Differential Effects on Cell Fusion Activity of Mutations in Herpes Simplex Virus 1 Glycoprotein B (gB) Dependent on Whether a gD Receptor or a gB Receptor Is Overexpressed

Abstract

Glycoprotein B (gB) of herpes simplex virus (HSV) is one of four glycoproteins essential for viral entry and cell fusion. Recently, paired immunoglobulin-like type 2 receptor (PILR␣) was identified as a receptor for HSV type 1 (HSV-1) gB. Both PILR␣ and a gD receptor were shown to participate in HSV-1 entry into certain cell types. The purpose of this study was to determine whether insertional mutations in gB had differential effects on its function with PILR␣ and the gD receptor, nectin-1. Previously described gB mutants and additional newly characterized mutants were used in this study. We found that insertional mutations near the N terminus and C terminus of gB and especially in the central region of the ectodomain reduced cell fusion activity when PILR␣ was overexpressed much more than when nectin-1 was overexpressed. Most of the insertions reduced the binding of gB to PILR␣, for at least some forms of gB, but this reduction did not necessarily correlate with the selective reduction in cell fusion activity with PILR␣. These results suggest that the regions targeted by the relevant mutations are critical for functional activity with PILR␣. They also suggest that, although both the binding of gB to a gB receptor and the binding of gD to a gD receptor may be required for HSV-induced cell fusion, the two receptor-binding activities may have unequal weights in triggering fusogenic activity, depending on the ratios of gB and gD receptors or other factors.

The Ig-Like V-Type Domain of Paired Ig-Like Type 2 Receptor Alpha Is Critical for Herpes Simplex Virus Type 1-Mediated Membrane Fusion

“…Previous results have shown that PILR␣ binds to gB and CD99 (3,26,40). The binding of either PILR␣ or PILR␤ to CD99 depends on the presence of sialyated O-linked glycans on CD99 (41).…”

“…Binding of PILR␣, PILR␤, the PILR␣ Ig-like V-type domains, and tryptophan mutants to gB. Previous studies have shown that when the PILR␣ ectodomain is fused to IgG Fc, the resulting fusion protein (PILR␣-Ig) can bind to gB on cell surfaces (3,26) and to its natural ligand, CD99 (41). To assess whether wild-type PILR␤ or mutant PILR␣ could bind to gB expressed on the cell surface of CHO-K1 cells, we made several chimeric constructs.…”

“…Plates of 293 PEAK Rapid cells were transfected with PILR-Ig plasmids using 293 Fectin (Invitrogen). The concentration of PILR␣-Ig in the culture supernatant was measured using a human IgG ELISA kit (Immunology Consultants Laboratory, Inc.) (3). The supernatants of PILR␤-Ig, PILR␣ V-Ig, PILR␣ W-Ig, and PILR␣ W-V-Ig were normalized according to concentration of PILR␣-Ig determined by Western blotting.…”

“…Supernatants from media of pME18S PILR␣-Ig-, PILR␣ V-Ig (pQF58)-, PILR␣ W-Ig (pQF57)-, PILR␣ W-V-Ig (pQF61)-, and PILR␤-Ig (pQF59)-transfected cells were prepared as described by Fan et al (3). Plates of 293 PEAK Rapid cells were transfected with PILR-Ig plasmids using 293 Fectin (Invitrogen).…”

Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

“…Reminiscent of mCD99/PILR␣ interactions, PILR␣ recognition of gB also requires two sialylated O-glycans on gB (17). PILR␣ associates with HSV-1 gB but not with other HSV-1 glycoproteins, despite the presence of O-glycosylation sites on these envelope proteins (18). PILR␣ and PILR␤ proteins do not strongly bind carbohydrate molecules in glycan microarrays (12).…”