JOURNAL OF VIROLOGY, Aug. 2002, p. 8011–8018 0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.16.8011–8018.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 16
Identification of Two Additional Translation Products from the Matrix (M) Gene That Contribute to Vesicular Stomatitis Virus Cytopathology Himangi R. Jayakar† and Michael A. Whitt* Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163 Received 13 November 2001/Accepted 3 May 2002
The matrix (M) protein of vesicular stomatitis virus (VSV) is a multifunctional protein that is responsible for condensation of the ribonucleocapsid core during virus assembly and also plays a critical role in virus budding. The M protein is also responsible for most of the cytopathic effects (CPE) observed in infected cells. VSV CPE include inhibition of host gene expression, disablement of nucleocytoplasmic transport, and disruption of the host cytoskeleton, which results in rounding of infected cells. In this report, we show that the VSV M gene codes for two additional polypeptides, which we have named M2 and M3. These proteins are synthesized from downstream methionines in the same open reading frame as the M protein (which we refer to here as M1) and lack the first 32 (M2) or 50 (M3) amino acids of M1. Infection of cells with a recombinant virus that does not express M2 and M3 (M33,51A) resulted in a delay in cell rounding, but virus yield was not affected. Transient expression of M2 and M3 alone caused cell rounding similar to that with the full-length M1 protein, suggesting that the cell-rounding function of the M protein does not require the N-terminal 50 amino acids. To determine if M2 and M3 were sufficient for VSV-mediated CPE, both M2 and M3 were expressed from a separate cistron in a VSV mutant background that readily establishes persistent infections and that normally lacks CPE. Infection of cells with the recombinant virus that expressed M2 and M3 resulted in cell rounding indistinguishable from that with the wild-type recombinant virus. These results suggest that M2 and M3 are important for cell rounding and may play an important role in viral cytopathogenesis. To our knowledge, this is first report of the multiple coding capacities of a rhabdovirus matrix gene. protein. M protein has numerous functions in infected cells. For example, M protein is the driving force behind the assembly and budding of virions. M protein interacts with the viral ribonucleoprotein core (RNP), resulting in the condensation of the RNP and subsequent inhibition of viral transcription (29). A fraction of the M protein (⬃10%) is also associated with the inner leaflet of the plasma membrane where virus assembly and budding takes place (6). Recent work has shown that a motif (PPPY) located within the first 30 amino acids of M contributes to this budding activity (11, 13). M protein is also responsible for most of the cytopathic effects of VSV infection. Expression of M protein by itself can cause inhibition of host gene expression, which occurs mostly at the transcriptional level (2, 3, 18). This inhibition appears to be mediated via inactivation of the TFIID protein (17). M protein, when expressed alone in the absence of other viral components, also causes cytoskeletal disorganization. Disassembly of microtubules by M protein ultimately leads to cell rounding (4, 23), which is a hallmark of VSV infection in cell culture. Recently it was shown that a fraction of M protein colocalizes with nuclear pore complexes (NPCs) at the nuclear rim (19). This nuclear fraction of M protein is thought to contribute to the host shutoff function of the protein by inhibiting RNA export from the nucleus. In this study, we show that the M mRNA encodes two additional polypeptides, which we refer to as M2 and M3. These proteins are synthesized from downstream methionines in the same reading frame that encodes the 229-aminoacid M protein (which is referred to as M1 protein in this report). We also show that M2 and M3 are important for cell
The nonsegmented, negative-strand RNA viruses have a relatively small genome size, ranging from 11 to 19 kb. To maximize their coding capacities, many of these viruses have evolved different strategies to express additional proteins. Increased coding capacity can occur either at the transcriptional level (mRNA processing or modification) or at the translational level, in which proteins are produced from alternative reading frames or from translation initiation at non-AUG or downstream AUG codons (1, 5, 7, 8, 10, 12, 24). The bestcharacterized examples are the use of multiple overlapping open reading frames (ORFs) within the P mRNAs of several paramyxoviruses (1, 10, 27) and the mRNA encoding the NA and NB glycoproteins of influenza B virus (22). A similar phenomenon has recently been described for Vesicular stomatitis virus (VSV), the prototype member of the Rhabdoviridae family. The VSV genome contains five genes, N, P, M, G, and L, and each, except the P gene, is thought to encode a single unique protein. The VSV P gene, like its counterpart in paramyxoviruses, has been shown to encode two additional proteins, C and C⬘, in a second ORF (14, 24) and a 7,000-molecular-weight (7K) polypeptide in the same ORF that encodes the P protein (12). The VSV (Indiana serotype) M gene is transcribed into a single mRNA which encodes the 229-amino-acid matrix (M)
