We will focus on three areas of poxvirus molecular biology: (1) the mechanism by which these viruses selectively mobilize nuclear components to the cytoplasm and whether proteins to be mobilized are modified; (2) the enzymology of late viral gene transcription and the role in this process of host cell RNA polymerase II (Pol II); and (3) the identification and elimination of viral genes associated with various pathogenic properties of the virus. The sensitivity of poxvirus infections to alpha-amanitin and the failure of the virus to grow in enucleated cells suggest that cellular Pol II is required for virus production. We have shown that poxviruses induce translocation of the largest subunit of Pol II to the cytoplasm. A study of the mechanism by which poxvirus recruits nuclear components necessitates an assay for the ability of viral components to induce translocation. We hope to achieve this by: (1) microinjection into frog oocytes or mammalian cells; (2) fusion with yeast spheroplasts; or (3) the addition of viral components directly to isolated nuclei. To investigate the enzymology and the role of Pol II in late viral gene transcription, we will continue to prepare or obtain monoclonal antibodies to Pol II and the viral RNA polymerase. The antibodies, coupled with enzyme assays will allow us to follow the fates of viral and host RNA polymerase subunits during the later phases of infection to gain insight about the composition and function of the late transcription complex. Ultimately, we wish to purify a complex capable of late promoter recognition in vitro. Another major goal is to study the molecular basis of pathogenesis of poxviruses. Identification of genes associated with aspects of poxvirus pathogenesis is timely because of the interest in recombinant vaccinia viruses as vaccine vectors. We describe an avirulent virus deletion mutant which grows only locally during scarification and is incapable of growth within the CNS or viscera of animals. We will engineer this mutant to contain a unique site for reinsertion of the deleted regions and thereby identify the genes which permit the virus to grow within the CNS and viscera. We will try to correlate increases in pathogenesis with changes in host range in culture which might serve as a simple index of potential vaccine complications and side effects.
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