Production Of Rous Sarcoma Virus Research Articles

A single amino-acid substitution in gag p19 protein (MA) of Rous sarcoma virus suppresses virus production from infected cells.

Gag p19 protein (MA) of the transformation-defective Rous sarcoma virus mutant, tdPH2010, has a point mutation at nucleotide 376 (G to A) that results in an amino-acid change at residue 126 of p19 (Glu to Lys). This single amino-acid change is the cause of the aberrantly fast migration of this protein on SDS-polyacrylamide gels. To study the biological significance of the mutation, we introduced this mutation into a transformation-defective derivative of the molecularly cloned Rous sarcoma virus, SRA2, and examined its effect on virus replication. The virus possessing the mutation in its gag p19 gene had 50% slower replication as measured by the amount of reverse transcriptase as well as gag p27 protein (CA) in the culture media. Glu at position 126 appears to be important for efficient production of Rous sarcoma virus in vitro.

In vitro inhibition of HIV-1 proteinase by cerulenin

Retroviruses encode proteinases necessary for the proteolytic processing of the viral gag and gag-pol precursor proteins. These enzymes have been shown to be structurally and functionally related to aspartyl proteinases such as pepsin and renin. Cerulenin is a naturally occurring antibiotic, commonly used as an inhibitor of fatty acid synthesis. Cerulenin has been observed to inhibit production of Rous sarcoma virus and murine leukaemia virus by infected cells, possibly by interfering with proteolytic processing of viral precursor proteins. We show here that cerulenin inhibits the action of the HIV-1 proteinase in vitro, using 3 substrates: a synthetic heptapeptide (SQNYPIV) which corresponds to the sequence at the HIV-1 gag p17 p24 junction, a bacterially expressed gag precursor, and purified 66 kDa reverse transcriptase. Inhibition of cleavage by HIV-1 proteinase required preincubation with cerulenin. Cerulenin also inactivates endothiapepsin, a well-characterised fungal aspartyl proteinase, suggesting that the action of cerulenin is a function of the common active site structure of the retroviral and aspartic proteinases. Molecular modelling suggests that cerulenin possesses several of the necessary structural features of an inhibitor of aspartyl proteinases and retroviral proteinases. Although cerulenin itself is cytotoxic and inappropriate for clinical use, it may provide leads for the rational design of inhibitors of the HIV proteinase which could have application in the chemotherapy of AIDS.

Inhibition of rous sarcoma virus production by formycin

The effect of formycin, an adenosine analog, on the growth of chick embryo fibroblasts and on Rous sarcoma virus (RSV) production was studied. An adverse effect on cell proliferation was observed in the presence of 10 μM formycin. Treatment with 5 μM formycin for 24 hr reduced by a factor of about 1000 the yield of infections progeny whereas the cell growth remained unaltered. Moreover the few particles released in the presence of formycin showed a markedly decreased ability to synthesize viral cDNA. This impairment was shown to be related to a nonfunctional primer tRNA.

Dimethyl-10,12-benz(a)acridine: Evidence for differential effects on the synthesis of RNA of mammalian or avian fibroblasts and some RNA viruses

We have studied the differential effect of dimethyl-10,12-benz(a)acridine (DMBAcr) on the synthesis of RNA of chicken or mouse fibroblasts in culture and that of some RNA-containing viruses such as Rous sarcoma virus and Mengovirus. DMBAcr at low concentrations blocks the cell multiplication of both normal and Rous sarcoma virus-transformed chicken fibroblasts in culture; it affects transformed cells more than normal ones. The cell growth inhibiting effect of DMBAcr is reversible after short periods of incubation. DMBAcr depresses the synthesis of cellular DNA and RNA in parallel. Concurrently the synthesis of protein proceedes at a relatively high rate in DMBAcr-treated cultures. Its inhibitory effect on cellular RNA synthesis is mostly due to a block in the formation of 28 S and 18 S ribosomal RNA species; in contrast, the synthesis of 45 S ribosomal RNA precursor is proceeding at almost control rate. Also, the synthesis of heterogeneous nuclear RNA is not blocked by DMBAcr. The production of Rous sarcoma virus in transformed fibroblasts is not affected by DMBAcr. Since this is correlated with persisting high rates of protein and heterogenous nuclear RNA synthesis, the effects of DMBAcr suggest that the synthesis of Rous sarcoma virus-RNA shares the specificity of messenger and heterogeneous nuclear RNA. DMBAcr inhibits the synthesis of viral RNA of Mengovirus under conditions where the synthesis of total cellular RNA is not appreciably depressed, suggesting its differential effect on the DNA-directed and the RNA-directed RNA synthesis.

