Inhibition of sindbis virus replication in Aedes albopictus cells by virazole (Ribavirin) and its reversal by actinomycin: A correction

The antibiotic actinomycin interferes with the antiviral action of ribavirin in Sindbis virus-infected Aedes albopictus mosquito cells. In the absence of actinomycin, ribavirin inhibits viral nucleic acid synthesis whether assayed by infectivity, incorporation of [³H]uridine, or ethidium bromide staining.     

Viral interference by defective particles of vesicular stomatitis virus measured in individual cells

The exponential relationship usually observed between the yield of infectious vesicular stomatitis virus (VSV) as a function of multiplicity of interfering particles was interpreted by Bellett and Cooper to mean that in most cells infection by one interfering particle was sufficient to exclude totally the yield of infectious virus. We have tested directly this model for the action of defective-interfering particles (DIP) in blocking the replication of vesicular stomatitis virus by measuring the total yield of infectious virus from individual cells coinfected with several plaque-forming particles of vesicular stomatitis virus and various multiplicities of defective-interfering particles. Single cells were manipulated by the pêcher (“fishing”) technique, a procedure which eliminates sampling error and the need to correct single-cell-yield data for the fraction of samples without cells. We established conclusively that in most cells one or a few DIP sufficed to interfere totally with the yield of infectious virus. Thus, interference can be so efficient that a single cell capable of producing over 5000 infectious particles will not yield any infectious virus when simultaneously infected with a single defective-interfering particle. We also observed a decrease in the efficiency of interference in cells infected with higher multiplicities of DIP-an interference with interference. The pêcher procedure provides an extremely easy and rapid means for determining the effects of various conditions and reagents on the yield of virus from a single cell, and offers a simple technique for cloning defective-interfering particles.     

Superinfection exclusion by vesicular stomatitis virus

The infection of baby hamster kidney (BHK21) cells by the Indiana strain of vesicular stomatitis virus (VSV) causes a rapid loss of the ability of the cells to be superinfected by VSV virions or defective-interfering particles. This exclusion phenomenon is at the level of virus penetration and requires viral gene expression and a functional VSV transmembrane glycoprotein G. Infection with the New Jersey serotype of VSV also inhibits the uptake of the Indiana serotype. However, infection of BHK21 cells with either encephalomyocarditis, Newcastle disease, or influenza A viruses does not inhibit superinfection by VSV.     

Immunoprecipitating human antigens associated with vesicular stomatitis virus grown in HeLa cells

Vesicular stomatitis virus (VSV) preparations made in HeLa cells, VSV(HeLa), appeared to contain non-viral structural proteins. This was suggested by neutralization of the virus with homologous and heterologous antisera made against VSV prepared in different cells. Antisera against uninfected HeLa cells failed to neutralize VSV(HeLa) but did immunoprecipitate the virus in the presence of Staphylococcus aureus. These immunoprecipitated VSV(HeLa) retained their infectivity, despite the presence of antibody and bacteria. The anti-HeLa cell serum did not react with VSV grown in rodent cells nor did anti-Vero cells serum immunoprecipitate VSV(HeLa). When the anti-HeLa cell serum was absorbed with whole HeLa cells, it no longer specifically precipitated VSV(HeLa). Because over 98% of infectious VSV(HeLa) was neutralizable by anti-VSV serum and immunoprecipitable by anti-HeLa serum, these virions were called mosaics. Physical identification of HeLa cell determinants on the mosaics was accomplished by further purification and radioiodination followed by selective immunoprecipitations with antisera. Two to three major bands with molecular weights around 75,000 Da were identified as HeLa cell determinants associated with the mosaic VSV(HeLa).     

Studies on tumorigenicity of cells persistently infected with vesicular stomatitis virus in nude mice

Previous work has demonstrated that persistent infection of cultured cells (normally tumorigenic in the nude mouse) by any of a number of enveloped RNA viruses results in the loss of their ability to form tumors in nude mice. From the BHK 21/VSV persistently infected cell line which did not form tumors in these nude mice, we selected in vivo a subline which is tumorigenic even though it is still persistently infected. We report here a preliminary characterization of this subline. These virus-infected cells were consistently tumorigenic for more than six sequential passages in nude mice. They shed large amounts of virus and DI particles into the bloodstream and tissues of the mice; however, we found no evidence of the virus establishing a persistent infection in the mouse tissues. We demonstrated that virus isolated from this tumorigenic cell line was able to establish new persistently infected cell lines which were also tumorigenic, indicating that this is a characteristic of the virus. T₁ oligonucleotide mapping of the virion RNA recovered from this tumorigenic carrier subline demonstrates that the viral genome is undergoing continual mutation in vivo. This is similar to mutational changes occurring in vitro in the parental persistently infected cells. Very recently, the BHK 21 cell line persistently infected with VSV for over 5 years has spontaneously (with no in vivo selection) acquired the ability to form tumors in nude mice. In contrast to the other tumorigenic subline, this carrier line produces almost no mature virus or DI particles at present, and its tumorigenicity appears to be associated with very low virus expression in infected cells. Apparently, viruses can escape the natural killer (NK) cell response by a variety of means.     