* Corresponding author. Mailing address: Department of Molecular Sciences, 858 Madison Ave., University of Tennessee Health Science Center, Memphis, TN 38163. Phone: (901) 448-4634. Fax: (901) 4488462. E-mail:
[email protected]. † Present address: GTx, Inc., Memphis, TN 38163. 8011
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rounding and may play an important role in viral pathogenesis. MATERIALS AND METHODS Plasmid construction and design. To prevent the expression of the full-length M1 protein (229 amino acids), three consecutive stop codons were introduced immediately downstream of the first AUG codon in the Bluescript-MT (BSMT) plasmid, which encodes a wild-type (WT) VSV M protein (Indiana serotype) under the control of the bacteriophage T7 promoter. The resulting construct was termed BS-M1SC. The BS-M2SC construct was generated by introducing three stop codons immediately downstream of the second AUG, at amino acid position 33 in the BS-M1SC plasmid. The third AUG, at amino acid position 51 in the M2SC plasmid, was mutated to encode arginine instead of methionine, generating construct BS-M3SC. A schematic representation of the various constructs is shown in Fig. 2A. The three constructs were made by using a PCR-based mutagenesis strategy. For the pBS-M1SC construct, a forward primer containing appropriate base changes to introduce three consecutive stop codons immediately following the first AUG was used, along with a reverse primer downstream of the MunI site, to generate a PCR product by using BS-MT as the template. The 200-bp PCR product was then gel purified and digested with SmaI and Mun I restriction enzymes, and the resulting 170-bp fragment was gel purified and used to replace the corresponding WT region in the pBS-MT plasmid. The pBS-M2SC construct was generated in a similar manner except that the reverse primer downstream of the MunI site contained appropriate base changes to introduce two additional stop codons downstream of the second AUG at amino acid position 33. The pBS-M3SC construct was generated by using a forward primer overlapping the MunI site and containing base changes to generate the M513R mutation and a reverse primer downstream of the BglII site in a PCR with pBS-M2SC as the template. The resulting PCR product was then gel purified, digested with MunI and BglII, and used to replace the corresponding region in plasmid pBSM2SC. The plasmids were then sequenced by using the dideoxy sequencing method to ensure that only the specified mutations were introduced during PCR amplification. The pCAGGS-M constructs were derived from the pBS-M constructs by subcloning a KpnI-XbaI fragment containing the entire M coding region from either pBS-MT, pBS-M1SC, or pBS-M2SC into the multiple cloning site of the modified pCAGGS vector pCAGGS-MCS. Generation of minigenome and full-length VSV M2 and M3 mutants. Minigenome mutants were all derived from the parent plasmid MGF-WT, which consists of a genomic sense VSV cDNA containing the M, G, and green fluorescent protein (GFP) genes (previously referred to as pBS-GMF) (26). The M333A, M513A, and M513R mutations were generated by a PCR-based cloning strategy similar to that described previously (13). For the M33A M51A mutation, the template used in the first PCR was MGF M33A instead of MGFWT. The M33A M51A (M33,51A) double mutant was subcloned into a modified full-length VSV genome, called ⌬M-PLF, described elsewhere (13). Plasmid MGF M33,51A was digested with EcoRV and NheI enzymes to produce a 1.6-kb insert. Meanwhile, the parent plasmid, ⌬M-PLF, was cut with SmaI and NheI enzymes. The restriction fragments were then gel purified and ligated in a two-way ligation. Positive clones were identified by PCR screening as well as restriction digestion. Generation of the NCP-M1SC mutant. The parent plasmid used for the NCPM1SC construct encodes a noncytopathic VSV mutant named rNCP12.1 (13), which has a WT VSV backbone except that the M gene contains four mutations. Two of the mutations are M333A and M513A. The other two mutations are in the C terminus and will be described in detail elsewhere (unpublished data). Plasmid pVSV-M1SC, expressing M1SC (encoding the M2 and M3 proteins) as a separate cistron between the G and the L gene, was digested with NheI, located in the 3⬘ untranslated region of the G gene, and HpaI, which is found in the 5⬘ untranslated region of the L gene. The resulting 1.6-kb fragment was used to replace a corresponding region in plasmid pVSV-rNCP12.1 in a two-way ligation. Positive colonies were identified by using a PCR screen with a forward primer in the G gene and a reverse primer in the M1SC gene. Recovery and characterization of M2 and M3 mutants. Recoveries of minigenome mutants were performed as described previously (25) and monitored by detecting the expression of GFP from the reporter gene. All full-length viruses were recovered from their cDNAs as described earlier (13). Assays to determine the total virus yield, for the one-step growth curve and for inhibition of host gene expression with the M33,51A mutant, were performed as described previously (13).
