Mutations in gH at the interface with the gp42 HP affect membrane fusion.

<p>Cell surface expression and fusion assay results with B cells with gH mutants in the R488, S507 and D511 regions. Cell surface expression was measured using the anti-gHgL monoclonal antibody E1D1. (A) The gH D-II and D-III sequence observed proximal to the gp42 HP from our EM model has been highlighted to show the sequence and secondary structure indicating the mutual positions of the mutations in gH that would validate the EM model. The residue mutated to Asn (N) that gets potentially glycosylated is shown in bold text, the NX[S/T] motif is underlined. Cystine bridge between C278–C335 is highlighted; EBV gH D-II and D-III mutants to validate the position of residues in gH close to HP as revealed in our EM model are studied in the following panels (B) and (C); (B) gH G276N in the background of the disulfide bond mutant C278A/C335A does not have the glycosylation motif NX[S/T] and exhibits near wildtype fusion levels with B cells. By contrast, the G276N/C278S that introduces the glycosylation motif at 276 reduces fusion levels to lower than 40%. (C) The R488A mutation does not have a significant effect on gHgL expression or B cell fusion, while the R488N/K90T mutation reduces expression and membrane fusion. The D511N/F513S mutant and its control (F513S) show drastically reduced surface expression and B cell fusion. Both gH507N/A509S and the control gH A509S are expressed near wildtype levels and the gH S507N glycosylation mutant shows a reduction in membrane fusion activity. The mutants where the surface expression is as good as the gp42 wildtype but the fusion levels are down (below ∼40% or less) are highlighted in the rounded rectangular box. Mutants that result in potential glycosylation due to the introduced residue change are shown with a check mark.