Identification of Two Additional Translation Products from the Matrix ...

JOURNAL OF VIROLOGY, Aug. 2002, p. 8011–8018 Vol. 76, No. 160022-538X/02/$04.00�0 DOI: 10.1128/JVI.76.16.8011–8018.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of Two Additional Translation Products from the Matrix(M) Gene That Contribute to Vesicular Stomatitis

Virus CytopathologyHimangi 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 responsiblefor condensation of the ribonucleocapsid core during virus assembly and also plays a critical role in virusbudding. 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 disrup-tion of the host cytoskeleton, which results in rounding of infected cells. In this report, we show that the VSVM gene codes for two additional polypeptides, which we have named M2 and M3. These proteins are synthe-sized from downstream methionines in the same open reading frame as the M protein (which we refer to hereas M1) and lack the first 32 (M2) or 50 (M3) amino acids of M1. Infection of cells with a recombinant virusthat does not express M2 and M3 (M33,51A) resulted in a delay in cell rounding, but virus yield was notaffected. Transient expression of M2 and M3 alone caused cell rounding similar to that with the full-length M1protein, suggesting that the cell-rounding function of the M protein does not require the N-terminal 50 aminoacids. To determine if M2 and M3 were sufficient for VSV-mediated CPE, both M2 and M3 were expressed froma separate cistron in a VSV mutant background that readily establishes persistent infections and that normallylacks CPE. Infection of cells with the recombinant virus that expressed M2 and M3 resulted in cell roundingindistinguishable from that with the wild-type recombinant virus. These results suggest that M2 and M3 areimportant for cell rounding and may play an important role in viral cytopathogenesis. To our knowledge, thisis first report of the multiple coding capacities of a rhabdovirus matrix gene.

The nonsegmented, negative-strand RNA viruses have a rel-atively small genome size, ranging from 11 to 19 kb. To max-imize their coding capacities, many of these viruses haveevolved different strategies to express additional proteins. In-creased coding capacity can occur either at the transcriptionallevel (mRNA processing or modification) or at the transla-tional level, in which proteins are produced from alternativereading frames or from translation initiation at non-AUG ordownstream AUG codons (1, 5, 7, 8, 10, 12, 24). The best-characterized examples are the use of multiple overlappingopen reading frames (ORFs) within the P mRNAs of severalparamyxoviruses (1, 10, 27) and the mRNA encoding the NAand NB glycoproteins of influenza B virus (22).

A similar phenomenon has recently been described for Ve-sicular stomatitis virus (VSV), the prototype member of theRhabdoviridae family. The VSV genome contains five genes, N,P, M, G, and L, and each, except the P gene, is thought toencode a single unique protein. The VSV P gene, like itscounterpart in paramyxoviruses, has been shown to encode twoadditional proteins, C and C�, in a second ORF (14, 24) and a7,000-molecular-weight (7K) polypeptide in the same ORFthat encodes the P protein (12).

The VSV (Indiana serotype) M gene is transcribed into asingle mRNA which encodes the 229-amino-acid matrix (M)

protein. M protein has numerous functions in infected cells.For example, M protein is the driving force behind the assem-bly and budding of virions. M protein interacts with the viralribonucleoprotein core (RNP), resulting in the condensationof the RNP and subsequent inhibition of viral transcription(29). A fraction of the M protein (�10%) is also associatedwith the inner leaflet of the plasma membrane where virusassembly and budding takes place (6). Recent work has shownthat a motif (PPPY) located within the first 30 amino acids ofM contributes to this budding activity (11, 13). M protein isalso responsible for most of the cytopathic effects of VSVinfection. Expression of M protein by itself can cause inhibitionof host gene expression, which occurs mostly at the transcrip-tional level (2, 3, 18). This inhibition appears to be mediatedvia inactivation of the TFIID protein (17). M protein, whenexpressed alone in the absence of other viral components, alsocauses cytoskeletal disorganization. Disassembly of microtu-bules by M protein ultimately leads to cell rounding (4, 23),which is a hallmark of VSV infection in cell culture. Recentlyit was shown that a fraction of M protein colocalizes withnuclear pore complexes (NPCs) at the nuclear rim (19). Thisnuclear fraction of M protein is thought to contribute to thehost shutoff function of the protein by inhibiting RNA exportfrom the nucleus.