J. VIROL. Cell-rounding assay. BHK-21 cells in 35-mm-diameter dishes were infected with either WT VSV or the mutant viruses at a multiplicity of infection (MOI) of 10. After 1 h, the inoculum was removed, and the cells were washed once with serum-free medium and then incubated at 37°C for varying times. At each time point, the medium was removed, and cells were washed twice with phosphatebuffered saline (PBS) and fixed with 3% paraformaldehyde at room temperature for 20 min, followed by two washes with PBS containing 20 mM glycine. Cells were observed by phase-contrast microscopy (Axiophot; Zeiss, Thornwood, N.Y.). Transient expression of M2 and M3 mutant proteins. Baby hamster kidney (BHK-21) cells in 35-mm-diameter dishes were infected with vTF7-3 (9) at an MOI of 10 for 1 h at 37°C in serum-free Dulbecco’s minimal Eagle’s medium (DMEM) (GIBCO BRL) without antibiotics. Cells were then transfected with 5 g of the pBS-M WT, pBS-M1SC, pBS-M2SC, or pBS-M3SC plasmid construct and 15 l of the lipid reagent TransfectACE (20). Three hours posttransfection (p.t.), the transfection mixture was replaced with 2 ml of DMEM containing 5% fetal bovine serum (FBS) and antibiotics (streptomycin and penicillin), and cultures were incubated at 37°C for 24 h. For transient expression from pCAGGS plasmids, BHK-21 cells grown on glass coverslips were transfected with 2 g of either pCAGGS-M WT, pCAGGSM1SC, pCAGGS-M2SC, or pCAGGS-N WT by using 6 l of Lipofectamine (GIBCO BRL) according to the manufacturer’s instructions. The lipid-DNA mixture was replaced with DMEM containing 10% FBS after 3 h, and cultures were incubated for an additional 45 h. Expression of M and N proteins was detected by an indirect immunofluorescence assay with either an M-specific (23H12) or an N-specific (10G4) monoclonal antibody (15) and a rhodamine conjugated goat anti-mouse secondary antibody (Jackson Research Laboratories). Western blot analysis. To detect the presence of M2 and M3 proteins in transfected or virus-infected cells; Western blot assays were performed as follows. A total of 5 ⫻ 105 cells were either transfected with the appropriate plasmids or infected with either WT or mutant virus. Cells were washed once in PBS and lysed in 800 l of 1⫻ detergent solution (10 mM Tris [pH 7.4], 66 mM EDTA, 0.4% sodium deoxycholate, 1% Triton X-100, 0.05% sodium azide) containing the protease inhibitor aprotinin (U. S. Biochemicals) at a concentration of 200 U/ml for 5 min at room temperature. Aliquots of cell lysates were separated on a sodium dodecyl sulfate (SDS)–10% polyacrylamide gel followed by transfer to a nitrocellulose membrane (Protran; Midwest Scientific). Immunoblotting was carried out by using an M-specific monoclonal antibody (23H12) (15) followed by a horseradish peroxidase-tagged goat anti-mouse secondary antibody (Jackson Research Laboratories). Proteins were visualized by using a chemiluminescence kit (Amersham) according to the manufacturer’s instructions. Immunoprecipitation assay. BHK-21 cells in 35-mm-diameter dishes were infected with vTF7-3 at an MOI of 10 for 1 h in serum-free DMEM. The cells were then transfected with 5, 4, and 1 g of plasmids encoding N, P, and L proteins, respectively, by using TransfectACE. Five hours p.t., the transfection mix was removed and the cells were infected with supernatants containing either WT or mutant minigenome particles. At 6 h after minigenome infection, the cells were rinsed twice with methionine-free, serum-free DMEM and then pulsed with 50 Ci of [35S]methionine in 1 ml of the same medium by using protein labeling mix (Dupont, NEN) at 37°C for 1 h. Following the 1-h labeling period, the radioactive medium was removed, and the cells were rinsed once in PBS and lysed immediately with 1 ml of detergent solution at room temperature for 5 min. An aliquot of the lysate was immunoprecipitated by using an M-specific monoclonal antibody (23H12) at 4°C overnight, followed by antibody-protein A complex formation at 4°C for 3 h. Immunoprecipitated proteins were then analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.
