Interaction of L and NS Proteins of Vesicular Stomatitis Virus with its Template Ribonucleoprotein during RNA Synthesis in vitro

Thornton, George B.; De, Bishnu P.; Banerjee, Amiya K.

doi:10.1099/0022-1317-65-3-663

Cited by 13 publications

(13 citation statements)

References 18 publications

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“…The interpretation of these results is that lowering the salt increases de novo polymerase initiations, and since the polymerases can bind directly only to the 3' end, only AC synthesis is increased. However, when Thornton et al (1984) repeated the reconstitution experiments with an AC reaction, they found both leader and mRNA starts, although 60% of the label in the "m RNA" oligonucleotide band was in sequences corresponding to neither leader nor mRNA. Until further experiments are done, there is no convincing way to reconcile the aforediscussed data, and it may be useful to review numerous caveats.…”

Section: Transcription Modelsmentioning

confidence: 94%

Transcription of Vesicular Stomatitis Virus

Emerson

1987

The Rhabdoviruses

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Section: Transcription Modelsmentioning

confidence: 94%

Transcription of Vesicular Stomatitis Virus

Emerson

1987

The Rhabdoviruses

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“…Internal transcription initiation has been long suggested for VSV (31,32), and more recently, VSV replication and transcription were shown to be initiated at different positions on the VSV genome (35). Polymerase complexes with different protein contents that are active either in replication or in transcription have been described (17).…”

Section: Discussionmentioning

confidence: 99%

Dissociation of Rabies Virus Matrix Protein Functions in Regulation of Viral RNA Synthesis and Virus Assembly

Finke

Conzelmann

2003

J Virol

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Matrix (M) proteins of negative-strand RNA viruses play a crucial role throughout the virus life cycle. The functions of the M proteins in virus assembly and morphogenesis and in budding off enveloped virus particles have been extensively studied and well recognized. More recently, reverse genetics experiments with rabies virus (RV), a member of the Rhabdoviridae family, revealed an additional and unexpected function of rhabdovirus M proteins apart from virus assembly, namely, the regulation of RNA synthesis and profoundly affecting the balance of replication and transcription products (13).The viral RNA of nonsegmented negative-strand RNA viruses (order Mononegavirales) is associated with the viral nucleoprotein (N) to form a tight ribonucleoprotein (RNP) complex. Such RNPs are the template for transcription of subgenomic virus mRNAs and for replication of full-length virus RNAs. The latter probably involves concurrent encapsidation of the nascent RNA into RNPs. A constant supply of N protein is therefore a prerequisite for productive replication (1, 28). In contrast, transcription from the RNP does not need a supply of N. Both RNA replication and transcription are performed by the virus-encoded RNP-dependent RNA polymerase, which is composed of a large, catalytically active subunit (L) and a polymerase cofactor, the phosphoprotein P. P also has an important role during replication, as N-P complexes, rather than N alone, are required for proper and selective encapsidation of viral RNA (8,20). After infection, replication is therefore not possible until the so-called primary transcription and translation provide enough N protein.In spite of the fact that N is the most abundant viral protein in infected cells at later stages, transcription is the predominant RNA synthesis mode throughout infection, arguing against a simple and popular model in which the amount of available N protein or N-P complexes would determine the level of replication versus transcription. This is supported by reverse genetics experiments with RV (unpublished), vesicular stomatitis virus (VSV) (34), and respiratory syncytial virus (RSV) (9). The ratio of replication and transcription was found to be fixed and could not be altered by providing variable levels of N protein. Even with limiting amounts of N protein, only the overall RNA synthesis was reduced, suggesting that other trans-acting factors are responsible for balancing replication and transcription. Indeed, the RSV M2-2 protein, which is unique to members of the Pneumovirus genus and is associated with RNPs, was shown to be a trans-acting factor able to shift the balance of RNA synthesis from transcription toward RNA replication (2).Reverse genetics experiments with RV minigenomes, Mgene deletion mutants, and recombinant full-length virus with modified M expression more recently revealed an important function of the M protein in the regulation of rhabdovirus RNA synthesis. In the absence of M, replication of RV was downregulated, while the transcription efficiency was enhanced, resul...