In this study, we show that the M mRNA encodes twoadditional polypeptides, which we refer to as M2 and M3.These proteins are synthesized from downstream methioni-nes in the same reading frame that encodes the 229-amino-acid M protein (which is referred to as M1 protein in thisreport). We also show that M2 and M3 are important for cell

* Corresponding author. Mailing address: Department of MolecularSciences, 858 Madison Ave., University of Tennessee Health ScienceCenter, Memphis, TN 38163. Phone: (901) 448-4634. Fax: (901) 448-8462. E-mail: [email protected].

† Present address: GTx, Inc., Memphis, TN 38163.

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rounding and may play an important role in viral pathogen-esis.

MATERIALS AND METHODS

Plasmid construction and design. To prevent the expression of the full-lengthM1 protein (229 amino acids), three consecutive stop codons were introducedimmediately downstream of the first AUG codon in the Bluescript-M�T (BS-M�T) plasmid, which encodes a wild-type (WT) VSV M protein (Indiana sero-type) under the control of the bacteriophage T7 promoter. The resulting con-struct was termed BS-M1SC. The BS-M2SC construct was generated byintroducing three stop codons immediately downstream of the second AUG, atamino acid position 33 in the BS-M1SC plasmid. The third AUG, at amino acidposition 51 in the M2SC plasmid, was mutated to encode arginine instead ofmethionine, generating construct BS-M3SC. A schematic representation of thevarious 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 basechanges to introduce three consecutive stop codons immediately following thefirst AUG was used, along with a reverse primer downstream of the MunI site,to generate a PCR product by using BS-M�T as the template. The 200-bp PCRproduct was then gel purified and digested with SmaI and Mun I restrictionenzymes, and the resulting 170-bp fragment was gel purified and used to replacethe corresponding WT region in the pBS-M�T plasmid. The pBS-M2SC con-struct was generated in a similar manner except that the reverse primer down-stream of the MunI site contained appropriate base changes to introduce twoadditional stop codons downstream of the second AUG at amino acid position33. The pBS-M3SC construct was generated by using a forward primer overlap-ping the MunI site and containing base changes to generate the M513R muta-tion and a reverse primer downstream of the BglII site in a PCR with pBS-M2SCas the template. The resulting PCR product was then gel purified, digested withMunI and BglII, and used to replace the corresponding region in plasmid pBS-M2SC. The plasmids were then sequenced by using the dideoxy sequencingmethod to ensure that only the specified mutations were introduced during PCRamplification.

The pCAGGS-M constructs were derived from the pBS-M constructs bysubcloning a KpnI-XbaI fragment containing the entire M coding region fromeither pBS-M�T, pBS-M1SC, or pBS-M2SC into the multiple cloning site of themodified pCAGGS vector pCAGGS-MCS.

Generation of minigenome and full-length VSV M2 and M3 mutants. Mini-genome mutants were all derived from the parent plasmid MGF-WT, whichconsists of a genomic sense VSV cDNA containing the M, G, and green fluo-rescent protein (GFP) genes (previously referred to as pBS-GMF) (26). TheM333A, M513A, and M513R mutations were generated by a PCR-basedcloning strategy similar to that described previously (13). For the M33A M51Amutation, the template used in the first PCR was MGF M33A instead of MGF-WT.

