The genome of vesicular stomatitis virus is negative-stranded, nonsegmented RNA (MW 4 million). Complexed with the viral nucleoprotein N, it serves as a template for two coupled pathways of RNA synthesis: Transcription (the sequential synthesis of a 47 base leader and five capped, methylated, and polyadenylated monocistronic mRNAs) and Replication (the synthesis of genomic-length positive- and negative-stranded RNAs which are complexed with N protein). The mechanisms governing the transcriptive and replicative pathways and their coupled regulation are not fully understood. The viral proteins L, NS and N are multifunctional and involved in both pathways. However, the roles of these proteins are not known. Transcriptive functions to be assigned include: 1) polymerase, 2) polyadenylase, 3) guanylyltransferase, 4) guanine-7-methyltransferase, 5) nucleoside 2'-methyltransferase, 6) putative processing endonuclease. Replicative functions include antitermination, nucleocapsid assembly, and negative strand synthesis. The long-range goal of this research program is to assign transcriptive and replicative functions to the viral proteins, and to determine how the proteins and activities interact to promote and modulate the two pathways of RNA synthesis. The concept is based on the use of ts mutants with lesions affecting transcription and replication as sources of altered L, NS, and N proteins, the functional consequences of which will be determined by the following experimental approaches: 1) Transcription defects will be identified through analyses of the RNA products synthesized in mutant-directed in vitro transcription reactions for aberrant capping, methylation, polyadenylation, and processing-punctuation; 2) Replication defects affecting nucleocapsid assembly and genomic-length RNA synthesis will be analyzed in vivo with the aid of chimeric T particles coinfected with mutant and wild-type standard virus; 3) An in vitro replication system will be established for refined functional analyses of mutant proteins; 4) The functional significance of NS phosphorylation will be explored through coordinated functional and structural analyses of mutant and wildtype variant NS proteins. The significance of these studies arises from the striking similarity of the salient features of VSV RNA synthesis to that of other members of the nonsegmented negative-strand RNA virus group which are pathogenic to man.
