FIG 3.

The removal of individual N-glycans in HeV G modulates viral entry and oligomerization of G. (A) Viral entry assay. Vero cells were infected with serial dilutions of HeV/VSV-rLuc pseudotyped virions containing either WT or mutant HeV G in combination with or without HeV F (G only). Cells were lysed 20 to 24 h postinfection, and relative light units (RLU) were quantified and plotted against the number of viral genomes/ml. Virions produced with HeV F only or empty vector yielded viral entry levels similar to those of G-only virions (data not shown). Data shown are the average results ± SEM from at least three independent experiments. (B) Mutant HeV G virus incorporation and oligomerization. HeV/VSV-rLuc pseudotyped virions as used in the experiment whose results are shown in panel A were separated by reducing or nonreducing SDS-PAGE and immunoblotted against G (rabbit anti-HA antibody)- and F (mouse anti-AU1 antibody)-specific antibodies. Oligomeric forms of G are indicated. Note that no monomeric form was separated by nonreducing SDS-PAGE when samples were obtained from virions. The percentages of F₁ cleavage were determined by densitometry. The results of one representative experiment out of three independent experiments are shown.