The M33A M51A (M33,51A) double mutant was subcloned into a modifiedfull-length VSV genome, called �M-PLF, described elsewhere (13). PlasmidMGF M33,51A was digested with EcoRV and NheI enzymes to produce a 1.6-kbinsert. Meanwhile, the parent plasmid, �M-PLF, was cut with SmaI and NheIenzymes. The restriction fragments were then gel purified and ligated in atwo-way ligation. Positive clones were identified by PCR screening as well asrestriction digestion.

Generation of the NCP-M1SC mutant. The parent plasmid used for the NCP-M1SC 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 inthe C terminus and will be described in detail elsewhere (unpublished data).Plasmid pVSV-M1SC, expressing M1SC (encoding the M2 and M3 proteins) asa separate cistron between the G and the L gene, was digested with NheI, locatedin 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 toreplace 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 inthe G gene and a reverse primer in the M1SC gene.

Recovery and characterization of M2 and M3 mutants. Recoveries of mini-genome mutants were performed as described previously (25) and monitored bydetecting the expression of GFP from the reporter gene. All full-length viruseswere recovered from their cDNAs as described earlier (13). Assays to determinethe total virus yield, for the one-step growth curve and for inhibition of host geneexpression with the M33,51A mutant, were performed as described previously(13).

Cell-rounding assay. BHK-21 cells in 35-mm-diameter dishes were infectedwith 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 withserum-free medium and then incubated at 37°C for varying times. At each timepoint, the medium was removed, and cells were washed twice with phosphate-buffered saline (PBS) and fixed with 3% paraformaldehyde at room temperaturefor 20 min, followed by two washes with PBS containing 20 mM glycine. Cellswere 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 anMOI 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 constructand 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), andcultures were incubated at 37°C for 24 h.

For transient expression from pCAGGS plasmids, BHK-21 cells grown onglass coverslips were transfected with 2 �g of either pCAGGS-M WT, pCAGGS-M1SC, pCAGGS-M2SC, or pCAGGS-N WT by using 6 �l of Lipofectamine(GIBCO BRL) according to the manufacturer’s instructions. The lipid-DNAmixture was replaced with DMEM containing 10% FBS after 3 h, and cultureswere incubated for an additional 45 h. Expression of M and N proteins wasdetected by an indirect immunofluorescence assay with either an M-specific(23H12) or an N-specific (10G4) monoclonal antibody (15) and a rhodamineconjugated goat anti-mouse secondary antibody (Jackson Research Laborato-ries).

Western blot analysis. To detect the presence of M2 and M3 proteins intransfected or virus-infected cells; Western blot assays were performed as fol-lows. A total of 5 � 105 cells were either transfected with the appropriateplasmids or infected with either WT or mutant virus. Cells were washed once inPBS and lysed in 800 �l of 1� detergent solution (10 mM Tris [pH 7.4], 66 mMEDTA, 0.4% sodium deoxycholate, 1% Triton X-100, 0.05% sodium azide)containing the protease inhibitor aprotinin (U. S. Biochemicals) at a concentra-tion of 200 U/ml for 5 min at room temperature. Aliquots of cell lysates wereseparated on a sodium dodecyl sulfate (SDS)–10% polyacrylamide gel followedby transfer to a nitrocellulose membrane (Protran; Midwest Scientific). Immu-noblotting was carried out by using an M-specific monoclonal antibody (23H12)(15) followed by a horseradish peroxidase-tagged goat anti-mouse secondaryantibody (Jackson Research Laboratories). Proteins were visualized by using achemiluminescence kit (Amersham) according to the manufacturer’s instruc-tions.

