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

The pGS3217 and pQF297 BACs were transfected into Vero-Cre cells, and virus was harvested from the cells. Vero cells were infected with samples from these transfections are located on the same side of the molecule as the C-terminal region (red), giving a postfusion hairpin conformation. The domain V arm (red) packs against the coiled-coil core of domain III (yellow) in an antiparallel orientation. A closeup of the coil-arm region is shown, with the three residues that are mutated in gB 3A shown as red spheres. (B) HSV-1 genome with internal repeats (IR) and terminal repeats (TR) shown as white rectangles. BamHI sites are indicated by lollipops. A region of the genome is expanded to show the UL27 open reading frame (gB opening reading frame) and the neighboring UL26 and UL28 open reading frames (arrowheads indicate gene orientation). (C) HSV-1 genome with UL27 (gB) deletion. Dotted lines indicate the UL27 open reading frame deletion in BAC pQF282. (D) gB 3A viruses were made by recombining the gB open reading frame from plasmid pSG5-HSVgB-I671A/H681A/F683A into the gB-null BAC to create BAC pQF297. (E, F) Ethidium bromide-stained agarose gels of BamHI-digested BAC DNA. Digestion of the parental, intermediate, and final BACs generated to create gB-null BAC pQF282 (E) and gB 3A BAC pQF297 (F) are shown. The Kan r cassette is~1.0 kb and contains one BamHI site. Removal of the Kan r cassette changes the BamHI digestion pattern, as highlighted by the arrows. Samples shown together were run on a single gel, but the lanes were rearranged for clarity. (G) Incorporation of gB into virions. Cells were infected with WT (G3217) or gB 3A viruses and cell lysates or supernatants (sup.) were harvested. Supernatants were pelleted through 10% sucrose. As shown at the top, lysates were separated by SDS-PAGE, blotted, and probed for HSV-1 gB. As shown at the bottom, duplicate supernatant samples were separated by SDS-PAGE, blotted, and probed for VP5 or gB. Samples shown next to one another were run on a single gel, but the lanes were rearranged for clarity. The values to the left of panels E and F identify the kilobase pairs of the DNA ladder.