J Virol 64:902C906. 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. IMPORTANCE LATH antibody The 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 Varenicline Tartrate 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. INTRODUCTION Vesicular 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 RNA-dependent 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 a lipid bilayer which is derived from the host cell plasma membrane and which is acquired during the budding process. G is a transmembrane glycoprotein that is involved in virus entry. Most of the M protein is located beneath the viral membrane and bridges the NC and the lipid bilayer (1). M is a multifunctional protein involved in virus assembly and budding. In relationship with this structural role, it has been demonstrated that VSV M interacts with both artificial and cellular membranes (2,C5) and that it binds to the viral nucleocapsid (6, 7). It also self-associates into large multimers at physiological salt concentrations (8,C10). The flexible amino-terminal part of M has the ability to recruit cellular partners that assist with viral assembly and budding. The first 10 amino acids of M bind dynamin, and this interaction is required for efficient viral assembly (11). The amino-terminal part of M also contains two late domains, 24PPPY27 and 37PSAP40, which have the ability to recruit cellular partners that are involved in the ultimate step of the budding process. The 24PPPY27 domain has been shown to interact with the WW domains of Nedd4-related E3 ubiquitin ligases (12,C14), a feature which is essential for efficient viral budding. The 37PSAP40 domain recruits TSG101 (15), a component of the endosomal sorting complex required for transport (ESCRT) complexes that plays a key role in the biogenesis of multivesicular bodies (MVBs) (16). It has also been shown that M protein targets several cellular proteins to inhibit host gene expression at multiple levels, including transcription and nuclear cytoplasmic transport. M interacts with host proteins Nup98 (17) and Rae1 (18), which have been implicated in the regulation of mRNA nuclear-cytoplasmic transport (18) and in cellular transcription (19). Proteasomes are the major nonlysosomal machines involved in protein degradation (20, 21). They are classified into three subtypes on the basis of the nature of Varenicline Tartrate their catalytic subunits. The structure of all three subtypes is basically the same, consisting of the 20S core particle, which is composed Varenicline Tartrate of 28 subunits arranged in four heptameric rings. The upper and the lower rings are formed by subunits, while the two central rings contain the subunits (22). Varenicline Tartrate The.