Effects of inhibitors of lipid synthesis on the replication of rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholesterol, which markedly inhibits the incorporation of [1- 14C]acetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1- 14C]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [ 35S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examination of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.

Inhibition of infectious Rous sarcoma virus production by rifamycin derivative.

A new rifamycin derivative, rifazone-82 (R-82), an inhibitor of viral RNA-dependent DNA polymerase, is selectively toxic to transformed chicken cells in culture. R-82 has now been shown to possess antiviral activity as well. The relatively nontoxic properties of R-82 to nontransformed cells have permitted the execution of experiments examining the effect of a rifamycin derivative on virus reproduction. Addition of low concentrations of R-82 (15 mug/ml) to cultures soon after Rous sarcoma virus infection prevents the spread of infection thoroughout the culture. This inhibition is not dependent on concomitant cellular transformation as identical results were obtained with cells infected with a transformation-defective Rous sarcoma virus. Addition of R-82 to cultures in which all the cells are infected does not substantially affect the yield of physical particles as measured by RNA-dependent DNA polymerase activity and by (3H) uridine incorporation into viral RNA. However, the infectivity of the progeny virus, as measured by focus-forming ability, is decrreased 95 to 99% by R-82 treatment.

Selective inhibition of Rous sarcoma virus production in transformed chick fibroblasts by two rifamycin derivatives

Selective inhibition of Rous sarcoma virus production in transformed chick fibroblasts by two rifamycin derivatives

Synchronization of Rous sarcoma virus production in chick embryo cells

The growth of chick embryo fibroblasts transformed by and producing the Schmidt-Ruppin strain of Rous sarcoma virus was synchronized by exposure to serum-free medium. It was found that virus-specific RNA and protein synthesis and virus release are synchronized in these cells and occur in the early S and G 1 phases of the cell cycle, respectively. These findings support the hypothesis that RSV synthesis is dependent upon specific host cell processes. The duration of the phases of the cell cycle in transformed cells could not be distinguished from the pattern in uninfected cells. Evidence is presented which indicates that the synchrony of transformed cells is achieved by the low pH of the serum-free medium rather than by the absence of serum.

Rous sarcoma virus production in mixed cultures of mammalian Rous sarcoma cells and chick embryo cells.

AbstractThe induction of Rous sarcoma virus (RSV) production was analyzed in mixed cultures of mouse or hamster Rous sarcoma cells and chick embryo cells treated with ultraviolet inactivated hemagglutinating virus of Japan (UV‐HVJ). The pre‐treatment of UV‐HVJ with a certain concentration of anti‐HVJ serum increased the yield of infectious RSV in mixed cultures enabling a quantitative analysis of this phenomenon to be made. RSV‐inducing ability of a mixture of HVJ and anti‐HVJ serum was affected by the concentration of anti‐HVJ serum and showed changes in parallel with the cell fusion activity of the mixture. The effect of UV‐HVJ on RSV induction was not achieved by incubation of the cell mixture at 0° C as it was after shaking at 37° C. These findings suggest that the production of infectious RSV in mixed cultures may be initiated in heterokaryons of mammalian Rous sarcoma cells and chick embryo cells. The number of infective centers in mixed cultures was, however, as small as 1% of that of heterokaryons. It is possible that only a small fraction of the heterokaryon population is productive of infectious RSV.

Rous sarcoma virus production from clones of nontransformed chick embryo fibroblasts

When chick embryo cells are infected with high concentrations of Rous sarcoma virus (RSV) and cloned, most of the clones which produce RSV contain transformed cells. The remaining virus-producing clones, however, are normal in appearance. When cells from these nontransformed virus-producing clones are dispersed and recloned they give rise to clones, of which 90% are normal in appearance and produce no virus and 10% contain transformed cells and produce virus. It is suggested that nontransformed virus-producing clones arise from infected cells which grow into mixed clones. This occurs when virus is segregated to only one of the two daughter cells produced during the first divisions of a newly infected cell.

The effects of actinomycin D on growth of Rous sarcoma virus in vitro

Low concentrations of actinomycin D (0.1 μg/ml) reversibly inhibit production of Rous sarcoma virus (RSV) by Rous sarcoma cells. Higher concentrations inhibit irreversibly. Genetically resistant variants of RSV are not found. Infection of cells and initiation of virus production are also inhibited.