Localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription

Iverson, Linda E.; Rose, John K.

doi:10.1016/0092-8674(81)90143-4

Cited by 309 publications

(281 citation statements)

References 28 publications

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“…Surprisingly, little information is available on ratios of leader RNA transcripts to other transcripts in the infected cell. Work of Iverson & Rose (1981) predicts that the amount of leader transcripts, although not analysed directly in their study, should be greater than the amount of N mRNA. Since our data on the intracellular ratio of leader to N mRNA are the steady state levels of leader and N mRNA rather than synthesis levels, our results are not necessarily contradictory to their hypothesis.…”

Section: Discussionmentioning

confidence: 93%

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

Fernandez-Larsson

1989

Journal of General Virology

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SUMMARYWe have studied the effect of phosphorylated ribavirin on the vesicular stomatitis virus (VSV) in vitro polymerase reaction by analysis of kinetic data obtained by varying the concentration of nucleoside triphosphates. The wild-type VSV had previously shown a competitive inhibition with the four natural nucleoside triphosphates with the use of ribavirin diphosphate (RDP) or ribavirin triphosphate (RTP). In contrast, when RDP (or RTP) was added to a transcription assay system using the polR1 mutant of VSV, a non-competitive or mixed type of inhibition was observed when the concentration of ATP was varied. Our results indicate that polR1 has an altered ATP function in addition to the previously described phenotypic characteristics of this mutant, which include synthesis of readthrough products of the leader/nucleocapsid (N) gene junction and a decreased ATP requirement for transcription. We have also studied CsCl-purified in vitro transcription products by primer-extending leader or N mRNA transcripts and found that the ratio of leader/N mRNA for VSV polR1 (1.3:1) was lower than values obtained previously for wild-type (3.7:1).Vesicular stomatitis virus (VSV) is the prototype rhabdovirus containing a negative-stranded RNA genome 11 kb long. The transcription and replication events of this virus have been studied for several years, but many questions related to these processes and to the switch from the transcriptive to the replicative mode remain unanswered. The VSV polR mutants (Perrault et al., 1983) have unique properties that may help solve the intricacies of transcription and replication. The properties of the polR mutants include synthesis of leader/nucleocapsid (N) gene readthrough transcripts (Perrault et al., 1983), utilization of imido-ATP for transcription initiation (Perrault & McClear, 1984), and an altered ATP utilization , whereas wild-type (wt) VSV has a high ATP requirement as well as an obligatory cleavage of the beta-gamma bond of this nucleotide for transcription (Testa & Banerjee, 1979;Green & Emerson, 1984; Perrault & McClear, 1984). We have used ribavirin to investigate by enzyme kinetic procedures and product analysis the mechanism of VSV mRNA synthesis, in an effort to understand the in vitro transcription of the virus.We have already reported that ribavirin 5'-monophosphate, 5'-diphosphate (RDP) and 5'-triphosphate (RTP) possess a significant direct suppressive effect upon viral polymerase activity. Inhibition by RDP or RTP could be reversed by addition of GTP, CTP and UTP, but not by the addition of GDP or ATP (Toltzis et al., 1988). The effective reversal of inhibition by all nucleoside triphosphates except ATP suggested that the ribavirin molecule does not act only as a guanine analogue as generally believed, but rather may interact with the polymerase complex on a site specific for at least three of the precursors of RNA synthesis. The fact that the effect of phosphorylated ribavirin could not be reversed with the addition of ATP could be explained if ATP had an additional inte...

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Section: Discussionmentioning

confidence: 93%

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

Fernandez-Larsson

1989

Journal of General Virology

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“…The encapsidation in the normal virus life cycle is thought to initiate at, or very close to, the 5 0 end of the nascent RNA chain, and then to progress strictly coupled with the chain elongation under the continuous supply of NP (Lamb & Kolakofsky 1996). This highly coordinated encapsidation probably does not occur in the initial round of transcription catalysed by T7 pol, because it elongates the chain much faster than mononegavirus polymerases (Chamberlin & Ryan 1982;Iverson & Rose 1981;Radecke et al 1995). The initial transcripts could thus remain naked for some time, or sparsely bound with NP, and hence would be readily attacked by RNases (Radecke et al 1995).…”

Section: Introductionmentioning

confidence: 99%

Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense

et al. 1996

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Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome. Recentsuccessintherecoveryofinfectiousvirusfroma transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense ( ) RNA. Starting with genomic negative sense (ÿ) RNA has been unsuccessful. Furthermore, the recovery efficiency has often been extremely low.

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“…Hybridizations were performed as described (22,23) with some modifications (no alkaline denaturation of the single stranded probes was required). Nitrocellulose filters (Schleicher & SchUll, BA 85 0.45u) were rinsed once with H20 and once with 6 x SSC ( 1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate) prior to filtration of DNA diluted in 6 x SSC.…”

Section: Methodsmentioning

confidence: 99%

Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus

Thierry

Danos

1982

Nucl Acids Res

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Specific single stranded DNA probes have been obtained for both influenza virion RNA (vRNA) and complementary RNA (cRNA) by cloning a hemagglutinin gene fragment in the single stranded DNA phage M13. These probes were used for hybridization with the total labeled RNA from cytoplasmic extracts of infected cells. MDCK cells were infected with temperature-sensitive mutants of Influenza HK/68 and the production of the virus specific RNA species was analysed at both permissive and restrictive temperatures. Results show that two NP mutants which undergo intracistronic complementation exhibit two different phenotypes at the non permissive temperature: ts2C is poly A cRNA and vRNA negative whereas ts463 is RNA positive. Two mutants of P genes were also analysed and we discuss the relationship existing between the synthesis of the three RNA species especially between poly A and non poly A cRNA.

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Localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription

Cited by 309 publications

References 28 publications

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin

Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense

Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus

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