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.
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 phage T7 promoter. Run-off transcripts were synthesized and translated in vitro to provide [35S]methionine-labeled complete NS or deletion mutant NS proteins. Immune coprecipitation assays involving these proteins were developed to map the regions of the NS protein responsible for binding to the structural viral nucleocapsid protein N and the catalytic RNA polymerase protein L. The data indicate the NS protein is a bivalent protein consisting of two discrete functional domains. Contrary to previous suggestions, the negatively charged amino-terminal half of NS protein binds to L protein, while the carboxyl-terminal half of NS protein binds to both soluble recombinant nucleocapsid protein N and viral ribonucleocapsid template.
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids; D012313:RNA / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase; D011817:Rabbits; D012261:Ribonucleoproteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014758:Viral Core Proteins / Q000378:metabolism; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000378:metabolism*
D012261:Ribonucleoproteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D012313:RNA; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease