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Dependence of vesicular stomatitis virus polymerase function on structural flexibility and domain-domain interactions /

  • Author(s): Ruedas, John Barragan
  • et al.
Abstract

Gene expression and replication of nonsegmented negative strand (NNS) RNA viruses is performed by an RNA-dependent RNA polymerase. The polymerase large (L) protein catalyzes all functions including addition of a cap 1 (m⁷GpppNm) structure on nascent transcripts. Alignment studies show L proteins are modular consistent with a multi-domain protein. Reports of L functions being coupled suggest orchestrated interactions among its domains occur however specific domain interactions have not been experimentally confirmed. At the start of this dissertation virtually no structural information was known for NNS L proteins other than an EGFP-tolerant hinge region just upstream of a methyltransferase domain was discovered in the measles L protein. This dissertation shows a homologous hinge region is also present in the NNS prototype vesicular stomatitis virus (VSV) L protein and aims to gain fundamental insight into putative interactions involving domains near this hinge. Insertion of EGFP in VSV L produced a temperature sensitive polymerase in minigenome assays with minimal effects at 33°C but severe attenuation of RNA synthesis at 37°C. This phenotype was also observed in engineered recombinant virus encoding this modified polymerase. Moreover, this virus exhibited an inability to synthesize transcripts in vitro. The specific effect on RNA synthesis led to the proposal here that the insert restricts hinge movement thus impeding critical domain interactions. Four domains adjacent to the hinge were exchanged with VSV serotype counterparts to determine the effect on polymerase function. Exchanging the methyltransferase domain had no impact on L functions in minigenome assays and recombinant virus indicating it enjoyed a degree of autonomy. Exchange of the other domains however abolished RNA synthesis showing polymerase activity depends heavily on peptide sequences in these domains, supporting a model of interaction between these domains and other polymerase components. Combination exchanges of all four domains also yielded polymerase inactivity with one exception. Co- exchange of the methyltransferase and adjacent C-terminal domain partially rescued a defect that occurred by exchanging the C-terminal domain alone. This combination rescued replication activity substantially more than transcription (avg. 67% and 13%, respectively) providing evidence of an essential domain-domain interaction for RNA synthesis that is more favorable for replication

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