Despite the medical importance of negative strand RNA viruses, little is known about the essential viral specific mechanisms involved in transcription, replication and viral assembly. The following aspects are being studied using the five cloned genes of the rhabdovirus, vesicular stomatitis virus (VSV): 1. Identification of the multiple functions of the RNA dependent RNA polymerase L through site specific mutagenesis. 2. Localization of the functional domains within the polymerase complex. 3. Identification of the nucleotide sequences required for transcription initiation, polyadenylation, encapsidation, replication, cell killing and viral assembly using a novel, recombinant, defective RNA virus particle. Towards these goals, we have cloned, sequenced and expressed both polymerase proteins L and NS of VSV in eucaryotic cells. Surprisingly, overexpression of functional polymerase protein L in COS cells specifically arrests a wild-type virus infection. Unlike with L, overexpression of the N or NS proteins does not cause virus arrest. These data suggest that transcription of nonsegmented negative strand viruses is autoregulated by the highly conserved gene order and the sequential and attenuated mode of transcription. L overexpression also arrests VSV of a different serotype which sheds light on the potential mechanisms of heterotypic exclusion between serotypes as well as heterotypic autointerference. In order to generate a novel, recombinant, defective VSV particle, we constructed a 160 bp """"""""mini VSV genome"""""""" from eight overlapping synthetic oligonucleotides. We are planning to transcribe an RNA from this construct with precise VSV terminal sequences, and to encapsidate and to propagate it with the help of parental virus. It is anticipated that all of these studies will identify essential, viral specific mechanisms which can potentially be the target for the treatment or prevention of viral infections of the CNS.
