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

Pulse-labeled Saint Louis encephalitis viral ribonucleic acid (RNA) is found in the cytoplasm of infected cells associated with a membranous structure which sediments with an average value of 250S. The integrity of the complex is destroyed by detergents and ribonuclease; however, it is stable in ethylenediaminetetraacetic acid (EDTA) which differentiates this structure from cellular polyribosomes. With cultures in which cellular RNA was highly labeled prior to infection, ribosomal RNA could not be demonstrated in the complex isolated from EDTA-sucrose gradients. Single-stranded 43S and the 26S and 20S forms of viral RNA were found in the complex. Viral RNA polymerase activity in sucrose-gradient fractions sedimented in the same region as the fractions which contained the pulse-labeled viral RNA. The polymerase incorporated 8H-guanosine triphosphate into acid-precipitable material in the absence of added template. It was also found that the replication complex contains viral-specific proteins. was added, and cultures were incubated at 37 C. Cell fractionation. Cultures were pulse-labeled for 15 min with 3H-uridine (50,uCi/ml) in single-label experiments. In dual-label experiments, 50,ACi of 3H-uridine/ml and 10,ACi of '4C-leucine/ml were used. After the radioactive pulse, cells were scraped off the glass and collected by centrifugation, and cytoplasmic extracts were prepared (7). In some experiments, cytoplasmic extracts were solubilized with Brij-58 or sodium deoxycholate (DOC; 0.5% for 10 min at 0 C) prior to sucrose gradient analysis. Extracts were layered over a 28-mi, 5 to 40% (w/v) sucrose gradient prepared in either reticulocyte standard buffer (RSB; reference 22) or ethylenediaminetetraacetic acid (EDTA) buffer (9) and centrifuged at 25,000 rev/min for 120 min at 4 C in a Beckman SW-27 rotor. Frac-565

Section: Resultssupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Saint Louis Encephalitis Viral Ribonucleic Acid Replication Complex

Qureshi

Trent

1972

“…Interferon treatment inhibits the formation of all species of ptogeny virus RNA (8). The present study, conducted early in the eclipse phase of the virus growth cycle in cells infected with radioactive virus, extends these observations by demonstrat-ing that interferon inhibits the formation of the specific product of the SFV RNA polymerase (12), a ribonuclease-resistant RNA form into which parental virus RNA enters after infection. Inhibitors of protein synthesis had a similar effect.…”

supporting

confidence: 72%

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

Friedman

Fantes

Levy

et al. 1967

Actinomycin D-treated chick fibroblasts were infected with purified 32P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 ,g/ml) or cycloheximide (200 ,g/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of 32P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.on July 6, 2020 by guest

“…In the case of Sindbis virus, reactions carried out with a partially purified mitochondrial fraction were reported to yield both 42S and 26S single-stranded RNA as well as RNA resistant to ribonuclease (29). However, with a crude mitochondrial fraction from SFV-infected cells only ribonucleaseresistant RNA appeared to be synthesized (16).…”

Section: Gradient Fraction 23mentioning

confidence: 99%

Analysis of Arbovirus Ribonucleic Acid Forms by Polyacrylamide Gel Electrophoresis

Levin

Friedman

1971

MATERIALS AND METHODS Materials. Actinomycin D was a gift from Merck, Sharp, and Dohme Research Laboratories (Div. of Merck & Co., Inc., Rahway, N. J.). Adenosine-2, 8-3H (7.0 Ci/mmole) and uridine-5-3H (19.9 Ci/mmole) were purchased from New England Nuclear Corp. (Boston, Mass.); 32P-phosphoric acid (carrier free) was obtained from Tracerlab (Waltham, Mass.); and 3H-guanosine triphosphate (1.0 Ci/ mmole) was purchased from Schwarz BioResearch Inc. (Orangeburg, N. Y.). Adenosine triphosphate, uridine triphosphate, and cytidine triphosphate were purchased from General Biochemicals, Inc. (Chagrin Falls, Ohio). Pancreatic ribonuclease, Pronase, phosphoenolpyruvate, pyruvate kinase, and dithiothreitol were obtained from Calbiochem (Los Angeles, Calif.). N, N, N', N' tetramethylethylenediamine, acrylamide, and N, N'-methylene bisacrylamide were purchased from Eastman Organic Chemicals (Rochester, N. Y.). The acrylamide and bisacrylamide were recrystallized according to Loening (14). Agarose (Seakem) was obtained from Bausch and Lomb Inc. (Rochester, N. Y.). Ribonuclease-free sucrose was purchased from Mann Research Laboratories (Orangeburg, N. Y.). Triton X-100 was obtained from Packard Instrument Co., Inc. (Downers Grove, Ill.) and toluene-Liquifluor was obtained from New England Nuclear Corp. Cells and virus pools. The procedures for prepara-504