Deletion Analysis of Four of Eighteen Late Gene Expression Factor Gene Homologues of the Baculovirus, BmNPV

Abstract Nucleotide sequence analysis of the genome of the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) identified 18 homologues of the Autographa californica NPV (AcNPV) lef s (late expression factor genes). These BmNPV lef s showed high (73–98%) amino acid sequence identities to AcNPV lef s and were localized to similar positions in the genome. One lef, p35, was previously characterized in AcNPV and BmNPV deletion experiments. Functional deletion of each of the BmNPV lef homologues was attempted here by insertion of a β-galactosidase gene cassette into the coding region of each lef. Four of 18 BmNPV lef ( 39K, ie-2, lef-7, and p35 ) deletion mutants were successfully isolated, indicating that the other 14 BmNPV lef s were likely essential for viral replication in cell culture. Further analysis showed that deletion of lef-7, p35, and ie-2 resulted in lower levels of viral DNA replication, indicating that the BmNPV lef-7, p35, and ie-2 products have stimulating effects on DNA replication. Deletion of 39K resulted in a significantly lower level of late gene transcription and extremely low (over 10 2 -fold less at 48–80 hr p.i.) production of progeny budded virus in BmN cells. In contrast, the deletion did not affect viral DNA replication, indicating that BmNPV 39K is involved in late gene transcription. Reduced late gene expression presumably affected production and/or release of progeny budded virus particles. This was corroborated by transmission electron microscopy, which showed that virus replication was abnormal in BmN cells infected with a BmNPV mutant lacking 39K and virion production was low. Even though 39K deletion resulted in a loss of oral infectivity, the 39K deletion mutant replicated in silkworm larvae when injected into the body cavity, as did the ie-2, lef7, and p35 deletion mutants. In addition, a BmNPV homologue of the baculovirus very late expression factor gene ( vlf-1 ) found in AcNPV was essential, implying an essential function of the BmNPV vlf-1 homologue at a step before the onset of very late gene expression.