<i>In Vitro</i>
            Synthesis of RNA That Contains Polyadenylate by Virion-Associated RNA Polymerase of Vesicular Stomatitis Virus

Banerjee, Amiya K.; Rhodes, Dennis P.

doi:10.1073/pnas.70.12.3566

Cited by 78 publications

(56 citation statements)

References 28 publications

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“…BHK-21 clone 13 (C1.13) and Vero cells (Flow Laboratories) were grown as monolayers in Dulbecco's modified and F15-modified minimal essential media respectively; BHK-21 C1.13 cells modified for suspension culture (Banerjee & Rhodes, 1973) were grown in Joklik's modified minimal essential medium. All media were supplemented with 5 ~ heat-inactivated calf serum and antibiotics.…”

Section: Methodsmentioning

confidence: 99%

Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme

Huszar

Beharry

Bacchetti

1983

Journal of General Virology

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SUMMARYWe have previously reported on the characterization of a novel ribonucleotide reductase induced in herpes simplex virus type 2 (HSV-2)-infected mammalian cells. The virus-induced enzyme was partially purified, free of the constitutive cell isozyme, by fractionation of infected cell extracts with ammonium sulphate. In this report we describe a further purification of the virus-induced enzyme and the development of a rabbit antiserum capable of specifically inhibiting its activity. Enzymically active salt fractions from infected cell extracts were sedimented through glycerol gradients; the virus-induced enzyme was found to sediment approximately 2.5 times faster than the constitutive, or control, enzyme and was separated from the majority of the protein in the sample. Immunization of rabbits with the partially purified enzyme preparation recovered from gradients resulted in the synthesis of antibodies which completely and specifically inhibited the virus-inducod reductase, and precipitated one major polypeptide and a few minor species from both HSV-1-and HSV-2-infected cells. The antibodies, however, exhibited much greater affinity for the HSV-2 than the HSV-1 antigen. These results demonstrate that the virus-induced enzyme differs antigenically, as well as biochemically, from the constitutive cell isozyme and lend further support to the hypothesis that the enzyme is virus-codod.

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Section: Methodsmentioning

confidence: 99%

Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme

Huszar

Beharry

Bacchetti

1983

Journal of General Virology

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“…Recent work in this laboratory (19) has shown that RNA species of two size classes, 31 S and 12-18 S, can be synthesized in vitro by the virion-associated RNA polymerase of VSV. The in vitro RNA products, similar in size to those found in vivo (10), are complementary to the genome and contain poly(A) segments (19)(20)(21), indicating that they could serve as messengers for the VSV proteins. In this report we show that the in vitro 12-18S RNA can be translated in cell-free extracts of wheat germ into proteins which seem identical to the viral polypeptides.…”

mentioning

confidence: 95%

“…[35S]methionine (20 ,Ci/ml) was added at 4 hr after infection. Five minutes after the addition of the label, a 100-fold excess of unlabeled methionine was added and the cell lysate was prepared (23 (25) following the methods of Laemmli (26), and electrophoresed at 125 V for 4 hr.…”

Section: Purification Of Virus and Preparation Of Lysates Of Infectedmentioning

confidence: 99%

Translation and identification of the mRNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

Both¹,

Moyer²,

Banerjee³

1975

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

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Vesicular stomatitis virus messenger RNA has been transcribed in vitro from the viral genome by the virion-associated RNA polymerase in quantities suitable for translation. Wheat germ cell-free extracts programmed with the isolated in vitro 12-18S RNA fraction synthesize polypeptides similar to the viral N, NS, and M proteins, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping of the in vitro products and the viral marker polypeptides. In addition, the RNA synthesized in vitro also codes for a protein of molecular weight 63,000 which may be a nonglycosylated form of the viral glycoprotein G. The 12-18S RNA has been partially separated into individual messenger species and these have been identified by the proteins for which they code. There are four monocistronic messenger species in the in vitro 12-18 S RNA and the coding capacity of three of these molecules agrees with the estimated molecular weight of the polypeptide assigned to it. Vesicular stomatitis virus (VSV) is a membrane-enveloped rhabdovirus (1) with a single-stranded RNA genome (2, 3) of molecular weight 3.6 to 4 X 10O (1,3). The virion also contains five structural polypeptides (4-6); the glycoprotein G (7, 8) is located in the virion surface projections, the matrix protein M is found in the virion envelope and the nucleoprotein N, together with the genome RNA and two minor proteins, NS and L, constitutes the ribonucleoprotein core (9).Polysomes of VSV-infected cells contain virus-specific messenger RNA (mRNA) which is complementary to the viral genome (10, 11). The mRNA can be resolved into two size classes by sucrose gradient sedimentation; a homogeneous species sedimenting at 28 S and heterogeneous RNAs sedimenting at 13-15 S (10). Further analysis of mRNA prepared from VSV-infected cells resolved the 13-15S component into at least three species (12-14) while the 28S RNA migrated as a homogeneous band (12). It was suggested, based on the molecular weights of these RNA species, that the 13-15S RNA could code for the G, N, NS, and M proteins (10) and that the 28S RNA could code for the L protein (6, 15). Indeed, subsequent work has shown that reticulocyte lysates programmed with 28S RNA isolated from VSV-infected cells can synthesize a protein which co-migrates with the viral L protein on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels (16). Similarly, it has also been shown that proteins which co-migrate with the viral N, NS, and M polypeptides can be synthesized in several different cell-free extracts programmed with 13-15S RNA, but it was not clear whether authentic G protein was made.Abbreviations: VSV, vesicular stomatitis virus; NaDodSO4, sodium dodecyl sulfate.VSV has a virion-associated RNA polymerase which is capable of synthesizing, in vitro, RNA complementary to the viral genome (17,18). Recent work in this laboratory (19) has shown that RNA species of two size classes, 31 S and 12-18 S, can be synthesized in vitro by the virion-associated RNA polymerase of VSV. The in vitro ...

