Low pH-induced conformational change in herpes simplex virus glycoprotein B

Abstract

Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.

FIG. 1.

Antibody reactivity of low pH-treated virions. Extracellular HSV-1 KOS virions (10⁵ PFU) were treated for 10 min at 37°C with medium buffered to the indicated pHs and were blotted immediately to a nitrocellulose membrane. Blots were probed at neutral pH with the indicated gB-specific antibodies, followed by horseradish peroxidase-conjugated goat secondary antibody. The exposure shown for MAb H126 highlights the pH threshold of conformational change.

FIG. 2.

Effect of low-pH treatment on the oligomeric state of gB. (A, top panel) Virions were solubilized with 1% Triton X-100 and subjected to sedimentation through sucrose (8 to 60%) buffered to pH 7.4 or 5.1. gB was immunoprecipitated from each collected fraction with MAb H1817 prior to SDS-PAGE and immunoblotting with MAb H1359 for detection of gB. In parallel experiments, protein standards were employed to approximate the range of molecular weights of proteins in each fraction. (A, bottom panel) Results were quantitated by densitometry. (B) Binding of gB oligomer-specific monoclonal antibody DL16 to low pH-treated virions. As described in the legend to panel A, HSV-1 KOS virions were treated with pH 7.2 or 5.5, and then 2-fold dilutions were blotted to a membrane. (C) Virions were treated with the indicated pH, solubilized with 1% SDS where indicated, and then analyzed by PAGE and immunoblotting for detection of gB. (D) HSV-1 KOS virions were treated at pH 7.4 or 5.1, solubilized with 1% SDS where indicated, and then analyzed by native PAGE and immunoblotting with polyclonal antibodies specific for gB, gC, gD, or gH-gL. Glycoprotein-specific bands are indicated with the name of the protein. Nonspecific bands that were detected in mock-infected, Vero cell-conditioned medium (not shown) are indicated by asterisks. Protein molecular weight standards are indicated on the left. α, anti.

FIG. 3.

Reversibility of pH-induced conformational changes in gB. Extracellular HSV-1 KOS virions were treated with medium buffered to pH 7.2 or 5.5. For the indicated samples, pH was neutralized back to 7.2 for 10 min at 37°C. Twofold dilutions were blotted immediately to nitrocellulose membranes. Membranes were probed at neutral pH with antibody H126, DL16, or R69, followed by the appropriate horseradish peroxidase-conjugated secondary antibody. The exposures shown document the reversibility of reactivity.

FIG. 4.

Effect of low-pH treatment on the conformation of purified gB. (A) HSV-1 KOS virions (10⁵ PFU) or s-gB (150 ng) in serum-free medium with 0.2% BSA was kept at pH 7.2 or adjusted to pH 5.5 for 10 min at 37°C with 0.05 N HCl. The pH of acidified samples was reneutralized to 7.2 with 0.05 N NaOH for 10 min at 37°C. Samples were analyzed by native PAGE and immunoblotting with gB-specific PAb R69 or MAb DL16. (B) Soluble gB derived from HSV-2 strain 333 (s-gB) was treated with pH 7.4 or 5.1 and then solubilized with 1% SDS either before or after neutralization of pH (as described in the legend to Fig. 2C). HSV-2 strain 333 virions were also treated at pH 7.4 or 5.1 and solubilized with 1% SDS. (C) s-gB was treated with a range of pH, as indicated, and then solubilized with 1% SDS. Samples were analyzed by PAGE and immunoblotting, with R69 for detection of gB.

FIG. 5.

Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 × g for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.

FIG. 6.

Effect of bafilomycin A1 on the conformation of HSV gB during viral entry. CHO-nectin-1 cells were mock treated (left) or treated with 25 nM bafilomycin A1 (right, +BFLA) for 15 min. HSV-1 KOS (MOI of 20) was bound to cells at 4°C for 1 h. Cultures were shifted to 37°C for 1 h in the constant presence of BFLA and 0.5 mM cycloheximide. Virion gB was visualized with MAb H126 or MAb H1817 followed by Alexa 488-labeled goat anti-mouse antibody. Nuclei were detected with DAPI (4′,6-diamidino-2-phenylindole) (not shown). Samples were visualized by confocal microscopy at 63× magnification. A total of 50 to 70 cells are shown per panel.