Alphavirus minus-strand RNA synthesis: identification of a role for Arg183 of the nsP4 polymerase.
A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused by a ts defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The ts defect was not due to a failure to form or accumulate nsP4 at 40 degrees C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.
Fata
Cori L
CL
Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614, USA.
Sawicki
Stanley G
SG
Sawicki
Dorothea L
DL
D000330:Aedes; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000502:physiology*; D002460:Cell Line; D002642:Chick Embryo; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D010641:Phenotype; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology*; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010948:Viral Plaque Assay
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D001120:Arginine; D012321:DNA-Directed RNA Polymerases