Immunoprecipitation assay. BHK-21 cells in 35-mm-diameter dishes wereinfected with vTF7-3 at an MOI of 10 for 1 h in serum-free DMEM. The cellswere then transfected with 5, 4, and 1 �g of plasmids encoding N, P, and Lproteins, respectively, by using TransfectACE. Five hours p.t., the transfectionmix was removed and the cells were infected with supernatants containing eitherWT or mutant minigenome particles. At 6 h after minigenome infection, the cellswere rinsed twice with methionine-free, serum-free DMEM and then pulsed with50 �Ci of [35S]methionine in 1 ml of the same medium by using protein labelingmix (Dupont, NEN) at 37°C for 1 h. Following the 1-h labeling period, theradioactive medium was removed, and the cells were rinsed once in PBS andlysed 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 mono-clonal antibody (23H12) at 4°C overnight, followed by antibody-protein A com-plex formation at 4°C for 3 h. Immunoprecipitated proteins were then analyzedby SDS-polyacrylamide gel electrophoresis followed by autoradiography.

RESULTS

VSV-infected cells produce two additional proteins, M2 andM3. The VSV genome has five genes that encode at least sevenproteins: N, P, C, C�, M, G, and L. In addition to these viralproteins, we have identified two additional polypeptides invirus-infected BHK cells, which were recognized by a mono-clonal antibody (23H12) specific for M protein (15). Theseproducts migrated faster than the full-length M protein on anSDS–10% polyacrylamide gel and had molecular sizes esti-mated at 20 to 25 kDa. The M-specific polypeptides were alsodetected in purified virions, although the amounts of the two

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smaller proteins incorporated into the virions relative to that ofM protein were less than those seen in cell extracts (Fig. 1A).Since the two smaller species were recognized by an M-specificmonoclonal antibody, we will refer to these as the M2 and M3proteins (whereas the full-length 229-amino-acid M protein isreferred to as the M1 protein). To determine if the expressionof M2 and M3 was cell type specific, we tested several othercell lines, including HeLa (human), D17 (canine), and QT6(avian) (Fig. 1B) as well as CV-1 (simian), Vero (simian), andHEK-293 (human) cell lines (data not shown). Both the M2and M3 proteins were made in most of the cell lines, except forHeLa and HEK-293, where the M2 protein was not detectable.The amount of M3 made in the QT6 line was less that thatmade in BHK or D17 cells. These results suggested that pro-duction of M2 and M3 was influenced by the properties of thehost cell.

M2 and M3 are products of downstream translation initia-tion events. Since M2 and M3 were recognized by the samemonoclonal antibody that detected the M1 protein, it is possi-ble that M2 and M3 are specific cleavage products of M pro-tein. Alternatively, M2 and M3 could be synthesized from theM mRNA by initiation at downstream methionine codons,namely, at M33 and M51, respectively. To test these possibil-ities, we made a construct named M1SC in which three con-

secutive stop codons were introduced immediately after thefirst AUG codon of the M mRNA (Fig. 2A). Western blotanalysis of extracts from BHK cells expressing the M1SC con-struct revealed that, as expected, the M1 protein was not made;however, M2 and M3 were synthesized, and in larger amountsthan those made during VSV infection (Fig. 2B; compare lanes2 and 5). Thus, the M2 and M3 proteins are not cleavageproducts of M protein. Instead, M2 and M3 are derived fromindependent translation initiation events at downstream AUGcodons. The first downstream AUG codon encountered in thesame ORF would be the AUG encoding Met-33, and the nextwould encode Met-51 (Fig. 2A). The predicted sizes forpolypeptides initiating from these methionines would be ap-proximately 24 and 21 kDa, respectively. Moreover, both of thedownstream AUG codons are in the context of a suboptimalKozak sequence, which would account for the fact that smalleramounts of M2 and M3 than of M1 are synthesized in VSV-infected cells. To examine the origin of M2 and M3 further, wemade additional mutations in M1SC to prevent the synthesis ofproteins initiating at methionine 33 (construct M2SC) andmethionine 51 (construct M3SC) (Fig. 2A). Transient expres-sion of these constructs in BHK cells revealed that when stopcodons were placed after M33 (plasmid M2SC), only the M3protein was made, whereas no M-related products were syn-thesized from plasmid M3SC as detected by antibody 23H12 ina Western blot assay (Fig. 2B, lanes 3 and 4, respectively).Collectively, these data support the conclusion that M2 andM3 are independent downstream translation products of the Mgene. Interestingly, in cells transiently expressing the WT Mprotein by use of the vaccinia virus-T7 expression system, onlythe full-length M1 protein was made (Fig. 2B, lane 1). Thisobservation suggested that the translation machinery might bemodified in VSV-infected cells such that there is increasedribosomal scanning, which results in initiation at the down-stream AUG codons.

