein-specific complexes, and studying their formation using live-cell imaging. It can be also used for the presentation of additional antigens or retargeting of the viruses to new receptors. In the present work, we generated recombinant SINV that expresses GFP in the nsp2 and isolated nsp2/gfp-specific protein complexes formed in the cytoplasm of the infected cells. These complexes contained viral nsps, all of the cellular proteins that we previously coisolated with SINV nsp3/gfp, and some other protein factors that were not found before at detectable concentrations. Moreover, SIN/nsP2GFP/nsP3Cherry, containing the insertions of two fluorescent proteins into different nsps, was also viable and provided additional information about fine structure of the nsp-containing protein complexes. Our present knowledge of structural and functional domains of alphavirus nsp2 is summarized in Fig. 8. This protein contains an RNA helicase, protease, and a putative S-adenosyl-Lmethionine-dependent RNA methyltransferase domains (Fig. 8A) (42). GFP insertions between these domains do not have a deleterious effect on virus replication but might affect the ratio of subgenomic to genomic RNA synthesis (Fig. 8B). The adaptive point mutations in the methyltransferase domain