Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes.
Replication of the influenza virus genome involves two discrete step reactions: vRNA-directed primer-independent (unprimed) synthesis of cRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts from both MDCK and HeLa cells infected with influenza virus A/PR8/34 exhibited unprimed synthesis of both cRNA and vRNA strands (a parameter of RNA replication). Ribonucleoprotein (RNP) complexes with the replication activity were isolated from these nuclear extracts by glycerol gradient centrifugation in the presence of 0.1 M KCl. At 0.5 M KCl, however, these complexes were dissociated into stripped RNP and soluble protein fractions. The soluble fraction contained the activity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to high salt concentrations. On the other hand, the high salt-treated RNP catalyzed only primer-dependent RNA synthesis, but regained a low level activity of exogenous template-dependent unprimed RNA synthesis by adding nuclear extracts from uninfected cells, suggesting that host factor(s) is involved in the functional interconversion of influenza virus RNA polymerase.
Department of Molecular Genetics, National Institute of Genetics, Shizuoka, Japan.
T
T
Toyoda
M
M
Kobayashi
A
A
Ishihama
D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D004285:Dogs; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000502:physiology; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics
D012367:RNA, Viral; D012261:Ribonucleoproteins; D012324:RNA-Dependent RNA Polymerase