Requirement for an aromatic amino acid or histidine at the N terminus of Sindbis virus RNA polymerase.
The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40 degrees C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30 degrees C and minute plaques at 40 degrees C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30 degrees C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.
Shirako
Y
Y
Division of Biology, California Institute of Technology, Pasadena 91125, USA. shirako@ims.u-tokyo.ac.jp
Strauss
J H
JH
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002642:Chick Embryo; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D010641:Phenotype; D012845:Sindbis Virus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010948:Viral Plaque Assay
D000596:Amino Acids; D017361:Viral Nonstructural Proteins; D006639:Histidine; D012321:DNA-Directed RNA Polymerases