A temperature sensitive VSV identifies L protein residues that affect transcription but not replication

To investigate the polymerase components selectively involved in transcription versus replication of vesicular stomatitis virus (VSV), we sequenced the polymerase gene of a conditionally RNA defective, temperature sensitive VSV: ts(G)114, which has a phenotype upon shift from permissive to non-permissive temperature of shut-down of mRNA transcription and unaffected genome replication. Sequence analysis of the ts(G)114 L gene identified three altered amino acid residues in the L protein. These three changes were specifically engineered individually and in combinations into a functional cDNA clone encoding the VSV genome and tested for association with the temperature sensitive and RNA defective phenotypes in the background of recovered engineered viruses. The data presented in this study show a specific amino acid substitution in domain II of the VSV L protein that significantly affects total RNA synthesis, but when in combination with two additional amino acid substitutions identified in the ts(G)114 L protein, leads to a specific reduction in mRNA transcription, but not replication.