Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene.

We have examined the expression and function of a gene, vlf-1, of Autographa californica nuclear polyhedrosis virus that is known to encode a regulator of very late gene transcription. Western blot analysis revealed that vlf-1 is expressed during the late phase of infection, primarily from 15 to 24 h postinfection. VLF-1 localized in the cell nucleus and was also present in the nucleocapsids of virus particles. Mapping of vlf-1 mRNA by primer extension showed that transcription initiates at a TAAG motif 71 bp upstream of the vlf-1 open reading frame. Disruption of this TAAG motif abolished the ability of vlf-1 to stimulate transcription from the very late polyhedrin gene (polh) promoter in transient expression assays, suggesting that vlf-1 expression is controlled by the TAAG motif. Using a highly efficient system to construct recombinant viruses with modifications in vlf-1, we confirmed that the TAAG motif was essential. Furthermore, efforts to construct null mutants of vlf-1 failed, suggesting that vlf-1 is an essential gene for virus replication. Computer-assisted sequence homology searches place vlf-1 in the lambda phage integrase family (McLachlin and Miller, 1994). None of the strictly conserved residues of this family which are found in vlf-1 could be changed in the viral genome, implying that the putative integrase activity of VLF-1 is associated with the essential function of vlf-1. However, mutation of a crucial active-site tyrosine did not affect the ability of vlf-1 to transactivate the polh promoter in transient expression assays, indicating that the very late transcriptional activity of VLF-1 does not require the integrase activity.