Effect of sulfhydryl reagents on the infectivity of vesicular stomatitis virus

The infectivity of vesicular stomatitis virus (VSV) was moderately affected by iodoacetic acid and drastically affected by N-ethylmaleimide; the antiviral effect of these sulfhydryl reagents was enhanced somewhat by the reducing agent, 2-mercaptoethanol. Reducing and/or alkylating reagents did not affect VSV hemagglutination and the impermeable sulfhydryl reagent, dextran-maleimide, did not significantly influence VSV infectivity. These data indicate that glycoprotein spikes are not the major sites for the antiviral activity of sulfhydryl reagents. [¹⁴C]Iodoacetic acid was able to penetrate the virion membrane to bind covalently to the free sulfhydryl groups of all five virion proteins, particularly the reduced disulfides of the L protein. The RNA polymerase activity of intact VSV was inhibited by iodoacetic acid and to a greater extent by N-ethylmaleimide, which probably accounts for the loss of viral infectivity caused by the permeable sulfhydryl reagents.     

RNA synthesis in standard and autointerfered vesicular stomatitis virus infections.

The levels of intracellular viral RNA have been measured in BHK-21 cells undergoing standard and autointerfered infections with vesicular stomatitis virus (VSV). The total amount of intracellular nucleocapsid RNA is almost unaffected by autointerference. However, the composition of this class of RNA changes with increasing autointerference: Control cells undergoing standard infections contain only the 140 S, full genomic length nucleocapsid, while autointerfered cells contain, in addition, a smaller nucleocapsid which corresponds in size to that found in the defective interfering (DI) particle. There is a systematic decrease in viral mRNA with increasing autointerference. This decrease in mRNA closely parallels the reduced yield of total progeny particles eventually released from these cells. A causal relationship between the two is proposed. These results are compatible with explanations of autointerference that depend upon a competition between the DI and VSV genomes for the viral replicase.     

Temporal regulation of the rate of vesicular stomatitis virus mRNA translation during infection of Chinese hamster ovary cells

Vesicular stomatitis virus (VSV) mRNAs on polysomes 3 hr after infection are not translated in vivo as efficiently as mRNAs 2 hr after infection. There are 14?, 12?, 11?, and 8-fold increases in the amounts of L, G, N, and M messengers, respectively, on polysomes between 2 and 3 hr after infection. However, there are only 3?, 5?, 3?, and 4-fold increases in the rates of synthesis of the respective proteins. The in vivo translational efficiences of these messengers, which is a measure of their capacity to act as template for protein synthesis, are therefore lower at 3 hr after infection than at 2 hr. Since the mRNAs isolated 3 hr after infection are as active in a wheat germ cell-free protein synthesizing system as the mRNAs isolated at 2 hr, the decreased efficiency of VSV translation in vivo at 3 hr is not due to changes in mRNA primary structure. The observed difference is more likely due to a reduced efficiency of the host cell translational system to translate VSV mRNA at 3 hr relative to 2 hr. It was found that at 2 hr after infection, the majority of VSV mRNA is not associated with polysomes, but at 3 hr, the majority of viral messages is polysome bound.     

Distribution of phosphoserine, phosphothreonine and phosphotyrosine in proteins of vesicular stomatitis virus

The two native phosphoproteins of vesicular stomatitis virus, NS and M, have a specific distribution of phosphoamino acids. The major one in each protein is phosphoserine. The NS NS tein is enriched for phosphothreonine, whereas the M protein contains most of the phosphotyrosine. Of the four VSV proteins phosphorylated in vitro, all have phosphotyrosine as well as phosphoserine. The NS protein appears to be the only one with phosphorylated threonine residues. When cellular and VSV proteins are compared, the relative abundance of phosphotyrosine is enriched in VSV proteins. This is further enhanced after in vitro phosphorylation. The presence of phosphotyrosine and the differential spectra of phosphoamino acids in individual VSV proteins may be crucial determinants of their intracellular location and function.