Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.     

Extreme heterogeneity in populations of vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) sequence evolution and population heterogeneity were examined by T1 oligonucleotide mapping. Individual clones isolated from clonal pools of wild-type Indiana serotype VSV displayed identical T1 maps. This was observed even after one passage at high concentrations of the potent viral mutagen 5-fluorouracil. Under low-multiplicity passage conditions, the consensus T1 fingerprint of this virus remained unchanged after 523 passages. Interestingly, however, individual clones from this population (passage 523) differed significantly from each other and from consensus sequence. When virus population equilibria were disrupted by high-multiplicity passage (in which defective interfering particle interference is maximized) or passage in the presence of mutagenic levels of 5-fluorouracil, rapid consensus sequence evolution occurred and extreme population heterogeneity was observed (with some members of these population differing from others at hundreds of genome positions). A limited sampling of clones at one stage during high-multiplicity passages suggested the presence of at least several distinct master sequences, the related subpopulations of which exhibit at least transient competitive fitness within the total virus population (M. Eigen and C.K. Biebricher, p. 211-245, in E. Domingo, J.J. Holland, P. Ahlquist, ed., RNA Genetics, vol. 3, 1988). These studies further demonstrate the important role of selective pressure in determining the genetic composition of RNA virus populations. This is true under equilibrium conditions in which little consensus sequence evolution is observed owing to stabilizing selection as well as under conditions in which selective pressure is driving rapid RNA virus genome evolution.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Molecular basis of fitness loss and fitness recovery in vesicular stomatitis virus

Viral populations subjected to repeated genetic bottleneck accumulate deleterious mutations in a process known as Muller's ratchet. Asexual viruses, such as vesicular stomatitis virus (VSV) can recover from Muller's ratchet by replication with large effective population sizes. However, mutants with a history of bottleneck transmissions often show decreased adaptability when compared to non-bottlenecked populations. We have generated a collection of bottlenecked mutants and allowed them to recover by large population passages. We have characterized fitness changes and the complete genomes of these strains. Mutations accumulated during the operation of Muller's ratchet led to the identification of two potential mutational hot spots in the VSV genome. As in other viral systems, transitions were more common than transversions. Both back mutation and compensatory mutations contributed to recovery, although a significant level of fitness increase was observed in nine of the 13 bottlenecked strains with no obvious changes in the consensus sequence. Additional replication of three strains resulted in the fixation of single point mutations. Only two mutations previously found in non-bottlenecked, high-fitness populations that had been adapting to the same environment were identified in the recovered strains.     

Inhibition of HIV-1 replication by vesicular stomatitis virus envelope glycoprotein pseudotyped baculovirus vector-transduced ribozyme in mammalian cells

The baculovirus has recently emerged as a promising vector for in vivo gene therapy. To investigate its potential as a delivery vector for an anti-virus ribozyme targeting HIV-1, we constructed recombinant baculovirus vectors bearing a ribozyme-synthesizing cassette driven by the tRNA(i)(Met) promoter with enhanced transduction efficiency by displaying vesicular stomatitis virus glycoprotein (VSV-G) on the viral envelope. Transduction of HeLa CD4(+) cells with a recombinant baculovirus delivering the HIV-1 U5 gene-specific ribozyme dramatically suppressed HIV-1 expression in this cell line. The VSV-G pseudotyped baculovirus vector-transduced ribozyme potently inhibited HIV-1 replication compared to a recombinant baculovirus vector-transduced ribozyme lacking VSV-G. The use of a baculovirus vector might be beneficial for application in gene therapy.     

pppA2''p5A'' blocks vesicular stomatitis virus replication in intact cells.

pppA2'p5'A blocked the production of infectious vesicular stomatitis virus in HeLa cells. When this compound was present from the beginning of infection, a selective inhibitory effect was observed in viral protein synthesis. Thus, cellular translation was not affected even after 10 h of incubation with this compound, and the bulk of viral proteins was not synthesized. However, this effect was not observed with ATP, GTP, or the core A2'p5'A. The step blocked by pppA2'p5'A is located early during virus infection, but adsorption, entry, and virus uncoating seemed to be unaffected by this compound. Analysis of the antiviral spectrum of pppA2'p5'A indicated that it is active against poliovirus, encephalomyocarditis virus, and Semliki Forest virus and shows no effect against herpes simplex virus type 1 and adenovirus type 5.     

Inhibition of adenovirus DNA replication by vesicular stomatitis virus leader RNA.

Vesicular stomatitis virus (VSV) leader RNA and a synthetic oligodeoxynucleotide of the same sequence were found to inhibit the replication of adenovirus DNA in vitro. In contrast, the small RNA transcribed by the VSV defective interfering particle DI-011 did not prevent adenovirus DNA replication. The inhibition produced by leader RNA was at the level of preterminal protein (pTP)-dCMP complex formation, the initiation step of adenovirus DNA replication. Initiation requires the adenovirus pTP-adenovirus DNA polymerase complex (pTP-Adpol), the adenovirus DNA-binding protein, and nuclear factor I. Specific replication in the presence of leader RNA was restored when the concentration of adenovirus-infected or uninfected nuclear extract was increased or by the addition of purified pTP-Adpol or HeLa cell DNA polymerase alpha-primase to inhibited replication reactions. Furthermore, the activities of both purified DNA polymerases could be inhibited by the leader sequence. These results suggest that VSV leader RNA is the viral agent responsible for inhibition of adenovirus and possibly cellular DNA replication during VSV infection.     

