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 baculovirusBombyx morinuclear polyhedrosis virus (BmNPV) identified 18 homologues of theAutographa californicaNPV (AcNPV)lefs (late expression factor genes). These BmNPVlefs showed high (73–98%) amino acid sequence identities to AcNPVlefs and were localized to similar positions in the genome. Onelef, p35,was previously characterized in AcNPV and BmNPV deletion experiments. Functional deletion of each of the BmNPVlefhomologues was attempted here by insertion of a β-galactosidase gene cassette into the coding region of eachlef.Four of 18 BmNPVlef(39K, ie-2, lef-7,andp35) deletion mutants were successfully isolated, indicating that the other 14 BmNPVlefs were likely essential for viral replication in cell culture. Further analysis showed that deletion oflef-7, p35,andie-2resulted in lower levels of viral DNA replication, indicating that the BmNPVlef-7, p35,andie-2products have stimulating effects on DNA replication. Deletion of39Kresulted in a significantly lower level of late gene transcription and extremely low (over 102-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 BmNPV39Kis 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 lacking39Kand virion production was low. Even though39Kdeletion resulted in a loss of oral infectivity, the39Kdeletion mutant replicated in silkworm larvae when injected into the body cavity, as did theie-2, lef7,andp35deletion 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 BmNPVvlf-1homologue at a step before the onset of very late gene expression.