Vesicular Stomatitis Virus RNA Replication: a Role for the NS Protein

Howard, Marybeth; Wertz, Gail W.

doi:10.1099/0022-1317-70-10-2683

Cited by 74 publications

(78 citation statements)

References 25 publications

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“…There is general agreement that the phosphoprotein (P) keeps N in a soluble form, able to support replication (Davis et al, 1986 ;Peluso, 1988 ;Masters & Banerjee, 1988 a, b ;Peluso et al, 1988 ;Howard & Wertz, 1989 ;Pre! haud et al, 1992).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…In contrast, the NP362 oligomers banded at a buoyant density of 1n27 g\cm$ (Fig. 5 E), reproducibly lighter than wt NP suggesting that they are not associated with RNA (Blumberg et al, 1983 ;Howard & Wertz, 1989). The population of NP362 molecules at the top of the gradient (fractions 2-4) most likely represents monomeric protein that has not reached equilibrium under these centrifugation conditions.…”

Section: Self-assembly Is Altered In the Np Mutantsmentioning

confidence: 93%

Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication.

Myers

Smallwood

Moyer

1999

Journal of General Virology

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Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication ; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP 0 -P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.

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“…The roles of P NTD in binding L and of P CTD in binding N-RNA were first recognized by biochemical and genetic studies, establishing a primary role of P as a mediator of L's access to the N-RNA template (11). A second essential role of P involves chaperoning the free N protein (N 0 ) via the extreme N terminus of P NTD , maintaining N 0 solubility and regulating its assembly on the nascent genomic RNA during replication (16,17).An expanding structural map of P is beginning to provide additional functional insight into its role in RNA synthesis. Analysis of the crystal structure of the VSV P CTD has shown that P CTD makes direct contact with the C-terminal lobes of two adjacent N protomers (18).…”

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confidence: 99%

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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Vesicular Stomatitis Virus RNA Replication: a Role for the NS Protein

Cited by 74 publications

References 25 publications

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

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

Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication.

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

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