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Vaccinia virus (VV) is the prototype of orthopoxviruse, which cause severe cyto-pathological effects in their hosts. CHO cells, though non-permissive for VVreplication, are readily killed by VV. To investigate cellular cytotoxicity due to virus infection, a mutant CHO clone, VV5-4, resistant to VV killing was isolated by retroviral insertional mutagenesis. Parental CHO cells die within 2 to 3 days after VV infection whereas VV5-4 cells survive this cytotoxic effect. Further studies demonstrate that in parental CHO cells, the life cycle of VV proceeds through expression of viral early genes, viral DNA replication, and expression of intermediate and late gene families. In contrast, viral DNAreplication appears to be blocked in VV5-4 cells. These results suggest that thegenetic mutation in VV5-4 alters cellular environment after viral infection and allows VV5-4 to survive. Besides VV, VV5-4 also survive shope fibroma virus andmyxoma virus killing but not cowpox virus. The ability of VV5-4 to survive VVcytotoxicity is not due to suppression of apoptosis which occurs to a same extent in both cell lines. To understand the molecular basis that counts for thesurvival phenotype, the locus integrated by retroviral insertion in VV5-4 was isolated. The cDNA, 1-2, was sequenced and revealed no homology with any known genes, suggesting it is a novel gene.CHO cell killing by VV could be mediated through necrosis as well as apoptosis.Apoptosis was thought to be a general mechanism to mediate host restriction during virus infection. We therefore investigated the role of apoptosis pathwayin virus life cycle. A recombinant VV expressing human bcl-2 gene was constructed. Expression of bcl-2 in VV-infected CHO cells suppressed apoptosis as expected. However, suppression of apoptosis is not sufficient to overcome host restriction. In contrast to bcl-2, a viral host range gene, CP77, could both suppress apoptosis and relieve host restriction. We therefore generated various CP77 mutants to investigate its structural and functional relationship.Some CP77 mutants rescue host restriction of VV in CHO cells can also suppressapoptosis. Those results indicate that the ability to suppress apoptosis isnecessary for CP77 to overcome host restriction.
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