Regulation of viral intermediate gene expression by the vaccinia virus B1 protein kinase

The B1 gene of vaccinia virus encodes a serine/threonine protein kinase tha t is expressed early after infection. Under nonpermissive conditions, tempe rature-sensitive mutants (ts2 and ts25) that map to B1 fail to efficiently replicate viral DNA, Our goal was to extend studies on the function of B1 b y determining if the kinase is required for intermediate or late gene expre ssion, two events that ordinarily depend on viral DNA replication. First, w e established that early viral gene expression occurred at the nonpermissiv e temperature. By using a transfection procedure that circumvents the viral DNA replication requirement, we found that reporter genes regulated by an intermediate promoter were transcribed only under conditions permissive for expression of active BI, To assay late gene expression, the T7 RNA polymer ase gene was inserted into the genome of ts25 to form ts25/T7, A DNA replic ation-independent late gene transcription system was established by cotrans fecting plasmids containing T7 promoter-driven late gene transcription fact ors and a late promoter reporter gene into ts25/T7-infected cells. Late gen es, unlike intermediate genes, were expressed at the nonpermissive temperat ure. Last, we showed that overexpression of B1 stimulated intermediate but inhibited late gene expression in tells infected with wild-type virus.