Symposium on replication of viral nucleic acids. 3. Replication of mengovirus ribonucleic acid.

The large-particle fraction from the cytoplasm of chick embryo fibroblasts infected with Semliki Forest virus was found to catalyze the incorporation of the 5'-triphosphates of guanosine, adenine, cytidine, and uridine into an acid-insoluble alkali-labile product. The conditions affecting the preparation and assay of this enzyme were investigated. The ribonucleic acid (RNA) polymerase was not present in uninfected cells, and it appeared in infected cells at the time of rapid viral RNA synthesis. The polymerase was found to catalyze the synthesis of a species of RNA MATERIALS AND MErHODS Growth and assay of virus. The origin of the Kumba strain of SFV and the chick embryo fibroblasts used in this work and the growth of the virus in monolayer cultures has already been described (E. Mecs et al., J. Gen. Virol., in press). For the routine preparation of polymerase, monolayer cultures in 20-oz flat bottles, containing about 2 X 108 cells in 32 ml of Gey's medium plus 0.25% lactalbumin hydrolysate, 2.5% calf serum, 0.005 M tris(hydroxymethyl)aminomethane(Tris) chloride (pH 7.6), and 2 gsg/ml of actinomycin D, were infected with SFV at a multiplicity of 50 to 100 plaque-forming units (PFU) per cell. Cultures were kept overnight at 4 C and then warmed to 37 C; this was taken as zero-time. Overnight preincubation at 4 C was used because it was convenient; previous work in this laboratory had shown that this treatment did not affect virus growth 97

Section: 70supporting

confidence: 55%

Ribonucleic Acid Polymerase Catalyzing Synthesis of Double-stranded Arbovirus Ribonucleic Acid

Martin

Sonnabend

1967

“…No decrease in activity was observed when phosphoenolpyruvate and pyruvate kinase were omitted from the reaction mixture, suggesting that an ATP-generating system is not required. This may be evidence that ATPases which are normally present in crudely prepared mitochondrial fractions were removed by sedimentation through 20% sucrose (25). The presence of 20 ,ug of actinomycin D per ml in the reaction failed to inhibit [3H]UMP incorporation, and therefore the possibility that the product was made from a DNA template is precluded.…”

Section: Absence Of An Rna-dependent Rna Polymer-mentioning

confidence: 99%

RNA-dependent RNA polymerase activity in coronavirus- infected cells

Dennis¹,

Brian²

1982

An enzymatic activity which incorporates [3H]UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with the mitochondrial fraction of infected cells, suggesting that the enzyme is membrane bound. The activity required the presence of all three ribonucleoside triphosphates in addition to [3H]UTP, and it was not inhibited by actinomycin D. The heated product was digested by RNase but not by DNase. Mg2+ was required for enzymatic activity, and its optimal concentration was approximately 5 mM. The size of the in vitro products was compared by electrophoresis with that of in vivo-synthesized virus-specified RNA to confirm the viral specificity of the polymerase activity. Virus-specified RNA from infected cells consisted of 10 species of single-stranded, polyadenylated RNA with molecular weights of 6.8 x 106, 6.2 x 106, 3.15 x 106, 1.40 x lop, 1.05 x 106, 0.94 x 106, 0.66 x 106, 0.39 x 106, 0.34 x 106, and 0.24 x 106. In vitrosynthesized RNA consisted of a high-molecular-weight species, of apparently higher molecular weight than genomic RNA, and two single-stranded species that electrophoretically comigrated with the species of 1.40 x 106 and 0.66 x 106 molecular weight made in vivo.

“…However, in the latter cases, cellular RNA polymerase activity continued to be depressed during the period of virus growth, whereas with FPV there was a rise in DNA-dependent RNA polymerase activity between 1 and 3 hr after infection. That this rise was not due to a new RNA-dependent polymerase is suggested by the dependence of the observed activity on DNA template, ammonium, sulfate, and Mn+ ions, and its inhibition by actinomycin D, in contrast to the new virusinduced RNA polymerases described so far (18,20). Thus, we have no information at present to suggest that the increase in enzyme activity which we observed represents anything more than the enhancement of normal cellular enzyme activity, and it could be that this is the result of some nonspecific derangement of cellular metabolism in the virus-infected cells.…”

Section: Resultsmentioning

confidence: 80%

“…There is a considerable body of information concerning the synthesis of virus-specific ribonucleic acid (RNA) in cells infected with small RNA viruses and RNA phages (18). Of the animal viruses, poliovirus and mengovirus have been particularly well studied, and it has been established that these viruses multiply within their host cells by inducing the formation of a virus-specific RNA synthesizing system in the cytoplasm, which is independent of the normal cell RNA synthesizing mechanism (20). In addition, it has been shown that the host cell nuclear deoxyribonucleic acid (DNA)-dependent RNA polymerase is inhibited shortly after infection with these viruses (2,14), and replaced by a new cytoplasmic RNA-dependent RNA polymerase (3).…”

mentioning

confidence: 99%

Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity in Cells Infected with Influenza Virus

Borland

Mahy

1968

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase activity was assayed on nuclear preparations of chick embryo fibroblast cells at various times after infection with an influenza A virus (fowl plague virus) and was compared with the activity of uninfected cells. Polymerase activity was increased by about 60% by 2 hr after infection, and this increase coincided with an increase in RNA synthesis in infected cells, as determined by pulse-labeling with uridine. No differences could be detected between the polymerases of infected and uninfected cells as to their requirements for DNA primer, divalent cations, and nucleoside triphosphates, and they were equally sensitive to addition of actinomycin D to the reaction mixture. It is possible that host cell DNA-dependent RNA polymerase is involved in the replication of influenza virus RNA.