Translation of in vitro synthesized reovirus messenger RNAs into proteins of the size of reovirus capsid proteins in a mouse L cell extract

Graziadei, W.D.; Lengyel, Péter

doi:10.1016/0006-291x(72)90056-3

Cited by 58 publications

(14 citation statements)

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“…dsRNA also causes a slight inhibition of the translation of reovirus mRNA in an extract from L cells [27] or EAT cells (unpublished data). Furthermore protein synthesis in extracts from interferontreated L cells or EAT cells is more susceptible to inhibition by dsRNA than that in extracts from control cells [26] (and unpublished data).…”

Section: Does Dsrna Enhance M R N a Degradation In Extracts From Intementioning

confidence: 82%

Interferon, Double‐Stranded RNA and RNA Degradation

Ratner

Sen

Brown

et al. 1977

European Journal of Biochemistry

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Extracts from interferon-treated Ehrlich ascites tumor cells differ in various biochemical characteristics from extracts from control cells. Thus, as reported earlier, double-stranded RNA (dsRNA) promotes the phosphorylation by ATP of at least two proteins in extracts from interferon-treated cells,but not,or to only a lesser extent, in extracts from control cells. Moreover reovirus mRNAs are degraded faster in reaction mixtures containing extracts from interferon-treated cells than in those containing extracts from control cells but only if the reaction mixtures are supplemented with dsRNA and ATP. The faster RNA degradation in extracts from interferon-treated cells is due to enhanced endonuclease action. We designated the agent(s) catalyzing this as endonuclease1NT. There is some enhancement of nuclease activity by dsRNA and ATP also in extracts from control cells. The extent of this enhancement is, however, much smaller than in extracts from interferon-treated cells. We report now that the promotion of mRNA cleavage in extracts from interferon-treated cells by dsRNA and ATP is apparently not a consequence of a possible impairment in the attachment of the mRNA to ribosomes since (a) the protein synthesis inhibitors (sparsomycin and edeine) do not affect the degradation and (b) dsRNA and ATP enhance RNA degradation also in the 200000 x g supernatant fraction from extracts of interferon-treated cells and this fraction is essentially free of ribosomes.(The ribosome concentration in our 200000 x g supernatant fraction is less than 1 % of that in the 30000 x g extract.) GTP or the ATP analogs AMP(CH2)PP or AMP-P(CH2)P do not substitute for ATP and dsDNA or DNA . RNA hybrids do not substitute for dsRNA in activating endonucleaseINT.The optimal concentration of ATP in activating endonucleaseINT is 1 mM or higher. The addition of dsRNA and ATP to extracts from interferon-treated cells affects the degradation of some RNAs much more than that of others. Thus it promotes the degradation of reovirus mRNA from the large size class strongly, the degradation of those from the medium size class to an intermediate extent and that of those from the small size class the least. Furthermore it enhances the degradation of mRNA from either control or interferon-treated Ehrlich ascites cells more than that of Ehrlich ascites cell ribosomal RNA or mouse globin mRNA. Reovirus dsRNA is not cleaved by endonucleaselNT. The partially purified mouse interferon preparation (specific activity 8 x lo8 NIH reference standard units/mg protein) used in several experiments was free of nuclease activity under our experimental conditions. Moreover the addition to extracts from control cells (or extracts from interferon-treated cells) of this interferon preparation together with dsRNA and ATP did not enhance nuclease activity.

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Section: Does Dsrna Enhance M R N a Degradation In Extracts From Intementioning

confidence: 82%

Interferon, Double‐Stranded RNA and RNA Degradation

Ratner

Sen

Brown

et al. 1977

European Journal of Biochemistry

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“…When added to extracts of uninfected mouse L cells, reovirus RNA stimulates the synthesis of low molecular weight polypeptides (27); very recently, this RNA has been shown to stimulate the synthesis of several complete viral polypeptides (28).…”

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confidence: 99%

Translation of Reovirus Messenger RNAs Synthesized In Vitro into Reovirus Polypeptides by Several Mammalian Cell-Free Extracts

McDowell

Joklik

Villa‐Komaroff

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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Single-stranded reovirus RNA, synthesized in vitro by reovirus cores, functioned as messenger RNA in cell-free extracts prepared from several mammalian cells: Krebs II mouse ascites cells, mouse L cells, Chinese hamster ovary cells, HeLa cells, and rabbit reticulocytes. As shown by acrylamide gel electrophoresis, all eight polypeptides known to be specified by reovirus were synthesized in the reticulocyte system. In the other extracts, from 5 to 7 complete virus proteins were made. Reticulocytes were obtained from New Zealand white rabbits as described (2), except that the rabbits were made anaemic by subcutaneous injection of 1.2% acetylphenylhydrazine according to the following schedule: 2 ml on day 1, 1.6 ml on day 2, 1.2 ml on day 3, 1.6 ml on day 4, and 2 ml on day 5.Preparation of Cell-Free Extracts. Liquid tumors of ascites cells were harvested from five mice and filtered through cheesecloth into cold isotonic buffer (35 mM Tris HCl, pH 7.5-146 mM NaCl-11 mM glucose). The cells were washed six times by differential centrifugation (80 X g for 5 min) in isotonic buffer to remove reticulocytes. Mouse L fibroblasts, HeLa cells, and CHO cells were each collected by centrifugation from 2 liters of suspension culture (1 to 2 X 109 cells), then washed three times with isotonic buffer. After washing, the procedure was identical for all four types of cells. One volume of packed cells was resuspended in 3 volumes of hypotonic buffer (10 mM Tris-HCl, pH 7.5-15 mM KCl-1.5 mM MgAc2-6 mM 2-mercaptoethanol). After 10 min at 00, the cells were disrupted in a Dounce homogenizer. 0.1 Volume of 10 X HEPES buffer (200 mM HEPES, pH 7.5-1200 mM KCl-50 mM MgAc2-60 mM 2-mercaptoethanol) was then added, the homogenate was centrifuged at 30,000 X g for 20 min, and the pellet was discarded. ATP was added to a final concentration of 1 mM, GTP to 0.2 mM, creatine phosphate to 8 mM, and creatine kinase to 0.2 mg/ml, and the extract was incubated at 370 for 30 min. The extract was then passed at 40 through a Sephadex G-25 column (3 X 30 cm) that had been equilibrated with 1 X HEPES buffer. The opalescent fractions were pooled and stored at -70°in small aliquots.Preparation of reticulocyte extract has been described (2).

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“…Evidence in support of a monocistronic function has been obtained for reovirus mRNA. RNA synthesized in vitro was separated according to size and translated in mammalian cell-free, protein synthesizing systems (153,154). Products similar in size to reovirus proteins were formed, and the size was a function of the molecular weight of the mRNA directing the system.…”

Section: Class 3: Influenza and Diplornaviruses (Segmented Genomes)mentioning

confidence: 99%

Animal RNA Viruses: Genome Structure and Function

Shatkin¹

1974

Annu. Rev. Biochem.

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Translation of in vitro synthesized reovirus messenger RNAs into proteins of the size of reovirus capsid proteins in a mouse L cell extract

Cited by 58 publications

References 14 publications

Interferon, Double‐Stranded RNA and RNA Degradation

Interferon, Double‐Stranded RNA and RNA Degradation

Translation of Reovirus Messenger RNAs Synthesized In Vitro into Reovirus Polypeptides by Several Mammalian Cell-Free Extracts

Animal RNA Viruses: Genome Structure and Function

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