CONTROL OF 2-5A PATHWAY BY TS MUTANT OF VACCINIA

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RNA turnover is central to the regulation of gene expression; however, the mechanisms involved are not well understood. The temperature sensitive mutant of vaccinia virus, ts22, provides a genetic tool for elucidating mechanism(s) involved in RNA turnover. The ts22 gene regulates the stability of RNA in vaccinia virus infected cells. ts22 virus has an abortive late phenotype. At the non-permissive temperature, ts22 infection proceeds normally in the early stages; however, at 8-10 h post infection, RNA is degraded and virus infection aborted (independent of exogenous interferon treatment). Our results indicate that the degradation of RNA is due to the activation of 2-5A dependent RNase. 2-5A synthetase-RNase pathway, initially discovered as one of the antiviral mechanisms of interferon, has been implicated in controlling RNA turnover during cell growth, hormone status and differentiation. The ability to modulate the 2- 5A pathway would be of value in assessing its role in RNA degradation. We hypothesize that the functional ts22 gene product inactivates the 2-5A system during productive infection. Inactivation of the 2-5A pathway by expression of the functional ts22 gene product, independent of virus infection, should help elucidate the role of 2-5A pathway in cellular RNA turnover. We propose to study the modulation of 2-5A pathway by ts22 gene product. This will be accomplished by determining the growth of ts22 vaccinia virus and rRNA cleavage (an indicator of activation of the 2-5A system) at the non-permissive temperature, in cells in which the 2-5A pathway has been rendered inoperative. Constitutive expression of the functional ts22 gene product will be tested for its ability to inactivate the 2-5A system. The step(s) at which the ts22 gene product inactivates the 2-5A system will be determined. Further use of vectors expressing the wild type ts22 gene or 2-5A synthetase antisense RNA should permit the assessment of the role of the 2-5A system in controlling RNA degradation during cell growth inhibition and we will determine effect of these vectors on the interferon or glucocorticoid induced growth inhibition and reduction in c-myc expression in Daudi cells.