Full-length viral rna synthesized in vitro by vesicular stomatitis virus-infected hela cell extracts

Batt-Humphries, S; Simonsen, Christian C.; Ehrenfeld, Ellie

doi:10.1016/0042-6822(79)90175-2

Cited by 27 publications

(15 citation statements)

References 27 publications

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“…Condra and Lazzarini (24) showed previously that intact VSV-infected cells made permeable with lysolecithin would carry out efficient VSV genome RNA replication that was coupled to protein synthesis; however, theirs was not a cell-free system. Batt-Humphries et at (25) and Hill et at (26) prepared cell-free extracts of VSV-infected cells by Dounce homogenization. In those two studies, 42S RNA synthesis occurred in vitro in the absence of de novo protein synthesis but at a very low rate consisting of only 1-2% of the total RNA product (25,26).…”

Section: Discussionmentioning

confidence: 99%

“…Batt-Humphries et at (25) and Hill et at (26) prepared cell-free extracts of VSV-infected cells by Dounce homogenization. In those two studies, 42S RNA synthesis occurred in vitro in the absence of de novo protein synthesis but at a very low rate consisting of only 1-2% of the total RNA product (25,26). Davis and Wertz (27) have coupled VSV genome replication (2-5% of the total RNA synthesized) to protein synthesis by supplying the VSV proteins necessary for replication; they added intracellular VSV nucleocapsid templates to rabbit reticulocyte lysates programmed with VSV mRNAs.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.

Peluso¹,

Moyer²

1983

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

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A system for studying the in vitro replication of the RNA genomes of both wild-type vesicular stomatitis virus (VSV) and its defective interfering particle MS-T has been developed. After lysolecithin treatment of cells infected with VSV or VSV plus MS-T, a cell-free cytoplasmic extract is prepared which will support VSV mRNA synthesis and the synthesis of the 42S wild-type or 19S MS-T Reproduction of the rhabdovirus vesicular stomatitis virus (VSV) requires both transcription and replication of the (-)-strand genome RNA. Transcription, but not replication, can be carried out by purified detergent-disrupted virus and, after initiation at the 3' end, results in the sequential synthesis of first leader RNA and then the monocistronic mRNAs for the N, NS, M, G, and L proteins (1). The intracellular replication of the VSV RNA involves the synthesis of a genome-length (+)-strand RNA which then serves as the template for the production of progeny (-)-strand virion RNA (2, 3). Both of these single-stranded templates are found within the cell as nucleocapsids containing the viral proteins L, N, and NS (2). Earlier studies (4,5) have suggested that VSV RNA replication, in contrast to transcription, requires viral protein synthesis, In this regard, a replication model has been proposed by Kolakofsky and his co-workers (6-8) which suggests that it is the VSV N protein association with the nascent RNA chains-i.e., nucleocapsid-formation-that is specifically required for replication to proceed.A complication in the study of (-)-strand RNA virus replication is that the genome RNA serves as the template for both mRNA synthesis and replication. In order to focus exclusively on the process of replication, we have used for these studies a defective interfering (DI) particle of VSV designated MS-T. This DI particle contains a genome of 1 x 106 daltons (19 S) that consists only of the 5' terminus of the virus, representing a portion of the L cistron with a 3' terminus complementary to the 5' terminus (7, 9, 10). Because MS-T virion RNA lacks the 3' initiation signal for transcription as well as most of the VSV genetic information, MS-T particles can neither synthesize mRNAs nor replicate their genome autonomously in vitro, although a 46-nucleotide DI leader RNA is produced (11). For its reproduction, the defective MS-T particle requires the presence of wild-type VSV as helper to provide the mRNAs and proteins necessary for RNA replication and particle maturation, while simultaneously inhibiting the replication of the helper VSV genome RNA in the mixed infection (10, 12). The use of intracellular MS-T nucleocapsids therefore should provide templates with 19S RNAs of both the (+)-and (-)-strand sense involved specifically in the process of replication. MATERIALS AND METHODS Growth and Purification of Virus. Monolayer cultures of baby hamster kidney (BHK) cells were used for all experiments.The HR strain of VSV (Indiana) (13) was grown as described (14). The MS-T (9) was propagated by using HR-VSV as helper virus and was puri...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.