RESULTS VSV-infected cells produce two additional proteins, M2 and M3. The VSV genome has five genes that encode at least seven proteins: N, P, C, C⬘, M, G, and L. In addition to these viral proteins, we have identified two additional polypeptides in virus-infected BHK cells, which were recognized by a monoclonal antibody (23H12) specific for M protein (15). These products migrated faster than the full-length M protein on an SDS–10% polyacrylamide gel and had molecular sizes estimated at 20 to 25 kDa. The M-specific polypeptides were also detected in purified virions, although the amounts of the two
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FIG. 1. Expression of M2 and M3 proteins during WT VSV infection. (A) Cells were infected with WT virus (rVSV-GFP) at an MOI of 10. At 8 h postinfection, the supernatant was harvested and the virus was concentrated by centrifugation. The cells were then lysed in a detergent buffer. An aliquot of pelleted virus and cell lysate was separated on an SDS–10% polyacrylamide gel, and the M proteins were detected by Western blotting using an M-specific monoclonal antibody (23H12). (B) BHK-21, D17, HeLa, and QT6 cells were infected with WT virus at an MOI of 10. At 8 h postinfection, cells were radioactively labeled with [35S]methionine for 1 h. Cell extracts were made, and proteins were immunoprecipitated by using monoclonal antibody 23H12. Immunoprecipitated proteins were analyzed on an SDS–10% polyacrylamide gel followed by autoradiography.
smaller proteins incorporated into the virions relative to that of M protein were less than those seen in cell extracts (Fig. 1A). Since the two smaller species were recognized by an M-specific monoclonal antibody, we will refer to these as the M2 and M3 proteins (whereas the full-length 229-amino-acid M protein is referred to as the M1 protein). To determine if the expression of M2 and M3 was cell type specific, we tested several other cell lines, including HeLa (human), D17 (canine), and QT6 (avian) (Fig. 1B) as well as CV-1 (simian), Vero (simian), and HEK-293 (human) cell lines (data not shown). Both the M2 and M3 proteins were made in most of the cell lines, except for HeLa and HEK-293, where the M2 protein was not detectable. The amount of M3 made in the QT6 line was less that that made in BHK or D17 cells. These results suggested that production of M2 and M3 was influenced by the properties of the host cell. M2 and M3 are products of downstream translation initiation events. Since M2 and M3 were recognized by the same monoclonal antibody that detected the M1 protein, it is possible that M2 and M3 are specific cleavage products of M protein. Alternatively, M2 and M3 could be synthesized from the M mRNA by initiation at downstream methionine codons, namely, at M33 and M51, respectively. To test these possibilities, we made a construct named M1SC in which three con-
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secutive stop codons were introduced immediately after the first AUG codon of the M mRNA (Fig. 2A). Western blot analysis of extracts from BHK cells expressing the M1SC construct revealed that, as expected, the M1 protein was not made; however, M2 and M3 were synthesized, and in larger amounts than those made during VSV infection (Fig. 2B; compare lanes 2 and 5). Thus, the M2 and M3 proteins are not cleavage products of M protein. Instead, M2 and M3 are derived from independent translation initiation events at downstream AUG codons. The first downstream AUG codon encountered in the same ORF would be the AUG encoding Met-33, and the next would encode Met-51 (Fig. 2A). The predicted sizes for polypeptides initiating from these methionines would be approximately 24 and 21 kDa, respectively. Moreover, both of the downstream AUG codons are in the context of a suboptimal Kozak sequence, which would account for the fact that smaller amounts of M2 and M3 than of M1 are synthesized in VSVinfected cells. To examine the origin of M2 and M3 further, we made additional mutations in M1SC to prevent the synthesis of proteins initiating at methionine 33 (construct M2SC) and methionine 51 (construct M3SC) (Fig. 2A). Transient expression of these constructs in BHK cells revealed that when stop codons were placed after M33 (plasmid