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 Auto grapha californica nuclear polyhedrosis virus that is known to encode a regulator of very late gene transcription. Western blot analysis rev ealed that vlf-1 is expressed during the late phase of infection, prim arily from 15 to 24 h postinfection. VLF-1 localized in the cell nucle us and was also present in the nucleocapsids of virus particles. Mappi ng of vlf-1 mRNA by primer extension showed that transcription initiat es at a TAAG motif 71 bp upstream of the vlf-1 open reading frame. Dis ruption of this TAAG motif abolished the ability of vlf-1 to stimulate transcription from the very late polyhedrin gene (polh) promoter in t ransient expression assays, suggesting that vlf-1 expression is contro lled by the TAAG motif. Using a highly efficient system to construct r ecombinant viruses with modifications in vlf-1, we confirmed that the TAAG motif was essential. Furthermore, efforts to construct null mutan ts of vlf-1 failed, suggesting that vlf-1 is an essential gene for vir us replication. Computer-assisted sequence homology searches place vlf -1 in the h phage integrase family (McLachlin and Miller, 1994). None of the strictly conserved residues of this family which are found in v lf-l 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 tran sient expression assays, indicating that the very late transcriptional activity of VLF-1 does not require the integrase activity, (C) 1998 A cademic Press.