Recovery of recombinant viruses lacking M2 and M3. Wenext asked whether virus lacking the M2 and M3 proteins wasviable. To address this we first generated minigenome mutantsin which M33 and/or M51 was changed to an alanine or anarginine. The MGF M33A and MGF M51R constructs wouldnot express M2 and M3, respectively, whereas MGF-M33,51Awould lack both M2 and M3 and would express only M1 pro-tein (Fig. 3A). All of the minivirus mutants were recoveredwith efficiencies similar to that of the WT minivirus (data not

FIG. 1. Expression of M2 and M3 proteins during WT VSV infec-tion. (A) Cells were infected with WT virus (rVSV-GFP) at an MOI of10. At 8 h postinfection, the supernatant was harvested and the viruswas concentrated by centrifugation. The cells were then lysed in adetergent buffer. An aliquot of pelleted virus and cell lysate was sep-arated on an SDS–10% polyacrylamide gel, and the M proteins weredetected by Western blotting using an M-specific monoclonal antibody(23H12). (B) BHK-21, D17, HeLa, and QT6 cells were infected withWT virus at an MOI of 10. At 8 h postinfection, cells were radioactivelylabeled with [35S]methionine for 1 h. Cell extracts were made, andproteins were immunoprecipitated by using monoclonal antibody23H12. Immunoprecipitated proteins were analyzed on an SDS–10%polyacrylamide gel followed by autoradiography.

FIG. 2. M2 and M3 proteins are made independently of M1 protein. (A) Schematic diagram showing mutations in the M gene. The positionsof 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 firstinfected with a recombinant vaccinia virus expressing T7 polymerase and then transfected with 2.5 �g of either pBS-M, containing the WT McDNA, 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 detectedby Western blotting. Cell extracts from a WT virus-infected cell were used as a positive control (lane 5).

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shown). Immunoprecipitation of M proteins from extracts ofBHK cells infected with either WT or mutant minivirus showedthat the M products were made as expected (Fig. 3B). Thesedata confirmed that M2 and M3 originated from the 33rd and51st amino acids, respectively. To determine if the lack of M2and M3 expression markedly affected virus budding, we askedwhether the MGF mutants could be passaged. To monitor cellinfection with the miniviruses, we examined the cells for GFPexpression by fluorescence microscopy. When supernatantsfrom transfected cells were consecutively passaged on cellsexpressing the VSV N, P, and L proteins, the number of GFP-positive cells increased with each passage (data not shown).These results indicated that the M2 and M3 proteins were notessential for virus assembly or budding.

Characterization of the M33,51A mutant. We next wantedto determine if the lack of M2 and M3 had any effect on thecytopathic effects seen during virus infection of cells. For thispurpose we subcloned the M33,51A mutant into a full-lengthvirus cDNA and recovered it successfully. The mutant madethe same amount of virus as WT VSV (Fig. 4A) and expressedsimilar amounts of all viral proteins, except that it lacked theM2 and M3 proteins. The kinetics of virus production was alsosimilar to that of WT VSV (Fig. 4B). To examine the effect ofdeleting M2 and M3 on inhibition of host gene expression,