M2SC), only the M3 protein was made, whereas no M-related products were synthesized from plasmid M3SC as detected by antibody 23H12 in a Western blot assay (Fig. 2B, lanes 3 and 4, respectively). Collectively, these data support the conclusion that M2 and M3 are independent downstream translation products of the M gene. Interestingly, in cells transiently expressing the WT M protein by use of the vaccinia virus-T7 expression system, only the full-length M1 protein was made (Fig. 2B, lane 1). This observation suggested that the translation machinery might be modified in VSV-infected cells such that there is increased ribosomal scanning, which results in initiation at the downstream AUG codons. Recovery of recombinant viruses lacking M2 and M3. We next asked whether virus lacking the M2 and M3 proteins was viable. To address this we first generated minigenome mutants in which M33 and/or M51 was changed to an alanine or an arginine. The MGF M33A and MGF M51R constructs would not express M2 and M3, respectively, whereas MGF-M33,51A would lack both M2 and M3 and would express only M1 protein (Fig. 3A). All of the minivirus mutants were recovered with efficiencies similar to that of the WT minivirus (data not
FIG. 2. M2 and M3 proteins are made independently of M1 protein. (A) Schematic diagram showing mutations in the M gene. The positions of the first three methionines are shown. XXX represents three consecutive stop codons, which were introduced downstream of the first AUG (M1SC) or the first and second AUGs (M2SC). (B) Transient expression of M1, M2, and M3. Approximately 5 ⫻ 105 BHK-21 cells were first infected with a recombinant vaccinia virus expressing T7 polymerase and then transfected with 2.5 g of either pBS-M, containing the WT M cDNA, or one of the mutant constructs. At 24 h p.t., cells were lysed in a detergent buffer. M-specific proteins in the cell lysates were detected by Western blotting. Cell extracts from a WT virus-infected cell were used as a positive control (lane 5).
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FIG. 3. Generation of M2 and M3 deletion mutants in the MGF minigenome. (A) Diagram of the MGF minigenome. The positions of the hepatitis delta virus ribozyme (HDV), the T7 terminator sequence (T), and the T7 promoter are shown. The symbols l and t denote leader and trailer sequences, respectively. Arrows indicate the direction of transcription for each gene. The solid box represents the sequence encoding the N-terminal region of M protein and is enlarged below. Numbering indicates the positions of the first, second, and third methionines in M protein. Alanine substitutions were made at the M33 and M51 residues, while an arginine substitution was made at the M51 residue to recreate the mutation in the ts082 mutant reported and characterized earlier (6a). (B) Immunoprecipitation of M1, M2, and M3 proteins expressed in WT and mutant MGF minigenome-infected cells. Cells expressing the N, P, and L proteins were infected with either the WT or a mutant MGF minivirus, and then the cells were labeled at 6 h postinfection with [35S]methionine for 1 h. Cells were lysed in detergent buffer, and M proteins were immunoprecipitated with monoclonal antibody 23H12 and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Control lanes, immunoprecipitates from cells either infected with VVT7 alone or infected with VVT7 and transfected with plasmids encoding the N, P, and L proteins (N,P,L) only.
shown). Immunoprecipitation of M proteins from extracts of BHK cells infected with either WT or mutant minivirus showed that the M products were made as expected (Fig. 3B). These data confirmed that M2 and M3 originated from the 33rd and 51st amino acids, respectively. To determine if the lack of M2 and M3 expression markedly affected virus budding, we asked whether the MGF mutants could be passaged. To monitor cell infection with the miniviruses, we examined the cells for GFP expression by fluorescence microscopy. When supernatants from transfected cells were consecutively passaged on cells expressing the VSV N, P, and L proteins, the number of GFPpositive cells increased with each passage (data not shown). These results indicated that the M2 and M3 proteins were not essential for virus assembly or budding.
