FIG. 1.

Site-directed mutagenesis of the AcMNPV gp64 gene. Two plasmids encoding full-length AcMNPV gp64 were used as the starting materials for mutagenesis of the consensus N-glycosylation sites in the gp64 protein. The BamHI fragment of pBS-BglEΔS was subcloned into M13 and used to mutagenize the consensus sites at positions 160, 198, 355, and 385, while the BamHI-PstI fragment of p64KTV1 was subcloned into M13 and used to mutagenize the consensus site at position 426. The mutagenized fragments then were excised from the M13 vectors and used to replace the corresponding wild-type fragments in p64KTV1 as described in Materials and Methods. Double and triple mutations were produced by using two oligonucleotides to simultaneously mutagenize M13 subclones, by performing a second round of mutagenesis on a single mutant in M13, or by combining different fragments from two mutants after each had been subcloned into p64KTV1. Ultimately, this strategy yielded a large set of transfer plasmids (shown in the two boxes) which was used to isolate a set of recombinant baculoviruses encoding every possible single, double, and triple N-glycosylation site mutation in AcMNPV gp64.