The polyhedrin gene of Autographa californica nuclear polyhedrosis virus is maximally expressed very late in infection, after the release of extracellular virus. It is believed that temporal regulation of polyhedrin requires the sequential expression of an immediate early gene(s) to activate delayed early gene expression; a delayed early gene(s) to turn on DNA synthesis and large gene expression; and a late gene(s) to stimulate polyhedrin, a very large gene. I have devised a complementation assay to map the viral factors which control the temporal regulation of polyhedrin expression. A plasmid, polcat, was constructed that contains the bacterial gene chloramphenicol acetyltransferase (CAT) under the control of the polyhedrin promoter. CAT is expressed upon cotransfection of permissive insect cells with polcat and viral DNA, but not with viral DNA which has previously been digested with restriction enzymes. The inability of digested DNA to support polcat expression is apparently due to inactivation of one or more essential genes by restriction digest. The location of these genes can be mapped by complemenation with cloned PstI-restriction fragments. Preliminary results indicated that twelve of the PstI clones complement different digests of viral DNA, indicating that at least twelve genes are involved in the temporal cascade. I intend to map and determine the function of several of these factors in temporal regulation of polyhedrin expression. The ultimate goal of this project is to define a limited set of viral genes which permits high level expression of polyhedrin in insect cells. Although it may not be possible to identify all of the factors during the grant period, this research should increase our understanding of the mechanisms of temporal regulation of baculovirus gene expression.
