Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA.
A gel mobility-shift assay was used to demonstrate the binding of the Sindbis virus transcriptase to the promoter for the synthesis of subgenomic (SG) RNA. The assay made use of a P15 fraction (the cell fraction that is pelleted at 15,000 x g) from cells infected with recombinant vaccinia virions expressing various Sindbis virus nonstructural proteins (nsPs) and a (32)P-labeled 24-mer oligoribonucleotide representing the minimal sequence with SG promoter activity. By itself, nsP4, the viral RNA-dependent RNA polymerase, did not bind to the SG promoter; rather, all four nsPs were required for the binding of the transcriptase to the promoter. UV crosslinking of the transcriptase to a thiouridine-containing SG promoter, followed by V8 protease digestion of the complex, generated a peptide fragment that was bound to the SG promoter. This peptide fragment contained a sequence that corresponded to residues 329-334 of nsP4. This peptide may be in the fingers domain of nsP4. The peptide that was identified contained Arg residues at positions 331 and 332. Another Arg is present at position 327. By changing each of the Arg residues to Ala, we demonstrated that only the Arg residues at positions 331 and 332 were required for binding nsP4 to the SG promoter.
Li
Mei-Ling
ML
Department of Molecular Genetics, Microbiology, and Immunology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Stollar
Victor
V
D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D011994:Recombinant Proteins / Q000378:metabolism; D012845:Sindbis Virus / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases