Conformational Changes in Herpes Simplex Virus Glycoprotein C

Abstract

Low endosomal pH facilitates herpesvirus entry in a cell-specific manner. Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. HSV-1 enters cells by low-pH and neutral-pH pathways. Low-pH-induced conformational changes in the HSV envelope glycoprotein B (gB) may mediate membrane fusion during viral entry. HSV-1 gC, a 511-amino acid, type I integral membrane glycoprotein, mediates HSV-1 attachment to host cell surface glycosaminoglycans, but this interaction is not essential for viral entry. We previously demonstrated that gC regulates low-pH viral entry independent of its known role in cell attachment. Low-pH-triggered conformational changes in gB occur at a lower pH when gC is absent, suggesting that gC positively regulates gB conformational changes. Here, we demonstrate that mildly acidic pH triggers conformational changes in gC itself. Low-pH treatment of virions induced antigenic changes in distinct gC epitopes, and those changes were reversible. One of these gC epitopes is recognized by a monoclonal antibody that binds to a linear sequence that includes residues within gC amino acids 33 to 123. This antibody inhibited low-pH entry of HSV, suggesting that its gC N-terminal epitope is particularly important. We propose that gC plays a critical role in HSV entry through a low-pH endocytosis pathway, which is a major entry route in human epithelial cells. IMPORTANCE Herpesviruses are ubiquitous pathogens that cause lifelong latent infections and are characterized by multiple entry pathways. The HSV envelope gC regulates HSV entry by a low-pH entry route. The fusion protein gB undergoes pH-triggered conformational changes that are facilitated by gC. Here, we report that gC itself undergoes a conformational change at low pH. A monoclonal antibody to gC that binds to a region that undergoes pH-induced changes also selectively inhibits HSV low-pH entry, corroborating the importance of gC in the low-pH entry pathway. This study illustrates the complex role of endosomal pH during HSV entry and provides novel insights into the functions of gC.

Keywords: conformational change; endocytosis; endosomal pH; gC; glycoproteins; herpes simplex virus; herpesviruses; membrane fusion; viral entry.

FIG 1

Low-pH-triggered conformational changes in HSV-1 gC. (A) Wild-type HSV-1 KOS was treated at a range of pH values for 10 min at 37°C. Samples were blotted to nitrocellulose membranes and probed with gC MAb 3G9, T96, or H1413 or gB MAb SS10 at neutral pH. Blots were developed by exposure to X-ray film. (B) Reactivity of at least two independent experiments was quantitated, and results are shown as a percentage of those for the corresponding pH 7.4-treated sample. *, P < 0.01, Student's t test. ns, not significant.

FIG 2

Low-pH-triggered conformational changes in HSV-1 gC are partially reversible. (A) Wild-type HSV-1 KOS was treated with a pH of 7.4 or 5.0 for 10 min at 37°C. pH 5.0-treated samples were neutralized to pH 7.4 for 10 min at 37°C. Samples were blotted to nitrocellulose membranes and probed with gC MAbs 3G9, T96, or H1413 or gB MAb H126 at neutral pH. Blots were developed with an Azure Biosystems imager. (B) Reactivity of at least three independent experiments was quantitated, and results are shown as a percentage of those for the corresponding pH 7.4-treated sample. Reversed samples were not significantly different (ns) from pH 7.4-treated samples (Student's t test).

FIG 3

HSV-1 gC is not a target of low-pH inactivation of virion infectivity. (A) HSV-1 ΔgC or gCR virions (100 PFU) were treated with the indicated pH values for 10 min. Samples were neutralized to pH 7.4 and added to Vero cells. At 18 to 24 hpi, infectivity was determined by plaque assay. Infectivity at pH 7.4 was set as 100%. (B) Virions were treated for 3 h at pH 7.4 or 5.0, the pH was neutralized to pH 7.4, and titers were determined. (C) Virions were treated for 10 min with pH 5.0 and then neutralized to pH 7.4 for 10 min (one time [1×]). This treatment was repeated for a total of two times (2×) or four times (4×). Data are the mean of four replicate samples with standard deviation. Results are representative of at least two independent experiments. ns, not significant (Student's t test).

FIG 4

gC-specific MAb inhibits low-pH entry. HSV-1 KOS virions were mixed with increasing concentrations of gC MAbs 3G9 (A) or H1413 (B), gB MAb SS55 (C), or H1359 (D) for 1 h at 37°C. Samples were added to CHO-HVEM or B78-nectin-1 cells. At 6 hpi, viral entry was determined via β-galactosidase reporter assay. No-antibody samples were set to 100%. Means with standard errors for three independent experiments are shown. *, P < 0.01; **, P < 0.05, Student's t test.

FIG 5

Characterization of MAbs to HSV-1 gC. (A) HSV-1 KOS virions were prepared either in modified Laemmli sample buffer containing only 0.2% SDS without reducing agent and, without heating (native [N]), or in Laemmli buffer containing 2% SDS and 200 mM DTT, with heating (denaturing [D]). Samples were resolved by SDS-PAGE, and Western blotting was performed with gC MAbs 3G9, H1413, or T96 or gC polyclonal antibody R47. (B) Epitope mapping of gC MAbs. CHO-K1 cells were transfected with plasmids encoding wild-type (WT) gC or the indicated gC deletion mutants. Cell lysates were resolved by SDS-PAGE, and Western blotting was performed with the indicated antibodies. Molecular weight standards (in kilodaltons) are indicated to the left (A) or right (B).