Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis.
Proteolytic processing of the Sindbis virus non-structural polyproteins (P123 and P1234) and synthesis of minus- and plus-strand RNAs are highly regulated during virus infection. Although their precise roles have not been defined, these polyproteins, processing intermediates or mature cleavage products (nsP1-4) are believed to be essential components of viral replication and transcription complexes. In this study, we have shown that nsP4 can function as the polymerase for both minus- and plus-strand RNA synthesis. Mutations inactivating the nsP2 proteinase, resulting in uncleaved P123, led to enhanced accumulation of minus-strand RNAs and reduced accumulation of genomic and subgenomic plus-strand RNAs. In contrast, no RNA synthesis was observed with a mutation which increased the efficiency of P123 processing. Inclusion of this mutation in a P123 polyprotein with cleavage sites 1/2 and 2/3 blocked allowed synthesis of both minus- and plus-strand RNAs. We conclude that nsP4 and uncleaved P123 normally function as the minus-strand replication complex, and propose that processing of P123 switches the template preference of the complex to minus-strands, resulting in efficient synthesis of plus-strand genomic and subgenomic RNAs and shut-off of minus-strand RNA synthesis.
Lemm
J A
JA
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110-1093.
Rümenapf
T
T
Strauss
E G
EG
Strauss
J H
JH
Rice
C M
CM
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D012313:RNA; D012367:RNA, Viral / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012845:Sindbis Virus / Q000254:growth & development*; D013997:Time Factors; D014158:Transcription, Genetic; D014452:Ubiquitins / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D012367:RNA, Viral; C039919:RNA, recombinant; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D017361:Viral Nonstructural Proteins; D012313:RNA; D012321:DNA-Directed RNA Polymerases