In vitro synthesis of full-length influenza virus complementary RNA.
Influenza virus-specific RNA has been synthesized in vitro, using cytoplasmic or microsomal fractions of influenza virus-infected MDCK cells. The RNA polymerase activity was stimulated 5-30 times by priming with ApG. About 20-30% of the product was polyadenylated. Most of the in vitro product was of positive polarity, as shown by hybridization to strand specific probes and by T1 fingerprinting of the poly(A)+ and poly(A)- RNA segments encoding haemagglutinin and nucleoprotein. The size of poly(A)- RNA segments, determined on sequencing gels, was indistinguishable from that of virion RNA, whereas poly(A)+ RNA segments contain poly(A) tails approximately 50 nucleotides long. The size of in vitro synthesized RNA segments was also determined by gel electrophoresis of S1-treated double-stranded RNAs, obtained by hybridization of poly(A)+ or poly(A)- RNA fractions with excess of unlabelled virion RNA. The results of these experiments indicate that poly(A)- RNA contains full-length complementary RNA. This conclusion is further substantiated by the presence of additional oligonucleotides in the T1 fingerprints of in vitro synthesized poly(A)- haemagglutinin or nucleoprotein RNA, selected by hybridization to cloned DNA probes corresponding to the 3' termini of the genes.
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del Río
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Martínez
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Domingo
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Ortín
D000818:Animals; D002478:Cells, Cultured; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004285:Dogs; D005814:Genes, Viral; D009980:Influenza A virus / Q000235:genetics / Q000378:metabolism*; D011061:Poly A / Q000096:biosynthesis; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D018075:RNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral / Q000096:biosynthesis*; D014158:Transcription, Genetic
D018075:RNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral; D011061:Poly A; D012313:RNA; D012321:DNA-Directed RNA Polymerases