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

Abstract

The 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. V esicular stomatitis virus (VSV) is the prototype rhabdovirus and for years has been used as a model to study many aspects of the virus life cycle. Its negative-strand RNA genome of 11,161 nucleotides successively encodes the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and the RNAdependent RNA polymerase (L). The N, P, and L proteins are associated with the RNA molecule and compose the transcriptionally active nucleocapsid (NC). The NC is enveloped by...

Mechanisms of Vesicular Stomatitis Virus Inactivation by Protoporphyrin IX, Zinc-Protoporphyrin IX, and Mesoporphyrin IX

“…VSV RNA is a negative-sense, single-stranded molecule that depends on viral RNA-dependent RNA polymerase, a complex formed by the L protein and the phosphoprotein (P), to replicate (32). The virion also contains the matrix (M) protein, located between the envelope inner surface and nucleocapsid (33). We demonstrate that PPIX, ZnPPIX, and MPIX inactivate VSV and that their antiviral activities are strongly enhanced by porphyrin photoactivation.…”

The host cellular protein Ndufaf4 interacts with the vesicular stomatitis virus M protein and affects viral propagation

“…To further identify the critical residues in the M protein involved in the M-Ndufaf4 interaction, all amino acids at the surface of the globular domain of the M protein were chosen for mutagenesis analysis and the following 27 point or double point mutations were designed: R73A, N75A, R79A/T80A, H93A/M94A, I96A, M98A, M98R, V122A/L123A, D125A/Q126A, E136A, P149A/P150A, L152D, V154Y, V154A/P155A, E156A, E156A/H157A, R159A/R160A, G165A/L166A, D180A/E181A, L183A/E184A, P187A/M188A, S199A/D200A, K214A/K215A, S217A, G218A, D223A, and V225A/S226A [18,19]. As shown by the results, all cells expressing the mutants grew as well as cells expressing wild-type M on SD/-4 medium, except for cells expressing E156A, E156A/H157A, or D180A/E181A M (Fig.…”

EIF3i affects vesicular stomatitis virus growth by interacting with matrix protein

“…The VSV M protein is composed of two domains, a globular carboxyterminal domain and a flexible amino-terminal domain (comprising the 57 first residues) (Gaudier et al, 2002;Beilstein et al, 2015) ( Fig. 2A).…”

“…To further determine the key resides of M protein participating in this interaction, amino acids exposed at the surface of the globular domain of M protein were chosen for mutagenesis analysis (Gaudier et al, 2002;Beilstein et al, 2015). As shown in the results, all other mutants grew as well as wild type M on SD/-4 medium except for V122A/L123A, D125A/Q126A, E136A, V154A/P155A, E156A, E156A/ H157A, R159A/R160A, G165A/L166A and D181A/E182A (Fig.…”

“…The polymerase L is an RNA-dependent RNA polymerase (RdRP) that associates with phosphoprotein (P), nucleocapsid (N) and genomic RNA to form the transcriptionally active nucleocapsid (Barr et al, 2002). This complex is condensed by matrix protein (M) to generate a coiled helical structure and then enclosed within a lipid bilayer directed by the integral transmembrane glycoprotein (G) (Ge et al, 2010;Beilstein et al, 2015).…”