FIG. 3. Generation of M2 and M3 deletion mutants in the MGFminigenome. (A) Diagram of the MGF minigenome. The positions ofthe hepatitis delta virus ribozyme (HDV), the T7 terminator sequence(�T), and the T7 promoter are shown. The symbols l and t denoteleader and trailer sequences, respectively. Arrows indicate the direc-tion of transcription for each gene. The solid box represents the se-quence encoding the N-terminal region of M protein and is enlargedbelow. Numbering indicates the positions of the first, second, and thirdmethionines in M protein. Alanine substitutions were made at the M33and M51 residues, while an arginine substitution was made at the M51residue to recreate the mutation in the ts082 mutant reported andcharacterized earlier (6a). (B) Immunoprecipitation of M1, M2, andM3 proteins expressed in WT and mutant MGF minigenome-infectedcells. Cells expressing the N, P, and L proteins were infected witheither the WT or a mutant MGF minivirus, and then the cells werelabeled at 6 h postinfection with [35S]methionine for 1 h. Cells werelysed in detergent buffer, and M proteins were immunoprecipitatedwith monoclonal antibody 23H12 and analyzed by SDS-polyacrylamidegel electrophoresis followed by autoradiography. Control lanes, immu-noprecipitates from cells either infected with VVT7 alone or infectedwith VVT7 and transfected with plasmids encoding the N, P, and Lproteins (N,P,L) only.

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 virusat an MOI of 10. Cells were continuously labeled from 7 to 15 hpostinfection with [35S]methionine. At 15 h postinfection, viruses wereharvested from the supernatants by centrifugation and analyzed bySDS-polyacrylamide gel electrophoresis followed by autoradiography.The positions of VSV proteins are indicated on the left. (B) Thegrowth kinetics of the M33,51A mutant was compared with that of WTvirus by taking aliquots of the supernatant at various times postinfec-tion and determining the virus titer by a standard plaque assay on BHKcells.



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infected cell extracts labeled with [35S]Met were analyzed bySDS-polyacrylamide gel electrophoresis followed by autora-diography. There appeared to be no difference between theabilities of the mutant and the WT virus to cause host shutoff(data not shown). Thus, M2 and M3 appeared to make nosignificant contribution to the inhibition of host gene expres-sion.

The M33,51A virus is defective in cell rounding. Interest-ingly, we found that during the infection of BHK cells, therewas a significant delay in the rounding of mutant virus-infectedcells compared to that of cells infected with the WT virus (Fig.5). About 50% of the mutant virus-infected cells remained flatand 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 wascell type specific, we examined four other cell lines (CV1,Vero, 293, and HeLa) after infection with the M33,51A mu-tant. The effects on CV1 and Vero cells were similar to thoseseen with BHK cells (Fig. 6A, C, and E). In contrast, themutant caused cell rounding with kinetics similar to that of WTvirus in HeLa and 293 cells (Fig. 6G and I). These data sug-gested that the M2 and M3 proteins may be involved in cellrounding during VSV infection and that this effect is cell typespecific.

Expression of M2 and M3 is sufficient to cause cell round-

ing. To examine directly the role of M2 and M3 in cell rounding,we first asked whether expression of the M2 and M3 proteinscould induce cell rounding when expressed alone. As shown inFig. 7, expression of either M2 or M3 caused cell rounding. Toexamine this further, we asked whether M2 and M3 could causecell rounding when expressed from the genome of a recombinantvirus (rVSV-NCP12.1) that is completely defective in the ability tocause cell rounding but can still make infectious virus particles.The mutations that result in the noncytopathic phenotype arefound in the M gene and include the M33A and M51A substitu-tions, as well as two additional mutations (T133A and S226G). Areport describing the isolation and characterization of this mutantand its derivatives is in preparation. A recombinant virus (rVSV-NCP12.1-M1SC) that expresses M2 and M3 from a separate cis-tron inserted between the G and L genes was successfully recov-

FIG. 5. Cell-rounding phenotype of the M33,51A mutant. BHK-21cells were infected with either WT virus or the M33,51A mutant at anMOI of 10. At each time point indicated, the medium was removedand cells were fixed with 3% paraformaldehyde. Cells were observedby phase-contrast microscopy using a Zeiss Axiophot microscope witha 10� objective, and images were captured using a Zeiss Axiocamdigital camera and Axiovision software.

