Membrane vesiculation function and exocytosis of wild-type and mutant matrix proteins of vesicular stomatitis virus

Abstract

Transfection of mammalian CV1 cells with a recombinant M-gene pTM1 plasmid, driven by vaccinia virus-expressed phage T7 polymerase, resulted in the expression of matrix (M) protein, which is progressively released from the exterior surface of the transfected-cell plasma membrane. Exocytosis of M protein begins 2 to 4 h posttransfection and reaches a peak by 10 to 16 h posttransfection; dye uptake studies reveal that >97% of cells are alive and have intact membranes at 16 h posttransfection. Density gradient centrifugation and labeling with radioactive palmitic acid revealed that the M protein is released from cells in association with lipid vesicles. Expression of M-gene deletion mutants suggests that exocytosis of M protein requires the presence of a membrane-binding site at N-terminal amino acids 1 to 50. Cells transfected with the pTM1 plasmid containing the M gene of the temperature-sensitive mutant tsO23 expressed ample quantities of the mutant M protein at permissive (31؇C) and restrictive (39؇C) temperatures, but the exocytosis of the mutant M protein occurred only at the permissive temperature. The tsO23 M gene has three site-specific mutations resulting in amino acid substitutions at residues 21, 111, and 227. Expression of wild-type and mutant M genes with mutations or revertants at each of these sites resulted in exocytosis of M protein at the nonpermissive temperature only when wild-type leucine was present at residue 111, but M-protein exocytosis was restricted (to some extent even at the permissive temperature) when mutant phenylalanine was present at residue 111. Past and present data indicate that a specific structural conformation of the M protein is responsible for the formation and budding of vesicles, a property of the M protein which probably also promotes vesicular stomatitis virus assembly and budding of virions from host cells.

The Glycoprotein Cytoplasmic Tail of Uukuniemi Virus ( Bunyaviridae ) Interacts with Ribonucleoproteins and Is Critical for Genome Packaging

“…In some enveloped viruses, an interaction between the viral RNP complex and envelope proteins drives and facilitates the budding of virus particles, as shown, e.g., for Semliki Forest virus (40), simian virus 5 (38), murine leukemia virus (27), and Ebola virus (22). For other enveloped viruses, the RNPs are dispensable for viral envelope formation and production of virus particles, as demonstrated, e.g., for coronavirus (43), Marburg virus (41), influenza virus (10), vesicular stomatitis virus (15), and human parainfluenza virus type 1 (5). It was also reported in the 1990s that for Hantaan virus, in the Bunyaviridae family, the nucleoprotein is essential for generating VLPs and for the initiation of budding (3).…”

Generation and Analysis of Infectious Virus-Like Particles of Uukuniemi Virus ( Bunyaviridae ): a Useful System for Studying Bunyaviral Packaging and Budding

“…Vaccinia virus is a lytic virus which expresses quite a lot of proteins that could interfere with VLP generation, compared to our plasmid-driven system. The presence of nucleocapsids to facilitate VLP generation has been shown to be important for some viruses (e.g., Semliki Forest virus [49], Hantaan virus [5], paramyxovirus simian virus 5 [45], murine leukemia virus [38], and Ebola virus [33]) but not for others (e.g., coronavirus [51], Marburg virus [50], influenza virus [20], vesicular stomatitis virus [25], and human parainfluenza virus type 1 [10]). As expected, no nucleoprotein was released into the supernatant when nucleoprotein was expressed alone (lane 5).…”

Naked Sendai virus vector lacking all of the envelope‐related genes: reduced cytopathogenicity and immunogenicity

“…The HN protein is known to be one of the major targets of the host humoral immune responses against SeV infection, and to induce NK and cytotoxic T lymphocyte responses 11, 12. The M protein promotes vesiculation of the membrane and the release of particles into the extracellular medium without the aid of other viral proteins 13, 14. We previously succeeded in the recovery of high titers of F‐gene‐deleted (SeV/ΔF) 15, M‐gene‐deleted (SeV/ΔM) 16, HN‐gene‐deleted (SeV/ΔHN), and both M‐ and F‐genes‐deleted (SeV/ΔMΔF) 17 SeV vectors in addition to other types of SeV vectors 18 by using packaging cell lines that express the respective proteins encoded by the deleted gene(s).…”