Vesicular stomatitis virus (VSV) is a single-stranded RNA virus belonging to the same family as rabies virus (the Rhabdoviridae). Virions contain an RNA-dependent RNA polymerase (transcriptase) which makes messenger RNAs which are complementary to genomic RNA. The mechanism whereby monocistronic, capped, methylated, polyadenylated mRNAs are generated is not understood. Three of the five virally-coded proteins are required (L, NS and N), host proteins may be involved, and at least one other virally-coded protein (M) may modulate the process. It is probable that replication also involves L, NS and N. We plan to use temperature-sensitive (ts) mutants of VSV to help to elucidate: the mechanism of transcription; the role of viral proteins in mRNA modification; and the relationship between transcription and RNA modification. We plan to survey in vitro RNA synthesis by ts mutants of VSV belonging to complementation groups I, II and IV (thought to correspond to mutations in the L, NS and N genes, respectively) to find mutants which exhibit a perturbation in RNA synthesis. Initially, RNA synthesized in vitro will be examined for size, ability to be translated and the nature of the translation products, capping, methylation and polyadenylation. Aberrant RNAs produced by ts mutants will be further characterized. In some cases, we will also examine intracellular RNAs, since this may enable us to more readily answer some of the questions raised by examination of RNAs made in vitro using purified virions. We will determine the defective moiety in mutants which make aberrant RNAs which is responsibility for the abnormal RNA phenotype. (This is necessary since assignment of mutations by complementation analysis is ambiguous; furthermore, some mutants may have multiple genetic changes, and if revertants are not readily generated (as is the case with some of the mutants we will survey) genetic analysis is not sufficient.) The defective moiety will be identified by methods involving, e.g., protein purification and reconstitution of transcriptase activity using homologous or heterologous systems, one- or two-dimensional gel electrophoresis, partial- or complete-proteolytic digestion, and examination of revertants (if they can be generated). These studies should reveal more of the nature of the transcription process and the role of viral proteins in that process. Since transcription and replication are closely related processes, such experiments may enable us to propose hypotheses about some of the details of the replication process.
