Self-propagating, infectious, virus-like vesicles (VLVs) are generated when an alphavirus RNA replicon expresses the vesicular stomatitis virus glycoprotein (VSV G) as the only structural protein. ...The mechanism that generates these VLVs lacking a capsid protein has remained a mystery for over 20 years. We present evidence that VLVs arise from membrane-enveloped RNA replication factories (spherules) containing VSV G protein that are largely trapped on the cell surface. After extensive passaging, VLVs evolve to grow to high titers through acquisition of multiple point mutations in their nonstructural replicase proteins. We reconstituted these mutations into a plasmid-based system from which high-titer VLVs can be recovered. One of these mutations generates a late domain motif (PTAP) that is critical for high-titer VLV production. We propose a model in which the VLVs have evolved in vitro to exploit a cellular budding pathway that is hijacked by many enveloped viruses, allowing them to bud efficiently from the cell surface. Our results suggest a basic mechanism of propagation that may have been used by primitive RNA viruses lacking capsid proteins. Capsids may have evolved later to allow more efficient packaging of RNA, greater virus stability, and evasion of innate immunity.
Significance All known membrane-enveloped RNA viruses have capsid proteins that encase their RNA genomes. This paper shows that infectious, membrane-enveloped, virus-like vesicles with RNA genomes can evolve in vitro to grow to high titers without a capsid protein. The infectious vesicles are apparently generated from RNA replication factories called spherules that bud from the cell surface. They evolve in vitro to bud with high efficiency through the acquisition of multiple mutations in the non-structural replicase proteins. One mutation generates a critical motif found in many viral structural proteins. This motif is involved in recruiting cellular machinery to drive efficient budding. Prior to the evolution of capsid proteins, primitive RNA viruses may have used this budding mechanism.
Chandipura virus (CHAV), a member of the vesiculovirus genus, is an emerging human pathogen. As for other rhabdoviruses, CHAV entry into susceptible cells is mediated by its single envelope ...glycoprotein G which is both involved in receptor recognition and fusion of viral and cellular membranes. Here, we have characterized the fusion properties of CHAV-G. As for vesicular stomatitis virus (VSV, the prototype of the genus) G, fusion is triggered at low pH below 6.5. We have also analyzed the biochemical properties of a soluble form of CHAV-G ectodomain (CHAV-Gth, generated by thermolysin limited-proteolysis of recombinant VSV particles in which the G gene was replaced by that of CHAV). The overall behavior of CHAV-Gth is similar to that previously reported for VSV-Gth. Particularly, CHAV-Gth pre-fusion trimer is not stable in solution and low-pH-induced membrane association of CHAV-Gth is reversible. Furthermore, CHAV-Gth was crystallized in its low pH post-fusion conformation and its structure was determined at 3.6Å resolution. An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure. Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses. Finally, local differences indicate that CHAV has evolved alternate structural solutions in hinge regions between PH and fusion domains but also distinct pH sensitive switches. Globally the comparison between the post fusion conformation of CHAV and VSV-G highlights several features essential for the protein's function. It also reveals the remarkable plasticity of G in terms of local structures.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We developed an AIDS vaccine based on attenuated VSV vectors expressing
env and
gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different ...VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4
+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.
Anti-retroviral therapy is useful to treat human immunodeficiency virus type 1 (HIV-1)-infected individuals, but has some major problems, such as the generation of multidrug-resistant viruses. To ...develop a novel supplemental or alternative therapeutic for CCR5-tropic (R5) HIV-1 infection, we generated a recombinant vesicular stomatitis virus (rVSV) in which the gene encoding its envelope glycoprotein (G) was replaced with the genes encoding R5 HIV-1 receptors (human CD4 and CCR5), designated VSVΔG-CC5. Our present data demonstrate that this rVSV specifically infects cells that are transiently expressing R5 HIV-1 envelope glycoproteins, but does not infect those expressing CXCR4-tropic HIV-1 envelope glycoproteins. Notably, after a CD4+CCR5+ T cell line or primary cells initially infected with R5 HIV-1 were inoculated with G-complemented VSVΔG-CC5, the rVSV significantly reduced the number of HIV-1-infected cells, probably through direct targeting of the rVSV and VSV-mediated cytolysis and/or through syncytium formation- or cell–cell fusion-dependent killing, and markedly inhibited HIV-1 production. Furthermore, G-complemented VSVΔG-CC5 also efficiently inhibited HIV-1 infection in R5 HIV-1-infected humanized immunodeficient mice. Taken together, our findings indicate that a cytolytic rVSV that targets and eliminates R5 HIV-1-infected cells potentially has therapeutic value for controlling R5 HIV-1 infection.
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