FIG. 2.

Methionine codon 77 is the initiation codon for the U_S3.5 protein. (A, B, and C) Electrophoretic profiles of U_S3 and U_S3.5 proteins in rabbit skin cells infected with HSV-1 viruses or baculoviruses expressing wild-type or mutant U_S3/U_S3.5 proteins. (A and C) Replicate cultures of rabbit skin cells in 25-cm² flasks were either mock infected (lane 1) or infected with 10 PFU of wild-type HSV-1(F) (lane 2). A second set was exposed to either insect cell medium (lane 3) or 10 PFU of BC2600 (expressing wild-type U_S3). The cells were harvested 24 h after infection, rinsed three times with phosphate-buffered saline containing protease inhibitor cocktail (Roche), and then solubilized in 150 μl of disruption buffer (50 mM Tris-HCl, pH 7, 2% sodium dodecyl sulfate, 710 mM β-mercaptoethanol, 3% sucrose). Fifty-microliter portions of lysates were boiled for 5 min, and the solubilized proteins were subjected to electrophoresis in an 11% denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, blocked with 5% nonfat milk, and reacted with polyclonal antibody to U_S3 as described in Materials and Methods. (B) Replicate cultures were either infected with 10 PFU of R7802 (ICP22 deleted; predominately expresses U_S3.5 protein) (lane 5) or transduced with baculoviruses BC2607, BC2608, and BC2609 expressing a U_S3 fragment initiating from methionine codon 77 (lanes 6 to 8). (C) Replicate cultures were transduced with either baculovirus BC2600 expressing wild-type U_S3 protein (lane 4) or baculoviruses (series BC2610 to BC2616) expressing proteins from the full-length U_S3 ORF in which the methionine initiation codon and two out-of-frame methionine codons upstream of methione codon 77 were mutated in all combinations (lanes 5 to 11).