scholarly journals Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

2015 ◽  
Vol 89 (23) ◽  
pp. 11750-11760 ◽  
Author(s):  
Timothy K. Soh ◽  
Sean P. J. Whelan
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Proteins ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Viral Particles ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Late Domains

ABSTRACTVesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

scholarly journals Phenotypes of vesicular stomatitis virus mutants with mutations in the PSAP motif of the matrix protein

2012 ◽  
Vol 93 (4) ◽  
pp. 857-865 ◽  
Author(s):  
Linda Obiang ◽  
Hélène Raux ◽  
Malika Ouldali ◽  
Danielle Blondel ◽  
Yves Gaudin
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Susceptibility Gene ◽  
Point Mutations ◽  
Binding Motif ◽  
Single Mutation ◽  
Amino Terminal ◽  
Vesicular Stomatitis ◽  
Late Domains

Vesicular stomatitis virus (VSV) matrix protein (M) has a flexible amino-terminal part that recruits cellular partners. It contains a dynamin-binding site that is required for efficient virus assembly, and two motifs, 24PPPY27 and 37PSAP40, that constitute potential late domains. Late domains are present in proteins of several enveloped viruses and are involved in the ultimate step of the budding process (i.e. fission between viral and cellular membranes). In baby hamster kidney (BHK)-21 cells, it has been demonstrated that the 24PPPY27 motif binds the Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) E3 ubiquitin ligase for efficient virus budding and that the 37PSAP40 motif, although conserved among M proteins of vesiculoviruses, does not possess late-domain activity. In this study, we have re-examined the contribution of the PSAP motif to VSV budding. First, we demonstrate that VSV M indeed binds TSG101 [tumour susceptibility gene 101; a component of the ESCRT1 (endosomal sorting complex required for transport 1)] through its PSAP motif. Second, we analysed the phenotype of several recombinant mutants. We show that a double mutant with point mutations in both the PSAP and the PPPY motifs is impaired compared with a single mutant in the PPPY motif, indicating that the PSAP motif partially compensates for the lack of the PPPY motif. Mutants’ phenotypes depend on cell lines: in CERA (chicken embryo-related, Alger clone) cells, a recombinant virus with a single mutation in the PSAP motif was impaired compared with the wild type, and a mutant with a single mutation in the dynamin-binding motif was much less impaired in Vero cells than in BSR (clones of BHK-21) cells. These results have implications for the VSV budding pathway that will be discussed.

scholarly journals Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

2009 ◽  
Vol 83 (6) ◽  
pp. 2611-2622 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Panda ◽  
Debasis Nayak ◽  
Asit K. Pattnaik
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Viral Envelope ◽  
M Protein ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

scholarly journals Genetic evidence for multiple functions of the matrix protein of vesicular stomatitis virus

1990 ◽  
Vol 71 (4) ◽  
pp. 991-996 ◽  
Author(s):  
P. Coulon ◽  
V. Deutsch ◽  
F. Lafay ◽  
C. Martinet-Edelist ◽  
F. Wyers ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Genetic Evidence ◽  
Multiple Functions ◽  
The Matrix ◽  
Vesicular Stomatitis

scholarly journals Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2

2015 ◽  
Vol 89 (21) ◽  
pp. 11019-11029 ◽  
Author(s):  
Frauke Beilstein ◽  
Linda Obiang ◽  
Hélène Raux ◽  
Yves Gaudin
Keyword(s):  
T Cells ◽  
Immune System ◽  
Mhc Class I ◽  
Vesicular Stomatitis Virus ◽  
Cd8 T Cells ◽  
Matrix Protein ◽  
Catalytic Subunit ◽  
The Matrix ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACTThe matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system.IMPORTANCEThe immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.

scholarly journals Double-Layered Membrane Vesicles Released from Mammalian Cells Infected with Sendai Virus Expressing the Matrix Protein of Vesicular Stomatitis Virus

Virology ◽  
1999 ◽  
Vol 263 (1) ◽  
pp. 230-243 ◽  
Author(s):  
Takemasa Sakaguchi ◽  
Tsuneo Uchiyama ◽  
Yutaka Fujii ◽  
Katsuhiro Kiyotani ◽  
Atsushi Kato ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Sendai Virus ◽  
Membrane Vesicles ◽  
The Matrix ◽  
Vesicular Stomatitis

scholarly journals Membrane-binding domains and cytopathogenesis of the matrix protein of vesicular stomatitis virus.

1994 ◽  
Vol 68 (11) ◽  
pp. 7386-7396 ◽  
Author(s):  
Z Ye ◽  
W Sun ◽  
K Suryanarayana ◽  
P Justice ◽  
D Robinson ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Membrane Binding ◽  
Binding Domains ◽  
The Matrix ◽  
Vesicular Stomatitis

scholarly journals Matrix protein of Vesicular stomatitis virus harbours a cryptic mitochondrial-targeting motif

2006 ◽  
Vol 87 (11) ◽  
pp. 3379-3384 ◽  
Author(s):  
Brian D. Lichty ◽  
Heidi McBride ◽  
Stephen Hanson ◽  
John C. Bell
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
M Protein ◽  
Mitochondrial Targeting ◽  
M Gene ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Green Fluorescent

Vesicular stomatitis virus (VSV) is a rhabdovirus that has attracted attention of late as an oncolytic virus and as a vaccine vector. Mutations in the matrix (M) gene of VSV yield attenuated strains that may be very useful in both settings. As a result of this interest in the M protein, this study analysed various M–green fluorescent protein (GFP) fusion constructs. Remarkably, fusion of the N terminus of the M protein to GFP targeted the fluorescent protein to the surface of mitochondria. Mutational analysis indicated that a mitochondrial-targeting motif exists within aa 33–67. Expression of these fusion proteins led to loss of mitochondrial membrane permeability and to an alteration in mitochondrial organization mirroring that seen during viral infection. In addition, a portion of the M protein present in infected cells co-purified with mitochondria. This work may indicate a novel function for this multifunctional viral protein.

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