J. VIROL.
FIG. 4. Virus yield and growth kinetics of the M33,51A mutant. (A) BHK-21 cells were infected with either the WT or M33,51A virus at an MOI of 10. Cells were continuously labeled from 7 to 15 h postinfection with [35S]methionine. At 15 h postinfection, viruses were harvested from the supernatants by centrifugation and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. The positions of VSV proteins are indicated on the left. (B) The growth kinetics of the M33,51A mutant was compared with that of WT virus by taking aliquots of the supernatant at various times postinfection and determining the virus titer by a standard plaque assay on BHK cells.
Characterization of the M33,51A mutant. We next wanted to determine if the lack of M2 and M3 had any effect on the cytopathic effects seen during virus infection of cells. For this purpose we subcloned the M33,51A mutant into a full-length virus cDNA and recovered it successfully. The mutant made the same amount of virus as WT VSV (Fig. 4A) and expressed similar amounts of all viral proteins, except that it lacked the M2 and M3 proteins. The kinetics of virus production was also similar to that of WT VSV (Fig. 4B). To examine the effect of deleting M2 and M3 on inhibition of host gene expression,
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FIG. 5. Cell-rounding phenotype of the M33,51A mutant. BHK-21 cells were infected with either WT virus or the M33,51A mutant at an MOI of 10. At each time point indicated, the medium was removed and cells were fixed with 3% paraformaldehyde. Cells were observed by phase-contrast microscopy using a Zeiss Axiophot microscope with a 10⫻ objective, and images were captured using a Zeiss Axiocam digital camera and Axiovision software.
infected cell extracts labeled with [35S]Met were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. There appeared to be no difference between the abilities of the mutant and the WT virus to cause host shutoff (data not shown). Thus, M2 and M3 appeared to make no significant contribution to the inhibition of host gene expression. The M33,51A virus is defective in cell rounding. Interestingly, we found that during the infection of BHK cells, there was a significant delay in the rounding of mutant virus-infected cells compared to that of cells infected with the WT virus (Fig. 5). About 50% of the mutant virus-infected cells remained flat and exhibited no cytopathic effects even at 24 h postinfection, whereas ⬎95% of WT virus-infected cells became rounded (Fig. 5E and F). To determine if the effect on cell rounding was cell type specific, we examined four other cell lines (CV1, Vero, 293, and HeLa) after infection with the M33,51A mutant. The effects on CV1 and Vero cells were similar to those seen with BHK cells (Fig. 6A, C, and E). In contrast, the mutant caused cell rounding with kinetics similar to that of WT virus in HeLa and 293 cells (Fig. 6G and I). These data suggested that the M2 and M3 proteins may be involved in cell rounding during VSV infection and that this effect is cell type specific. Expression of M2 and M3 is sufficient to cause cell round-
FIG. 6. Cell-rounding phenotype of the M33,51A mutant in different cell types. The cell-rounding activity of the M33,51A mutant was examined as described in the legend for Fig 5 in four different cell types: BHK-21, CV-1, HeLa, and HEK-293 cells.
ing. To examine directly the role of M2 and M3 in cell rounding, we first asked whether expression of the M2 and M3 proteins could induce cell rounding when expressed alone. As shown in Fig. 7, expression of either M2 or M3 caused cell rounding. To examine this further, we asked whether M2 and M3 could cause cell rounding when expressed from the genome of a recombinant virus (rVSV-NCP12.1) that is completely defective in the ability to cause cell rounding but can still make infectious virus particles. The mutations that result in the noncytopathic phenotype are found in the M gene and include the M33A and M51A substitutions, as well as two additional mutations (T133A and S226G). A report describing the isolation and characterization of this mutant and its derivatives is in preparation. A recombinant virus (rVSVNCP12.1-M1SC) that expresses M2 and M3 from a separate cistron inserted between the G and L genes was successfully recov-
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FIG. 7. Transient expression of M2 and M3 causes cell rounding. BHK-21 cells were transfected with plasmids encoding either M1 (WT M) (A), M2 and M3 (M1SC) (B), M3 (M2SC) (C and D), or VSV nucleocapsid (N) protein (E and F). Cells were stained with either the M-specific monoclonal antibody 23H12 (A, B, and C), or an N-specific antibody (E). Fluorescence (A, B, C, and E) and phase-contrast (D and F) images were captured using a Zeiss Axiophot microscope with a 40⫻ objective and a Zeiss Axiocam digital camera and associated Axiovision software.