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“…The RNP complex comprises a genomic RNA encapsidated by a nucleocapsid protein oligomer (N-RNA), associated with the RNA-dependent RNA polymerase (RdRP) consisting of a complex of the large polymerase protein (L) and a phosphoprotein (P) (1)(2)(3). The template RNA is buried between the N-and C-terminal lobes of each N protomer; nevertheless, the overall integrity of the N-RNA structure is maintained during copying by the RdRP (4,5).…”

mentioning

confidence: 99%

Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus

Rahmeh¹,

Morin²,

Schenk³

et al. 2012

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

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The RNA-dependent RNA polymerase (RdRP) of nonsegmented negative-sense RNA viruses consists of a large catalytic protein (L) and a phosphoprotein cofactor (P). During infection, the RdRP replicates and transcribes the viral genome, which resides inside an oligomer of nucleocapsid protein (N-RNA). The classical view of P as a cofactor for L assigns a primary role of P as a bridge mediating the access of L to the RNA template, whereby its N-terminal domain (P NTD ) binds L and its C-terminal domain (P CTD ) binds N-RNA. Recent biochemical and structural studies of a prototype nonsegmented negative-sense RNA virus, vesicular stomatitis virus, suggest a role for P beyond that of a mere physical link: P induces a structural rearrangement in L and stimulates polymerase processivity. In this study, we investigated the critical requirements within P mediating the functional interaction with L to form a fully functional RdRP. We analyzed the correlation between the impact of P on the conformation of L and its activity in RNA synthesis and the consequences of these events on RdRP function. We identified three separable elements of the P NTD that are required for inducing the conformational rearrangement of L, stimulating polymerase processivity, and mediating transcription of the N-RNA. The functional interplay between these elements provides insight into the role of P as a dynamic player in the RNA synthesis machine, influencing essential aspects of polymerase structure and function.large polymerase | replication and transcription | Mononegavirales | rhabdovirus T he functional unit necessary for transcription and replication of nonsegmented negative-sense (NNS) RNA viruses is a ribonucleoprotein (RNP) complex. The RNP complex comprises a genomic RNA encapsidated by a nucleocapsid protein oligomer (N-RNA), associated with the RNA-dependent RNA polymerase (RdRP) consisting of a complex of the large polymerase protein (L) and a phosphoprotein (P) (1-3). The template RNA is buried between the N-and C-terminal lobes of each N protomer; nevertheless, the overall integrity of the N-RNA structure is maintained during copying by the RdRP (4, 5). The L protein is the multifunctional catalytic core of the RNA synthesis machinery, harboring the RdRP as well as a capping enzyme and two methyltransferase activities that are required for mRNA synthesis (6-10). During RNA synthesis, L must gain access to the RNA, and its enzymatic activities must be regulated in accordance with a replicase or transcriptase mode of RNA synthesis. The access of L to the N-RNA is mediated by the noncatalytic cofactor P, which engages L and the N oligomer simultaneously (11,12).The functioning of an RNP as a highly regulated RNA synthesis machine requires an intricate, tight coordination of its individual components. The mechanisms that govern such functional coupling are largely unknown. Much of our understanding of the assembly, structure, and function of NNS RNA virus RNPs have come from studies of vesicular stomatitis virus (VSV). In part, this reflect...

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In Vitro Synthesis of RNA That Contains Polyadenylate by Virion-Associated RNA Polymerase of Vesicular Stomatitis Virus

Cited by 78 publications

References 28 publications

Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme

Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme

Translation and identification of the mRNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus

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