FIG. 6. Cell-rounding phenotype of the M33,51A mutant in differ-ent cell types. The cell-rounding activity of the M33,51A mutant wasexamined as described in the legend for Fig 5 in four different celltypes: BHK-21, CV-1, HeLa, and HEK-293 cells.



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ered. To ensure that the expression of an additional gene fromthe noncytopathic variant had no effect on cell rounding, we useda construct that contained the cDNA for GFP inserted betweenthe G and L genes as a control (Fig. 8A). As expected, M2 andM3 were expressed in cells infected with WT VSV or rVSV-NCP12.1-M1SC but not in cells infected with the rM33,51A orrNCP12.1-GFP virus (Fig. 8B). The rVSV-NCP12.1-M1SC virusgrew to WT titers (data not shown) and caused cell rounding withkinetics similar to that of WT VSV (Fig. 9). Thus, expression of

M2 and M3 was able to rescue the cell-rounding defect ofrNCP12.1. These studies supported our hypothesis that M2 andM3 are involved in cell rounding and may play an important rolein cytopathogenesis during virus infection.

DISCUSSION

Owing to a compact genome size, most negative-strandRNA viruses have evolved different strategies to maximizetheir coding capacity. The multiple reading frames within the P

FIG. 7. Transient expression of M2 and M3 causes cell rounding.BHK-21 cells were transfected with plasmids encoding either M1 (WTM) (A), M2 and M3 (M1SC) (B), M3 (M2SC) (C and D), or VSVnucleocapsid (N) protein (E and F). Cells were stained with either theM-specific monoclonal antibody 23H12 (A, B, and C), or an N-specificantibody (E). Fluorescence (A, B, C, and E) and phase-contrast (Dand F) images were captured using a Zeiss Axiophot microscope witha 40� objective and a Zeiss Axiocam digital camera and associatedAxiovision software.

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 GFPgene 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 withdetergent 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.

FIG. 9. Cell-rounding phenotypes of mutant viruses. BHK-21 cellswere infected with either WT or mutant viruses at an MOI of 10. Atthe indicated time points, cells were fixed and observed by phase-contrast microscopy (magnification, �125).



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genes of paramyxoviruses and rhabdoviruses have been wellcharacterized (1, 8, 10, 24, 27). Overlapping ORFs are alsoused to generate more than one glycoprotein in influenza Bvirus (22). However, there is no evidence so far that the Mgene of a nonsegmented, negative-strand RNA virus encodesmore than one protein. In this report we describe the existenceof multiple protein products expressed from the M gene ofVSV. We provide direct evidence that the M gene encodes atleast three polypeptides, which we refer to as the M1, M2, andM3 proteins. These three proteins are made from the sameORF and share a common C-terminal amino acid sequence(Fig. 2).

The M1 protein is the full-length 229-amino-acid polypep-tide that is synthesized from the first AUG in the M mRNA.Using site-directed mutagenesis we show that M2 and M3 aremade by initiation at downstream AUG codons encoding me-thionines at positions 33 and 51, respectively. It was reportedpreviously that during VSV infection of Chinese hamster ovary(CHO) cells, the 26-kDa M protein (or M1) is specificallycleaved by an unidentified protease to produce a 17.5-kDaproduct called M� (21). This degradation product of M proteinwas proposed to play a role in the regulation of M proteinlevels in the infected cell. The M� protein is similar to the M3protein reported here. However, our results using transientexpression of the M gene (Fig. 2B) as well as expression fromrecombinant viruses (Fig. 8B) show that M2 and M3 are madeindependently of the M1 protein and that they are generatedfrom downstream AUG codons. The region between the firstand second AUG codons does not have the characteristics ofan internal ribosome entry site (IRES) sequence, which istypically 200 to 300 nucleotides long. Moreover, all of the VSVtranscripts except for leader RNA are capped and methylated.Therefore, we hypothesize that the synthesis of M2 and M3proteins results from cap-dependent translation initiation andprobably occurs by a leaky ribosomal scanning mechanism.

