FIG. 1.
(A) Schematic representation of the HSV-1(F) US3 fragment expressed by recombinant baculoviruses and the construction of baculoviruses expressing US3.5 proteins. M1, M77, etc., refer to methionine codons in the US3 ORF. In lines 1 to 4 and 12, the filled circles represent in-frame methionine codons, and the arrow in line 2 indicates the location of a PstI restriction endonuclease site upstream of methionine codon 77 of the US3 ORF. Line 1, wild-type HSV-1(F) US3 protein expressed by recombinant baculovirus BC2600. The fragment was originated from pRB5970, which contains the HSV-1(F) US3 ORF encoding a 481-amino-acid protein with six in-frame methionine codons, as shown. The US3 protein expressed by BC2820 has a replacement of Cys-136 by Arg, and that of BC2602 (line 12) contains a C-terminal Flag epitope tag. Lines 2 to 4, US3.5 proteins initiating from Met-77 and expressed by baculoviruses BC2609, BC2608, and BC2607. In BC2609 (line 2), the fragment was derived from full-length US3 sequence (pRB5970) by collapse of the N-terminal portion bounded by EcoRI and PstI sites. In BC2608 (Flag tagged; line 4) and BC2607 (no tag; line 3), the fragments were generated by PCR using HSV-1(F) viral DNA as a template (see Materials and Methods for details). Lines 5 to 11, US3 fragments expressed by baculoviruses with ATG codons upstream of methionine codon 77 (initiation codon ATG-1 and out-of-frame ATG codons ATG-2 and ATG-3) mutated to GGC or GCG (line 5). Mutations are indicated by open circles in lines 5 to 11. The triple-ATG mutation in BC2616 (line 5) and the mutation of the initiation codon (ATG-1) in BC2613, BC2614, and BC2615 (lines 9 to 11) resulted in expression of the US3.5 protein (Fig. 2). (B) Schematic representation of BAD expressed by baculoviruses BC2800 and BC2808. BC2800 encodes wild-type murine BAD. In BC2808, a GST tag was inserted at the amino terminus of BAD and three serine codons (S) at regulatory positions 112, 136, and 155 were mutated to alanine codons (A).