Peluso¹,

Moyer²

1983

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

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“…The conditions used for the in vitro transcriptase assay were described previously (Batt-Humphries et al, 1979), except that melittin was used in place of Triton N101 in some experiments; Triton N101 was used in the reaction mixtures for the experiments presented in Fig. 4.…”

Section: Cells and Virusmentioning

confidence: 99%

Phosphorylation of Vesicular Stomatitis Virus Proteins as a Possible Contributing Factor in Virion Uncoating

Witt¹,

Naeve²,

Summers³

1981

Journal of General Virology

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SUMMARYThe relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of y-[32p]ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence of melittin and deoxyadenosine triphosphate, the virion envelope was disrupted based on the accessibility of the internal ribonucleoprotein core (RNP) to the heavy metal stain, uranyl acetate, as determined by electron microscopic observati~n. The RNP structure was not observed in unphosphorylated virions treated with melittin and uranyl acetate. Phosphorylated virions treated with uranyl acetate subsequently lost the capacity for transcription whereas unphosphorylated virions treated with the stain retained transcriptase activity. These observations suggest that phosphorylation of VSV proteins may contribute to virion uncoating by disrupting the virus envelope.

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Section: Introductionmentioning

confidence: 99%

“…However, the complete complement of the negative-strand genome RNA (the 42S plus strand), which is the required intermediate of replication, has not been detected in vitro although it has been identified in the infected cell (6, 7). Thus, it appears that transcription of the full-length VSV genome RNA in vitro possibly requires modified experimental conditions or mediation by host or intracellular viral proteins (8,9).We have recently shown that ATP plays a significant role during initiation of VSV mRNA synthesis in vitro (10). The apparent Km for ATP during the initiation step was significantly higher (0.5 mM) than that observed for the same nucleotide during the period of chain elongation (25MuM).…”

mentioning

confidence: 99%

In vitro synthesis of the full-length complement of the negative-strand genome RNA of vesicular stomatitis virus.

Testa¹,

Chanda²,

Banerjee³

1980

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

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Under the normal conditions of in vitro RNA synthesis, the virion-associated RNA polymerase of vesicular stomatitis virus synthesizes five monocristronic mRNAs and a 48-nucleotide-long leader RNA that represents the exact 3'-terminal region of the genome RNA [Colpnno, R. J. & Banerjee, A. K. (1978) Cell, 15, 93-101]. When the transcribing core was preincubated with ATP and CTP, reisolated, and then incubated in the presence of the fty imido analoguq of ATP (AdoPFIINMI) and the three normal ribonucleoside. triphosphates, the fulllength complementary strand of the getlotne RNA was synthesized in vitro. The results suggest that specific phosphorylated states of regulatory proteins may control transcription in vitro to generate the full-length plus strands.The virion-associated RNA polymerase of vesicular stomatitis virus (VSV) transcribes the singlestranded genome RNA of negative polarity in vitro into five"mbnocistronic mRNA species(1, 2). The transcription process appears to be sequential, with the synthesis of a "leader RNA" representing the 3'-terminal 48-nucleotide region of the genome RNA (3) followed by the synthesis of the mRNA species coding for the structural proteins in the order N, NS, M, G, and L (4,5). The sum of the molecular weights of the mRNA species approximately corresponds to the total coding potential of the genome RNA, indicating that the entire genome is transcribed in vitro (2). However, the complete complement of the negative-strand genome RNA (the 42S plus strand), which is the required intermediate of replication, has not been detected in vitro although it has been identified in the infected cell (6, 7). Thus, it appears that transcription of the full-length VSV genome RNA in vitro possibly requires modified experimental conditions or mediation by host or intracellular viral proteins (8,9).We have recently shown that ATP plays a significant role during initiation of VSV mRNA synthesis in vitro (10). The apparent Km for ATP during the initiation step was significantly higher (0.5 mM) than that observed for the same nucleotide during the period of chain elongation (25MuM). These results coupled with the observation that the first RNA'product, the leader RNA, terminates with a 5'-terminal diphosphate ppA-C-G sequence (3) suggested that the removal of the y-phosphate of ATP may be directly involved in the initiation step of RNA synthesis. This MATERIALS AND METHODS Purification of VSV. VSV (Indiana serotype) was grown in baby hamster kidney cells (BHK-21, clone 13) adapted to suspension culture and purified as described (11).Conditions for In Vitro Synthesis of Full-Length Positive Strand. Purified VSV (1.6 mg) was disrupted with Triton N-101 (0.05%) in 0.05 M Tris-HCl, pH 8/0.1 M NaCl/5 mM MgCl2/4 mM dithiothreitol/I mM ATP/0.5 mM CTP (total volume, 2 ml). The mixture was incubated for 30 min at 30'C. The reaction mixture was then chilled to 40C and layered onto 2 ml of solution containing 20% (wt/vol) glycerol in 0.01 M Tris.HCl, pH 7.4/0.01 M KCI/0.1 mM MgCl2/0.1 M NaCl/1 m...

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Full-length viral rna synthesized in vitro by vesicular stomatitis virus-infected hela cell extracts

Cited by 27 publications

References 27 publications

Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.

Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.

Phosphorylation of Vesicular Stomatitis Virus Proteins as a Possible Contributing Factor in Virion Uncoating

In vitro synthesis of the full-length complement of the negative-strand genome RNA of vesicular stomatitis virus.

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