Plus and minus strand leader rnas in negative strand virus-infected cells

Leppert, M.; Rittenhouse, Linda; Perrault, Jacques; Summers, Donald F.; Kolakofsky, Daniel

doi:10.1016/0092-8674(79)90127-2

Cited by 209 publications

(162 citation statements)

References 23 publications

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“…In vitro transcription products synthesized in the presence or absence of RDP or RTP were purified by CsC1 centrifugation as described by Leppert et al (1979) and primer-extended with oligonucleotides specific for primer or N mRNA transcripts. The first primer was complementary to 15 nucleotides of leader RNA to give a 30-mer extended product, and the second primer was complementary to 15 nucleotides of nucleocapsid mRNA to give a 50-met extended product.…”

Section: Discussionmentioning

confidence: 99%

“…The first primer was complementary to 15 nucleotides of leader RNA to give a 30-mer extended product, and the second primer was complementary to 15 nucleotides of nucleocapsid mRNA to give a 50-met extended product. As controls for primer extension, VSV-and mock-infected cell RNAs were prepared as previously described (Leppert et al, 1979). The two specific primers, labelled at their 5' ends with [y-32p]ATP and T4 polynucleotide kinase, were added to each of the RNA samples and extended with 16 units of avian myeloblastosis virus reverse transcriptase in a reaction mixture containing 0.5 mM each of dATP, dGTP, dCTP and dUTP, 80 mM-NaC1, 50 mM-Tris-HC1 pH 8.3 and 8 mM-MgCI2 for 90 rain at 42 °C.…”

Section: Discussionmentioning

confidence: 99%

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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: 99%

Section: Discussionmentioning

confidence: 99%

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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“…In most cases, the viral nucleocapsids are functional in vitro, either by just opening up the virus by detergent and adding NTPs (plus a primer for influenza virus) or after purification of the nucleocapsids (Baltimore et al, 1970 ;Leppert et al, 1979 ;Vidal & Kolakofsky, 1989 ;Honda et al, 1986 ;Ishihama et al, 1986). For VSV and Sendai virus it has been possible to separately purify the nucleoprotein-RNA complexes and L plus P (either from virus or from recombinant sources) and recover activity after reconstitution in vitro (Emerson & Wagner, 1972 ;Emerson & Yu, 1975 ;De & Banerjee, 1984 ;Masters & Banerjee, 1986 ;Helfman & Perrault, 1989 ;Emerson, 1982Emerson, , 1987Canter et al, 1993 ;Horikami et al, 1992 ;Curran et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures.

Iseni

Barge

Baudin

et al. 1998

Journal of General Virology

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“…This feature is inherent in a model (Kingsbury, 1974;Leppert et al, 1979;Blumberg et al, 1981Blumberg et al, , 1983Kolakofsky & Blumberg, 1982) in which the viral polymerase is assumed to function constitutively, being switched between its alternative transcriptase and replicase activities in response to the intracellular concentration of free viral nucleocapsid (N) protein. In this model (Fig.…”

Section: A Model For Virus Replication In Mixed Infectionsmentioning

confidence: 99%

Review Article: An Analytical Review of Defective Infections of Vesicular Stomatitis Virus

1984

Journal of General Virology

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Plus and minus strand leader rnas in negative strand virus-infected cells

Cited by 209 publications

References 23 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

Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures.

Review Article: An Analytical Review of Defective Infections of Vesicular Stomatitis Virus

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