Translation of reovirus messenger ribonucleic acids synthesized in vitro into reovirus proteins in a mouse L cell extract

Graziadei, W.D.; Roy, Debdutta; Konigsberg, William H.; Lengyel, Péter

doi:10.1016/0003-9861(73)90621-8

Cited by 17 publications

(3 citation statements)

References 22 publications

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“…2) may indicate that a ring position of the 5'-terminal guanosine can also be substituted .Messenger Activity of Methylated RNAA. Unmethylated, single-stranded reovirus RNA synthesized in vitro has been translated with fidelity in cell-free systems from several sources (7,8), including wheat germ extracts (Both, Lavi, and Shatkin, unpublished results), which are notable for their low endogenous activity (15 (3)(4)(5)17). In addition to the genome RNA, reovirus cores consist of 1 minor and 3 major polyleptides (3,18), and they may correspond to the RNA polymerase, methylase, and l)hosphohy(lrolase activities.…”

Section: Methodsmentioning

confidence: 99%

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Methylated Messenger RNA Synthesis In Vitro by Purified Reovirus

Shatkin¹

1974

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

101

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. Purified human reovirus contains RNA methylase activity in addition to an RNA polymerase. Virions incubated under appropriate conditions in the presence of S-adenosyl-L-methionine synthesize mRNA that is specifically methylated in the 5'-terminal guanosine. Alkaline digestion of the methylated RNA released a 5'-terminal dinucleotide, ppG'pCp, indicating that the guanosine contains 2'-O-methylribose. The possible roles of methylation in viral and cellular mRNA function is discussed.The genome of human reovirus consists of ten segments of double-stranded RNA (1), each containing the same two 5'-terminal sequences: GpApUp in the nminus (-) strand and G'pCp in the plus (+) strand (2). G' presumably is 2'-Omethylated guanosine, since the G'pCp sequence is resistant to hydrolysis by alkali or T1 RNase (2). A virion-associated RNA polymerase transcribes only the (-) strand of each reovirus duplex segment (3-6). The resulting ten (+) species of single-stranded RNA function as viral mRNA (7,8). In addition, they serve as precursors to the double-stranded genome segments (9), suggesting that reovirus mRNA may also be methylated at the 5'-termini. A similar situation exists for another double-stranded RNA virus, cytoplasmic polyhedrosis virus (CPV) of insects (10, 11). Furuichi recently found that the single-stranded viral RNA synthesized in vitro by the cytoplasmic polyhedrosis virus-associated transcriptase in the presence of S-adenosyl-L-methionine contains 1.2 methyl groups per RNA molecule of average chain length 2300 nucleotides (12). In the view of the above results and the report that mouse L cell mRNA contains 2.2 methyl groups per 1000 nucleotides (13), it was of interest to test the possibility that reovirus mRNA is methylated. MATERIALS AND METHODSReovirus type 3, Dearing strain, was grown in mouse L cells in suspension culture and purified as described (3).RNA was synthesized by the virion-associated transcriptase by incubating 70 u~g of purified reovirus with 30,ug of chymotrypsin for 30 min at 450 in a standard reaction mixture con- tinued at 450, and samples of 0.02 ml were removed to 5 ml of trichloroacetic acid-sodium pyrophosphate (10% each) at the' indicated times. After 30 min at 00, they were filtered through Millipore filters, washed, and counted in toluene-based scintillation fluid.Analysis of RNA products by velocity sedimentation in sucrose gradients (3) and chromatography oIn DEAE-cellulose in the presence of 7 AI urea has been described (14).[ (Fig. 1). In the absence of GTP, no RNA synthesis occurred, and there was no incorporation of [3H]methvl group. To test whether exogenous mRNA can be methylated in the absence of RNA synthesis, I added purifiecl single-stranded RNA made in vitro in the absence of S-adenosylmethionine to the incubation mixture lacking GTP. No incorl)oration of [3H]methyl group was found (Fig. 1). [methyl-3H ]Methionine did not replace S-adenosylmethionine as a methyl donor. As described for the reovirus RNA polymerase (3-5), methylase activity is also associated...

