Regulation of Viral Intermediate Gene Expression by the Vaccinia Virus B1 Protein Kinase

Abstract

The B1 gene of vaccinia virus encodes a serine/threonine protein kinase that is expressed early after infection. Under nonpermissive conditions, temperature-sensitive mutants ( ts 2 and ts 25) that map to B1 fail to efficiently replicate viral DNA. Our goal was to extend studies on the function of B1 by determining if the kinase is required for intermediate or late gene expression, two events that ordinarily depend on viral DNA replication. First, we established that early viral gene expression occurred at the nonpermissive 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 B1. To assay late gene expression, the T7 RNA polymerase gene was inserted into the genome of ts 25 to form ts 25/T7. A DNA replication-independent late gene transcription system was established by cotransfecting plasmids containing T7 promoter-driven late gene transcription factors and a late promoter reporter gene into ts 25/T7-infected cells. Late genes, unlike intermediate genes, were expressed at the nonpermissive temperature. Last, we showed that overexpression of B1 stimulated intermediate but inhibited late gene expression in cells infected with wild-type virus.