ered. To ensure that the expression of an additional gene from the noncytopathic variant had no effect on cell rounding, we used a construct that contained the cDNA for GFP inserted between the G and L genes as a control (Fig. 8A). As expected, M2 and M3 were expressed in cells infected with WT VSV or rVSVNCP12.1-M1SC but not in cells infected with the rM33,51A or rNCP12.1-GFP virus (Fig. 8B). The rVSV-NCP12.1-M1SC virus grew to WT titers (data not shown) and caused cell rounding with kinetics similar to that of WT VSV (Fig. 9). Thus, expression of
FIG. 9. Cell-rounding phenotypes of mutant viruses. BHK-21 cells were infected with either WT or mutant viruses at an MOI of 10. At the indicated time points, cells were fixed and observed by phasecontrast microscopy (magnification, ⫻125).
M2 and M3 was able to rescue the cell-rounding defect of rNCP12.1. These studies supported our hypothesis that M2 and M3 are involved in cell rounding and may play an important role in cytopathogenesis during virus infection. DISCUSSION Owing to a compact genome size, most negative-strand RNA viruses have evolved different strategies to maximize their coding capacity. The multiple reading frames within the P
FIG. 8. Expression of both M2 and M3 from the rNCP-M1SC construct. (A) Schematic diagrams of the rNCP12.1 and rNCP12.1-M1SC cDNAs. The symbols T, l, t, and HDV are as explained in the legend to Fig. 3A. The rNCP-M1SC virus is derived from rNCP12.1 by replacing the GFP gene with a second M gene, M1SC, which expresses only the M2 and M3 proteins. (B) Expression of M2 and M3 from the NCP-M1SC construct. BHK-21 cells were infected with either WT virus or the M33,51A, rNCP12.1, or NCP-M1SC mutant and grown for 8 h. Cells were then lysed with detergent buffer, and viral proteins were separated on an SDS–10% polyacrylamide gel followed by Western blot analysis. The positions of the M1, M2, and M3 proteins are indicated on the right.
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genes of paramyxoviruses and rhabdoviruses have been well characterized (1, 8, 10, 24, 27). Overlapping ORFs are also used to generate more than one glycoprotein in influenza B virus (22). However, there is no evidence so far that the M gene of a nonsegmented, negative-strand RNA virus encodes more than one protein. In this report we describe the existence of multiple protein products expressed from the M gene of VSV. We provide direct evidence that the M gene encodes at least three polypeptides, which we refer to as the M1, M2, and M3 proteins. These three proteins are made from the same ORF and share a common C-terminal amino acid sequence (Fig. 2). The M1 protein is the full-length 229-amino-acid polypeptide that is synthesized from the first AUG in the M mRNA. Using site-directed mutagenesis we show that M2 and M3 are made by initiation at downstream AUG codons encoding methionines at positions 33 and 51, respectively. It was reported previously that during VSV infection of Chinese hamster ovary (CHO) cells, the 26-kDa M protein (or M1) is specifically cleaved by an unidentified protease to produce a 17.5-kDa product called M⬘ (21). This degradation product of M protein was proposed to play a role in the regulation of M protein levels in the infected cell. The M⬘ protein is similar to the M3 protein reported here. However, our results using transient expression of the M gene (Fig. 2B) as well as expression from recombinant viruses (Fig. 8B) show that M2 and M3 are made independently of the M1 protein and that they are generated from downstream AUG codons. The region between the first and second AUG codons does not have the characteristics of an internal ribosome entry site (IRES) sequence, which is typically 200 to 300 nucleotides long. Moreover, all of the VSV transcripts except for leader RNA are capped and methylated. Therefore, we hypothesize that the synthesis of M2 and M3 proteins results from cap-dependent translation initiation and probably occurs by a leaky ribosomal scanning mechanism. In contrast to the situation in virus-infected cells, where we observed the synthesis of M1, M2, and M3 proteins, expression of the M gene from a plasmid using the vaccinia virus-T7 expression system resulted in the synthesis of only the M1 protein, not M2 or M3 (Fig. 2B, lane1). The preferential synthesis of M1 would be predicted, because the first AUG in the M mRNA has a very strong Kozak sequence, whereas the AUG codons specifying amino acids M33 and M51 have suboptimal Kozak sequences. However, when synthesis of the M1 protein was prevented by introducing stop codons immediately after the first AUG codon in the M gene, both the M2 and M3 proteins were expressed (Fig. 2B, lanes 2 and 3). The observation that all three proteins are