Synergistic Attenuation of Vesicular Stomatitis Virus by Combination of Specific G Gene Truncations and N Gene Translocations

Abstract

A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their "wild-type" progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD 50 ) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10-to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD 50 of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.Vesicular stomatitis virus (VSV) is a member of the Vesiculovirus genus of the family Rhabdoviridae. The negative-sense virus genome is 11,162 nucleotides long and contains five genes in the order 3Ј N-P-M-G-L 5Ј, encoding the five major viral proteins (1, 3). The bullet-shaped VSV particle (160 nm by 80 nm) contains a ribonucleoprotein core (nucleocapsid) composed of genomic RNA closely associated with N protein and a RNA polymerase composed of a complex of L and P proteins enveloped in a host cell-derived plasma membrane (4,18,19,44,53,56). Following uptake of the virus particle by susceptible cells, nucleocapsid and viral RNA polymerase are released into the cytoplasm and viral mRNA transcription ensues. A 3Ј-5Ј gradient of viral mRNA transcription leads to abundant N protein expression and successively decreasing levels of P, M, G, and L proteins (1,3,15,19,27,57). This gene expression gradient provides virus proteins in a suitable ratio for subsequent viral genome replication and assembly of mature virus particles. Virus replication in cell culture is rapid, and virus progeny are detectable 5 to 6 h postinfection.Since the initial recovery of infectious recombinant VSV (rVSV) from genomic cDNA (39, 61), effort has been directed towards the development of rVSV as a vaccine vector targeting a variety of different human pathogens, including human immu...

Re-engineering Vesicular Stomatitis Virus to Abrogate Neurotoxicity, Circumvent Humoral Immunity, and Enhance Oncolytic Potency

“…To directly evaluate neurotoxicity, escalating doses of rVSV (GP), rVSV, or the attenuated matrix protein-mutated rVSVDM51 virus were injected into the brains of CD-1 and Balb/c mice. Consistent with previous reports (21,22), all cohorts treated with VSV-G-expressing strains (doses as low as 10 1 PFU) succumbed to neurologic symptoms including significant weight loss by 9 days postinfection (dpi) (Fig. 1A-C).…”

“…We removed the VSV glycoprotein G, the key neurovirulence determinant (20)(21)(22), and replaced it with the arenavirus glycoprotein LCMV-GP (23)(24)(25)(26), thereby generating rVSV(GP). While there are no doses at which wtVSV can be safely introduced into rodent brains, we found that rVSV(GP) caused no significant neurotoxicity even at doses of 10 8 plaque-forming units (PFU).…”

Peripheral Immunization Blocks Lethal Actions of Vesicular Stomatitis Virus within the Brain

“…A number of VSV mutants have been described (7,11,15,32,33). Attenuation of the VSV phenotype here was done by using either a virus with the cytoplasmic portion of the G protein truncated from 29 amino acids to a single amino acid (VSV-CT1) or by combining an M51 deletion with a partially trun- (2,23,33).…”

Single-Dose Trivalent VesiculoVax Vaccine Protects Macaques from Lethal Ebolavirus and Marburgvirus Challenge

“…1C). For control vaccine, an attenuated rVSV-N4CT1 HIVgag1 design (20) expressing HIV-1 gag in the first position of the genome was used in animal studies (Fig. 1D).…”

“…Attenuation was achieved by combining VSV N gene translocation (N4) with truncation of the VSV G protein cytoplasmic tail domain (CT1) (20). These modifications reduced the intracellular abundance of the major structural N protein (21) and diminished the interaction between the VSV G protein CT domain and the viral nucleocapsid core during particle maturation (22).…”