The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules.
Overlapping cDNAs representing the complete L segment of Rift Valley fever virus were assembled, and the L protein was expressed via a recombinant vaccinia virus. The transcriptase activity of the L protein was assayed with two types of templates: natural ribonucleoproteins (RNPs) and artificial genome-like RNAs. RNPs purified in a CsCl gradient did not retain the RNA polymerase function, but the activity was restored when the L cDNA was expressed in mammalian cells via a recombinant vaccinia virus. Indeed, after transfection of transcriptase-depleted RNPs in cells infected with the recombinant vaccinia virus expressing the L protein, the mRNAs coding for the N and NSs proteins and to a lesser extent, those coding for the glycoproteins were synthesized as well as the corresponding proteins. The transcriptase activity of the recombinant L protein was then investigated by using synthetic templates containing the reporter chloramphenicol acetyltransferase gene in the antisense orientation flanked by the 3' and 5' noncoding region of the S genomic segment. Our results indicate that after transfection of the RNA templates, transcription was achieved in cells coexpressing both the L and N proteins. Together, the experiments demonstrate that the two proteins N and L are absolutely required and sufficient to reconstitute the transcriptase activity.
Lopez
N
N
Laboratoire des Bunyaviridés, Institut Pasteur, Paris, France.
Muller
R
R
Prehaud
C
C
Bouloy
M
M
D001483:Base Sequence; D016679:Genome, Viral; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000494:pharmacology; D012261:Ribonucleoproteins / Q000378:metabolism*; D012296:Rift Valley fever virus / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*; D014764:Viral Proteins / Q000494:pharmacology*
D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014764:Viral Proteins