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. 2002 Aug;76(16):8011-8.
doi: 10.1128/jvi.76.16.8011-8018.2002.

Identification of two additional translation products from the matrix (M) gene that contribute to vesicular stomatitis virus cytopathology

Himangi R Jayakar  1 , Michael A Whitt
Affiliations

Identification of two additional translation products from the matrix (M) gene that contribute to vesicular stomatitis virus cytopathology

Himangi R Jayakar et al. J Virol. 2002 Aug.

Abstract

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.

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Figures

FIG. 1.
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.
FIG. 2.
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 μg 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).
FIG. 3.
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 (φT), 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.
FIG. 4.
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.
FIG. 5.
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.
FIG. 6.
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.
FIG. 7.
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.
FIG. 8.
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 φT, 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.
FIG. 9.
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 phase-contrast microscopy (magnification, ×125).
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