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“…The presence of such a virion-associated replicase can be established by purification of the associated protein components (L, P, and N) from the RNP. (iii) The multiple initiation of RNA (leader RNA and mRNA) observed during transcription (30,31) is, thus, carried out by two polymerase entities, replicase and transcriptase, respectively, and both must initially be located on the leader template at separate promoter sites. Recent observations, in fact, tend to support these contentions (7a, 18, 32; S. P. Whelan and G. W. Wertz, 20th Annu.…”

Section: Vol 77 2003 Notes 735mentioning

confidence: 99%

Identification of a Novel Tripartite Complex Involved in Replication of Vesicular Stomatitis Virus Genome RNA

Gupta

Shaji

Banerjee

2003

J Virol

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Vesicular stomatitis virus (VSV), the prototype rhabdovirus, contains a single-stranded negative-sense genome RNA consisting of 11,161 nucleotides (nt) (26, 27) which is encapsidated by the nucleocapsid protein (N) and associated with the RNAdependent RNA polymerase consisting of a large protein (L) and the phosphoprotein (P) along with specific host factors (1). In the transcription process, phosphorylation of P protein by cellular casein kinase II at specific sites is required for its activity (3, 4, 5). The virion-associated RNA polymerase synthesizes sequentially from the 3Ј end of the genome RNA, a leader RNA (47 nt long) and five mRNAs in order, both in vitro and in vivo, encoding the N, P, M (matrix protein), G (glycoprotein), and L proteins. During the replication reaction, however, a critical attenuation of transcription occurs at the gene junctions, resulting in the synthesis of a full-length antigenome-sense RNA which is concomitantly enwrapped with the N protein. The positive-sense N-RNA complex then serves as a template for the RNA polymerase to produce more genome-sense progeny N-RNA, leading to mature virus synthesis. Since the encapsidation by N protein is a prerequisite during the replication process (1,7,12,15,22,23), it has been proposed that the N protein somehow modifies the RNA polymerase, thus facilitating the attenuation process (1, 7). It was subsequently shown that the N protein in the infected cells remains complexed with the P protein (6, 24), and this soluble complex, not N by itself, is directly involved in the replication process. It is also generally believed that the RNA polymerase, which is composed of L and P proteins, carries out both the RNA synthetic events, i.e., transcription and replication, with N-P complex initiating the latter process. The exact mechanism of the replication reaction, however, remains unclear.Some recent observations from our laboratory have brought to light a possible scenario where the transcriptase and replicase may, in fact, be two distinct entities. Using a reverse genetics system and utilizing a transcribing minigenome and defective interfering particle, we have shown that both phosphorylation-negative mutants (21) as well as mutation at the C-terminal basic domain of the P protein (11) are transcriptionally inactive but are fully active in replication of a defective interfering particle. These mutants, however, are capable of forming the soluble N-P complex, the required intermediate for the replication reaction (28). Since these mutant P proteins (Pmut) cannot provide the transcriptive function to the L protein but support replication, it was speculated that the L protein must interact directly with the N-P complex to form a functionally active tripartite complex L-(N-Pmut) which may serve as the putative replicase to carry out the replication reaction. These studies raised the possibility that replicase and transcriptase are two distinct entities with distinctly different subunit compositions.To gain insight into the structure and function of t...

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Interaction of L and NS Proteins of Vesicular Stomatitis Virus with its Template Ribonucleoprotein during RNA Synthesis in vitro

Cited by 13 publications

References 18 publications

Transcription of Vesicular Stomatitis Virus

Transcription of Vesicular Stomatitis Virus

Dissociation of Rabies Virus Matrix Protein Functions in Regulation of Viral RNA Synthesis and Virus Assembly

Identification of a Novel Tripartite Complex Involved in Replication of Vesicular Stomatitis Virus Genome RNA

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