Faithful and efficient in vitro reconstitution of vesicular stomatitis virus transcription using plasmid-encoded L and P proteins

Abstract

We demonstrate here that plasmid-expressed polymerase proteins of a negative-strand RNA virus can faithfully reconstitute all aspects of the transcription process carried out by virion cores in vitro. The assay is based on adding purified nucleocapsid templates of vesicular stomatitis virus to extracts of cells expressing L and P viral polymerase proteins via the vaccinia-T7 RNA polymerase recombinant virus. No significant differences were seen between the native virion core reaction and the optimally reconstituted system including ratio of transcripts produced, polyadenylation, net synthesis per template, amounts of polymerase proteins and template, and competence to initiate infection in vivo. Reconstitution was not dependent on cotranslation of P and L proteins in the same cell since nearly as much activity was obtained by mixing extracts expressing each protein individually. Cotransfection with P plasmid, however, stimulated L protein accumulation two- to fivefold relative to transfection with L alone. Surprisingly, deleting a small region in the C-terminal half of the L polymerase protein (amino acids 1638 to 1673) abolished transcription as well as stimulation by P coexpression. Since the polymerase domain of L presumably lies in the N-terminal half of the protein, these results suggest that the putative nucleotide-binding motif in the deleted segment may be involved in an accessory function essential for the transcription process.