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Section: Methodsmentioning

confidence: 99%

“…A virion-associated RNA polymerase transcribes only the (-) strand of each reovirus duplex segment (3-6). The resulting ten (+) species of single-stranded RNA function as viral mRNA (7,8). In addition, they serve as precursors to the double-stranded genome segments (9), suggesting that reovirus mRNA may also be methylated at the 5'-termini.…”

mentioning

confidence: 99%

Methylated Messenger RNA Synthesis In Vitro by Purified Reovirus

Shatkin¹

1974

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

101

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“…The polycistronic RNA from encephalomyocarditis virus makes a large polypeptide in vitro that resembles the precursor polypeptide known to be cleaved in a cascade of post-translation proteolysis that leads to the formation of the specific virion proteins (1)(2)(3)(4)(5)(6)(7). In contrast, the monocistronic mRNAs isolated from reovirus-infected cells direct the synthesis of eight polypeptides in a reticulocyte cell-free system that are indistinguishable from the proteins formed invivo (8,9). Morerecently, mRNA isolated from cells infected with a paramyxovirus has been shown to generate virion capsid protein in vitro (10).…”

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confidence: 99%

Formation of Sindbis Virus Capsid Protein in Mammalian Cell-Free Extracts Programmed with Viral Messenger RNA

Cancedda¹,

Schlesinger

1974

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

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Extracts from Krebs II ascites cells and rabbit reticulocytes effectively synthesize viral proteins with Sindbis viral mRNA isolated from Sindbis-infected BHK cells. The major product is identical to Sindbis capsid protein on the basis of its electrophoretic mobility in sodium dodecyl sulfate-acrylamide gels and two-dimensional tryptic-peptide fingerprints. Various amounts of several additional discrete polypeptides are formed, depending on the components of the cell-free extracts. One of these polypeptides may be a prematurely terminated part of the viral-capsid protein, while another is larger in molecular weight than capsid protein but contains the capsid tryptic peptides. Several of the proteins formed in vitro also are detected in extracts of Sindbis-infected BHK cells labeled with [IIS]methionine.The three proteins found in Sindbis virions are postulated to originate by proteolytic cleavage from a larger molecular weight polypeptide precursor that is translated from a polycistronic mRNA presumed to contain a single site for initiation of protein synthesis. The two in vitro systems appear to translate this polycistronic viral mRNA to yield specific viral capsid although no evidence was found for post-translational proteolysis. Other mechanisms for production of the capsid protein in the cell-free extracts are considered, and some of these may function in the viral-infected cell where unusually large amounts of viral capsid proteins are frequently detected.In vitro synthesis of proteins directed by mRNAs derived from animal viruses can provide important information about the processing of viral proteins and the role of various RNA species observed in virions and viral-infected cells. There are now a number of examples in which viral-specific RNAs act as messenger RNAs in cell-free systems, and several of these are reported to make proteins closely related or identical to viral proteins isolated from viral-infected cells. The polycistronic RNA from encephalomyocarditis virus makes a large polypeptide in vitro that resembles the precursor polypeptide known to be cleaved in a cascade of post-translation proteolysis that leads to the formation of the specific virion proteins (1-7). In contrast, the monocistronic mRNAs isolated from reovirus-infected cells direct the synthesis of eight polypeptides in a reticulocyte cell-free system that are indistinguishable from the proteins formed invivo (8,9). Morerecently, mRNA isolated from cells infected with a paramyxovirus has been shown to generate virion capsid protein in vitro (10).In the work presented, we have examined two mammalian cell-free systems for their ability to translate RNA from Sindbis virions and Sindbis-infected BHK cells. Sindbis is a relatively simple enveloped virus with a RNA genome of molecular weight about 4 X 108 and sedimentation coefficient of 42-49 S (11, 12). Infection of BHK cells by Sindbis shuts off host protein synthesis and leads to the predominant appearance of a 26S RNA that is believed to function as a polycistronic mRNA for ...

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Interferons: direct effects upon viral replication

Hovanessian

1985

Approaches to Antiviral Agents

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Translation of reovirus messenger ribonucleic acids synthesized in vitro into reovirus proteins in a mouse L cell extract

Cited by 17 publications

References 22 publications

Methylated Messenger RNA Synthesis In Vitro by Purified Reovirus

Methylated Messenger RNA Synthesis In Vitro by Purified Reovirus

Formation of Sindbis Virus Capsid Protein in Mammalian Cell-Free Extracts Programmed with Viral Messenger RNA

Interferons: direct effects upon viral replication

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