Inhibition of vesicular stomatitis virus replication by prostaglandin A1 in Aedes albopictus cells

Cyclopentenone prostaglandins (PGs) exhibit antiviral activity against RNA and DNA viruses in mammalian cell lines, and this effect has been associated with the induction of a heat shock protein (hsp70). We investigated the effect of prostaglandin A1 (PGA1) on the replication of vesicular stomatitis virus (VSV) in Aedes albopictus (mosquito) cells. PGA1 was found to inhibit VSV replication dose dependently. Virus yield was reduced to 50% (3 microg PGA1/ml) and to 95% with 8 microg PGA1/ml. Even with the dramatic reduction of virus production observed in cells treated with PGA1, VSV-specific protein synthesis was unaltered. Treatment of cells with PGA1 (5 microg/ml) stimulated the synthesis of a polypeptide identified as a heat-shock protein (hsp) by immunoblot analysis. PGA1 induced hsp70 synthesis in uninfected cells. However, in VSV-infected cells the induction of hsp70 by PGA1 was reduced. This is the first report of antiviral effects of PGs affecting the replication of VSV in a mosquito cell line.     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.     

Inhibitory effect of acylphloroglucinol derivatives on the replication of vesicular stomatitis virus.

The antiviral activity of natural phloroglucinols and of synthesized mono- and diacylphloroglucinols, and 2,6-diacyl-4,4-dialkylcyclohexa-1,3,5-triones was investigated. A correlation between the acyl chain length and inhibitory activity against vesicular stomatitis virus (VSV) was observed. Potent antiviral activity was found in di-isovalerylphoroglucinol. 2,6-Diacyl-4,4-dialkylcyclohexa-1,3,5-triones inhibited replication of the virus with low cytotoxicity.     

Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells.

Glycosylation of the envelope glycoprotein of vesicular stomatitis virus was examined using virus-infected HeLa cells that were pulse-labeled with radioactive sugar precursors. The intracellular sites of glycosylation and the stepwise elongation of the carbohydrate side chains of the G protein were monitored by membrane fractionation and gel filtration of Pronase-digested glycopeptides. The results with short pulses of sugar label (5 to 10 mtein linkage (glucosamine and mannose) are added to G which was associated with the rough endoplasmic reticulum-enriched membrane fraction, whereas the more distal sugars (galactose, sialic acid, fucose, and possibly more glucosamine) are added in the light-density internal membrane fraction. Accumulation of mature G was observed in the plasma membrane-enriched fraction. The gel filtration studies indicated that the initial glycosylation event may be the en bloc addition of a mannose and glucosamine oligomer, followed by the stepwise addition of the more distal sugars.     

Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.     

Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II Activity of defective interfering particles.

Purified defective interfering (DI) particles of vesicular stomatitis virus (VSV) inhibit the replication of a heterologous virus, pseudorabies virus (PSR), in hamster (BHK-21) and rabbit (RC-60) cell lines. In contrast to infectious B particles of VSV, UV irradiation of DI particles does not reduce their ability to inhibit PSR replication. However, UV irradiation progressively reduces the ability of DI particles to cause homologous interference with B particle replication. Pretreatment with interferon does not affect the ability of DI particles to inhibit PSR replication in a rabbit cell line (RC-60) in which RNA, but not DNA, viruses are sensitive to the action of interferon. Under similar conditions of interferon pretreatment, the inhibition of PSR by B particles is blocked. These data suggest that de novo VSV RNA or protein synthesis is not required for the inhibition of PSR replication by DI particles. DI particles that inhibit PSR replication also inhibit host RNA and protein synthesis in BHK-21 and RC-60 cells. Based on the results described and data in the literature, it is proposed that the same component of VSV B and DI particles is responsible for most, if not all, of the inhibitory activities of VSV, except homologous interference.     

Pseudotypes of vesicular stomatitis virus with the envelope properties of mammalian and primate retroviruses.

By employing improved techniques it has been possible to produce and characterize a representative spectrum of mammalian and primate retrovirus pseudotypes of vesicular stomatitis virus (VSV). Selection of appropriate cell lines for both the production and subsequent detection of the VSV pseudotypes has been the most important factor in permitting their demonstration. The host range for penetration of these retrovirus pseudotypes of VSV has been defined and found to differ from that reported for the replication of the corresponding retroviruses. Additionally, retroviruses having an identical host range for replication were distinguishable by differences in their host range for penetration, implying that restriction of replication may be occurring by different mechanisms. Studies of the plaque-forming efficiency of retrovirus pseudotypes of VSV in cell lines nonpermissive for replication of the corresponding retroviruses permitted a distinction to be made between the restriction of replication occurring as a consequence of postpenetration events and that occurring as a consequence of a block of penetration itself. The demonstration of primate retrovirus pseudotypes of VSV permits the use of VSV as a probe for the detection of this group of viruses.