Structure-Based Mutations in the Herpes Simplex Virus 1 Glycoprotein B Ectodomain Arm Impart a Slow-Entry Phenotype

Fan, Qing; Kopp, Sarah J.; Connolly, Sarah A.; Longnecker, Richard

To further investigate the role of the gB arm region in virus entry, we generated and characterized HSV-1 strains carrying the gB 3A mutations. Addition of the gB 3A mutations to the viral genome permitted analysis of multistep growth curves and plaque sizes, data that cannot be collected by using a virus that is complemented phenotypically. Our results showed that a gB 3A mutant virus had impaired growth and significantly delayed penetration of cells. We found that elevated temperatures could partially rescue the penetration of cells by gB 3A virus. The ability of heat to overcome the penetration defect is consistent with the hypothesis that the gB 3A mutations favor the prefusion conformation. The mutations did not impact virus binding to cells, and increasing the expression of gH/gL did not compensate for the gB 3A fusion defect in a cell-cell fusion assay. pQF282 ( Fig. 1B and C) . The gB 3A-encoding gene was then recombined into BAC pQF282 to generate BAC pQF297 (Fig. 1D) . At each step of these constructions, intermediate BACs (with kanamycin resistance [Kan r ]-encoding gene insertions) and final BACs (with the Kan r -encoding gene insertions removed) were confirmed by at least four restriction enzyme digestions. For example, BamHI digestions of the BACs show the expected band shifts for pQF282 and pQF297 ( Fig. 1E and F) . Band 1 is present only in the wild-type (WT) BAC, and band 2 is present only in the intermediate BAC with Kan r -encoding gene insertions but not in the BAC with UL27 deleted or the WT BAC (Fig. 1E) . Similarly, band 3 is present only in the intermediate BAC with Kan r -encoding gene insertions, band 4 is present only in the final gB 3A BAC, and band 5 (consisting of two bands that migrate as one because of the similarity of their molecular weights) is present only in the WT BAC and the BAC with UL27 deleted (Fig. 1F) .