In vitro Phosphorylation of NS Protein by the L Protein of Vesicular Stomatitis virus

Sánchez, Alejandro; De, Bishnu P.; Banerjee, Amiya K.

doi:10.1099/0022-1317-66-5-1025

Cited by 80 publications

(38 citation statements)

References 18 publications

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“…Whether NNSV L proteins have kinase activity has been controversial. There are reports indicating that L of vesicular stomatitis virus (New Jersey serotype) and Sendai virus may have intrinsic kinase activity (2,3,12,17,39). However, other reports suggest that the L-associated kinase activity may come from a host kinase that interacts with L (15,30).…”

Section: Discussionmentioning

confidence: 99%

AKT1-Dependent Activation of NF-κB by the L Protein of Parainfluenza Virus 5

Luthra

Sun

Wolfgang³

et al. 2008

J Virol

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Section: Discussionmentioning

confidence: 99%

AKT1-Dependent Activation of NF-κB by the L Protein of Parainfluenza Virus 5

Luthra

Sun

Wolfgang³

et al. 2008

J Virol

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“…6). Although both cellular protein kinases and L protein are reported to phosphorylate NS protein (24,25), the importance of phosphorylation to NS function is still open to debate. Both the NS3 and NS5 deletions affect L binding and both eliminate potentially relevant phosphorylation sites.…”

Section: Discussionmentioning

confidence: 99%

Location of the binding domains for the RNA polymerase L and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus.

Emerson¹,

Schubert²

1987

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

100

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Recombinant DNA techniques were used to delete regions of a cDNA clone of the phosphoprotein NS gene of vesicular stomatitis virus. The complete NS gene and four mutant 'genes containing internal or terminal deletions were inserted into a modified pGem4 vector under the transcriptional control of the ph.Age T7 promoter. Run-off transcripts were synthesized and translated in Vitro to provide onine-labpled complete NS or deletion mutant NS proteins. (9), an extension function of uncoiling the helical nucleocapsid (11), and a displacement function to temporarily shift N protein from the template RNA during transcription (12). During replication, in addition to serving as a noncatalytic subunit of the polymerase, the NS protein forms complexes with newly synthesized N protein that apparently prevent nonspecific N protein aggregation prior to assembly into nucleocapsids (13).In an effort to better define the structural functions of NS protein, we have altered the structure of the protein through molecular cloning techniques and have analyzed the effects of these alterations on the ability of the mutant NS polypeptides to interact with the other two viral proteins involved in RNA synthesis. Both internal and terminal deletions were constructed such that the set of proteins encoded by the deleted genes fractionated the NS protein into six regions. In this communication we present data derived from binding studies which demonstrate that NS protein can be functionally divided into two independent domains that mediate binding to L and N proteins, respectively. MATERIALS AND METHODSPlasmid Constructions. pGem4 XB was used for the transcription of the NS mutant genes and the N gene. It is identical to pGem4 (Promega Biotec, Madison, WI) except that we introduced unique Xho I and BssH2 cloning sites between the HindIII and Sph I sites by using synthetic oligonucleotides. The NS gene clones pLH7 and pLH3 were previously isolated and sequenced by Hudson et al. (12). The complete NS gene insert of pLH7 was used for the construction of the deletion mutants NS3, -4, and -5. The NS inserts of LH7 and the deletion mutant LH3 were inserted into pGem4 XB and were designated pGNS1 and pGNS2, respectively. Details on the construction of pGNS3, -4, and -5 will be described elsewhere. pGN2 contains the complete N gene of VSV Indiana serotype, which was excised from pJS223 (14) with Xho I.Abbreviations: VSV, vesicular stomatitis virus; RNP, ribonucleocapsid.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5655

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“…We propose that this site could be associated with polyadenylation or with protein kinase activities. It could correspond to one of the two binding sites, with substantially different affinity for ATP, that have been identified in VSV (S~nchez et al, 1985;Massey & Lenard, 1987). The other binding site could be associated with the specific phosphorylation of the NS protein, a function reported to be encoded by the L protein only in the Indiana strain of VSV (S~nchez et al, 1985).…”

Section: Search For Functional Motifsmentioning

confidence: 99%

Sequence Comparison of Five Polymerases (L proteins) of Unsegmented Negative-strand RNA Viruses: Theoretical Assignment of Functional Domains

Poch

Blumberg

Bougueleret

et al. 1990

Journal of General Virology

478

386

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The large (L) protein subunit of unsegmented negativestrand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.

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In vitro Phosphorylation of NS Protein by the L Protein of Vesicular Stomatitis virus

Cited by 80 publications

References 18 publications

AKT1-Dependent Activation of NF-κB by the L Protein of Parainfluenza Virus 5

AKT1-Dependent Activation of NF-κB by the L Protein of Parainfluenza Virus 5

Location of the binding domains for the RNA polymerase L and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus.

Sequence Comparison of Five Polymerases (L proteins) of Unsegmented Negative-strand RNA Viruses: Theoretical Assignment of Functional Domains

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