AnAutographa californicaNucleopolyhedroviruslef-2 Mutant: Consequences for DNA Replication and Very Late Gene Expression

Abstract

In order to define factors involved in very lateAutographa californicanucleopolyhedrovirus (AcMNPV) gene function, random mutagenesis of a baculovirus recombinant (AcUW1.lacZ) by 5′-bromodeoxyuridine treatment was performed. Five viruses were selected with deficiencies in very late gene expression. These were characterized by complementation analysis. One mutant virus, VLD1, was found to be completely deficient in very late gene function. This virus could be complemented by a helper virus to express the very late genes, suggesting that the mutant virus was defective in an activator of very late gene expression. Further studies revealed that the replication of VLD1 was temporally delayed when compared to wild-type virus. The mutation in VLD1 was mapped to a subfragment of theEcoRI-I region of the AcMNPV genome between 0 and 5 map units. Sequence analysis revealed the presence of point mutations in ORF2 and inlef-2. Further mapping experiments demonstrated that only replacement of the point mutation inlef-2 with a wild-type sequence could restore VLD1 to a normal phenotype. Previous studies have suggested that thelef-2 gene product is involved in DNA replication. This was investigated by comparison of DNA replication in wild-type- and VLD1-infected cells. It was found that the mutation in thelef-2 gene of VLD1 did not have an effect on DNA replication. It is proposed thatlef-2 may play a dual role, both in DNA replication and very late gene expression.