3 BACULOVIRUS GENES INVOLVED IN LATE AND VERY LATE GENE-EXPRESSION - IE-1, IE-N, AND LEF-2

We developed a method to identify baculovirus genes required for late and very late gene expression that is based on subtraction of clones from an Autographa californica nuclear polyhedrosis virus genomic library which is able to trans activate promoters of reporter plasmids in transient expression assays. Using this assay, we found that three genes located between 83.7 and 7.5 map units of the Autographa californica nuclear polyhedrosis virus genome were involved in expression from the late capsid protein (vp39) and very late polyhedrin (polh) gene promoters. Two of these genes, ie-1 and ie-n, trans regulate early genes in transient expression assays. Although ie-1 was necessary and sufficient for expression from the early promoter in our assay, it was necessary but not sufficient for expression from the vp39 and polh promoters. The presence of ie-n increased expression from the early, late, and very late classes of promoters tested. However, a third gene identified in this region was specifically required for expression from the vp39 and polh promoters. This gene, a previously sequenced 630-nucleotide open reading frame, was renamed lef-2 for late expression factor 2. We also found that other genes in the region between 83.7 and 7.5 map units were not required for expression from the promoters used in this assay, although we did not eliminate the possibility that they subtly modify expression. These genes include pe-38 and me53, early genes with zinc finger-like motifs, and the upstream exon of ie-0, which specifies an alternate form of IE-1.