De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation.
The replicase activity of rotavirus open cores has been used to study the synthesis of (-) strand RNA from viral (+) strand RNA in a cell-free replication system. The last 7 nt of the (+) strand RNA, 5'-UGUGACC-3', are highly conserved and are necessary for efficient (-) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (-) strand synthesis. By preincubating open cores with (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (-) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (-) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (-) strand synthesis initiated at the 3'-terminal C residue of the (+) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 3' end of the (+) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (-) strand synthesis and that the synthesis of pGpG may occur by a template-independent process. From these data, we conclude that initiation of rotavirus (-) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (+) strand RNA, and possibly a 5'-phosphorylated dinucleotide, that is, pGpG or ppGpG.
Chen
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Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Patton
J T
JT
D001483:Base Sequence; D002474:Cell-Free System; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases; D005814:Genes, Viral / Q000235:genetics; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012401:Rotavirus / Q000201:enzymology* / Q000235:genetics*; D012965:Sodium Chloride / Q000037:antagonists & inhibitors / Q000494:pharmacology; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects; D014764:Viral Proteins; D014779:Virus Replication / Q000187:drug effects
D017931:DNA Primers; D009711:Nucleotides; D012367:RNA, Viral; D014764:Viral Proteins; D012965:Sodium Chloride; C062210:bacteriophage T7 RNA polymerase; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases