Expression and purification of vesicular stomatitis virus N-P complex from Escherichia coli: role in genome RNA transcription and replication in vitro.
The nucleocapsid protein (N) and phosphoprotein (P) genes of vesicular stomatitis virus (VSV), Indiana serotype, were coexpressed in Escherichia coli BL21(DE3) by using the expression vector pET-3a. The coexpression resulted in the formation of N-P complex. The purified N-P complex was found to inhibit transcription in vitro mediated by viral ribonucleoprotein (RNP) complex in a dose-dependent manner. However, addition of uninfected mammalian cell extracts together with the N-P complex to the transcribing RNP resulted in the synthesis of full-length negative-strand genome RNA. These results indicate that the N-P complex regulated transcription and a cellular factor(s) in combination with the N-P complex may switch the RNA polymerase from transcription to replication mode.
Gupta
A K
AK
Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Banerjee
A K
AK
D003001:Cloning, Molecular; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral; D046911:Macromolecular Substances; D019251:Nucleocapsid / Q000235:genetics*; D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D011485:Protein Binding; D011994:Recombinant Proteins; D012261:Ribonucleoproteins / Q000378:metabolism; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000737:chemistry / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*; D014779:Virus Replication
D046911:Macromolecular Substances; D019590:Nucleocapsid Proteins; C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D015678:Viral Structural Proteins