In contrast to the situation in virus-infected cells, where weobserved the synthesis of M1, M2, and M3 proteins, expressionof the M gene from a plasmid using the vaccinia virus-T7expression system resulted in the synthesis of only the M1protein, not M2 or M3 (Fig. 2B, lane1). The preferential syn-thesis of M1 would be predicted, because the first AUG in theM mRNA has a very strong Kozak sequence, whereas theAUG codons specifying amino acids M33 and M51 have sub-optimal Kozak sequences. However, when synthesis of the M1protein was prevented by introducing stop codons immediatelyafter the first AUG codon in the M gene, both the M2 and M3proteins were expressed (Fig. 2B, lanes 2 and 3). The obser-vation that all three proteins are made in virus-infected cells,but not when expressed transiently, suggests that the hosttranslation machinery may be altered during a VSV infectionsuch that more ribosomes fail to initiate at the first AUGcodon and instead scan and initiate at the two downstreammethionine codons to generate the M2 and M3 proteins. Thealteration in AUG utilization could result from some viralfactor or virus-induced host component that modifies the hosttranslational machinery. Alternatively, this could result fromsequestration of host components needed for efficient transla-tional initiation. Whether or not this phenomenon is unique toVSV, an understanding of the basis for the differential expres-sion of M2 and M3 will likely provide a better understanding of

virus-host interactions and how VSV modifies the host machin-ery 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 proteininvolved in virus assembly and budding has been mapped tothe N-terminal 30 amino acids (3, 11, 30). The M2 (amino acids33 to 229) and M3 (amino acids 51 to 229) proteins lack thisregion and would be predicted to play little to no role in virusassembly and budding. This prediction was confirmed by twoobservations: (i) the recombinant M33,51A virus, which lacksM2 and M3, was fully proficient in virus assembly and budding(Fig. 4A), and (ii) full-length VSV mutants lacking M1 butexpressing the M2 and M3 proteins could not be recoveredfrom plasmids (unpublished data). The recombinant M33,51Avirus was also able to inhibit host gene expression similarly tothe WT virus but was defective in cell rounding. This defecteither could be due to a slight conformational change in theM1 protein resulting from the substitution of M33 and M51with alanines or it could be due to the lack of the M2 and M3proteins. The observation that the cell-rounding defect of anoncytopathic VSV mutant which does not express the M2 andM3 proteins could be rescued by the expression of M2 and M3argues that the lack of M2 and M3, and not just a conforma-tional change, was an important contributing factor to thecell-rounding defect in the M33,51A and rNCP12.1 viruses.

It has been shown previously that the ability of M protein toinduce cell rounding is correlated with its ability to inhibit hostgene expression but not with the virus assembly function (16).Since the process of host shutoff requires about 10-fold-smalleramounts of M protein than those required to induce cellrounding (16), it is possible that enough M1 protein is made tocompensate for the lack of M2 and M3 proteins in inhibitinghost gene expression but that this is still not enough to causerapid and extensive cell rounding. Alternatively, it is possiblethat the two cytopathic functions of M protein, i.e., host shutoffand disorganization of the host cytoskeleton, are independentof each other and that we have now obtained a tool with whichto dissect these two functions. Recently it was demonstratedthat the region between amino acids 51 and 59 is important forinhibition of nucleocytoplasmic transport, which also can in-fluence host gene expression (19, 28). Since M2 and M3 alsocontain this region, they would be predicted to cause both hostshutoff and inhibition of nucleocytoplasmic transport. We arecurrently investigating the abilities of M2 and M3 to carry outthese functions.

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