made in virus-infected cells, but not when expressed transiently, suggests that the host translation machinery may be altered during a VSV infection such that more ribosomes fail to initiate at the first AUG codon and instead scan and initiate at the two downstream methionine codons to generate the M2 and M3 proteins. The alteration in AUG utilization could result from some viral factor or virus-induced host component that modifies the host translational machinery. Alternatively, this could result from sequestration of host components needed for efficient translational initiation. Whether or not this phenomenon is unique to VSV, an understanding of the basis for the differential expression of M2 and M3 will likely provide a better understanding of
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virus-host interactions and how VSV modifies the host machinery during the course of an infection. The M protein is a key player in virus assembly and budding, as well as in cytopathogenesis. The region of the M protein involved in virus assembly and budding has been mapped to the N-terminal 30 amino acids (3, 11, 30). The M2 (amino acids 33 to 229) and M3 (amino acids 51 to 229) proteins lack this region and would be predicted to play little to no role in virus assembly and budding. This prediction was confirmed by two observations: (i) the recombinant M33,51A virus, which lacks M2 and M3, was fully proficient in virus assembly and budding (Fig. 4A), and (ii) full-length VSV mutants lacking M1 but expressing the M2 and M3 proteins could not be recovered from plasmids (unpublished data). The recombinant M33,51A virus was also able to inhibit host gene expression similarly to the WT virus but was defective in cell rounding. This defect either could be due to a slight conformational change in the M1 protein resulting from the substitution of M33 and M51 with alanines or it could be due to the lack of the M2 and M3 proteins. The observation that the cell-rounding defect of a noncytopathic VSV mutant which does not express the M2 and M3 proteins could be rescued by the expression of M2 and M3 argues that the lack of M2 and M3, and not just a conformational change, was an important contributing factor to the cell-rounding defect in the M33,51A and rNCP12.1 viruses. It has been shown previously that the ability of M protein to induce cell rounding is correlated with its ability to inhibit host gene expression but not with the virus assembly function (16). Since the process of host shutoff requires about 10-fold-smaller amounts of M protein than those required to induce cell rounding (16), it is possible that enough M1 protein is made to compensate for the lack of M2 and M3 proteins in inhibiting host gene expression but that this is still not enough to cause rapid and extensive cell rounding. Alternatively, it is possible that the two cytopathic functions of M protein, i.e., host shutoff and disorganization of the host cytoskeleton, are independent of each other and that we have now obtained a tool with which to dissect these two functions. Recently it was demonstrated that the region between amino acids 51 and 59 is important for inhibition of nucleocytoplasmic transport, which also can influence host gene expression (19, 28). Since M2 and M3 also contain this region, they would be predicted to cause both host shutoff and inhibition of nucleocytoplasmic transport. We are currently investigating the abilities of M2 and M3 to carry out these functions. REFERENCES 1. Bellini, W. J., G. Englund, S. Rozenblatt, H. Arnheiter, and C. D. Richardson. 1985. Measles virus P gene codes for two proteins. J. Virol. 53:908–919. 2. Black, B. L., and D. S. Lyles. 1992. Vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo. J. Virol. 66:4058–4064. 3. Black, B. L., R. B. Rhodes, M. McKenzie, and D. S. Lyles. 1993. The role of vesicular stomatitis virus matrix protein in inhibition of host-directed gene expression is genetically separable from its function in virus assembly. J. Virol. 67:4814–4821. 4. Blondel, D., G. G. Harmison, and M. Schubert. 1990. Role of matrix protein in cytopathogenesis of vesicular stomatitis virus. J. Virol. 64:1716–1725. 5. Cattaneo, R., K. Kaelin, K. Baczko, and M. A. Billeter. 1989. Measles virus editing provides an additional cysteine-rich protein. Cell 56:759–764. 6. Chong, L. D., and J. K. Rose. 1993. Membrane association of functional vesicular stomatitis virus matrix protein in vivo. J. Virol. 67:407–414. 6a.Coulon, P., V. Deutsch, F. Lafay, C. Martinet-Edelist, F. Wyers, R. C. Herman, and A. Flamand. 1990. Genetic evidence for multiple functions of the matrix protein of vesicular stomatitis virus. J. Gen. Virol. 71:991–996.
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