Conformational Changes in Herpes Simplex Virus Glycoprotein C.

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.

Herpes Simplex Virus Glycoprotein C Regulates Low-pH Entry.

Herpes simplex viruses (HSVs) cause significant morbidity and mortality in humans worldwide. Herpesviruses mediate entry by a multicomponent virus-encoded machinery. Herpesviruses enter cells by endosomal low-pH and pH-neutral mechanisms in a cell-specific manner. HSV mediates cell entry via the envelope glycoproteins gB and gD and the heterodimer gH/gL regardless of pH or endocytosis requirements. Specifics concerning HSV envelope proteins that function selectively in a given entry pathway have been elusive. Here, we demonstrate that gC regulates cell entry and infection by a low-pH pathway. Conformational changes in the core herpesviral fusogen gB are critical for membrane fusion. The presence of gC conferred a higher pH threshold for acid-induced antigenic changes in gB. Thus, gC may selectively facilitate low-pH entry by regulating conformational changes in the fusion protein gB. We propose that gC modulates the HSV fusion machinery during entry into pathophysiologically relevant cells, such as human epidermal keratinocytes.IMPORTANCE Herpesviruses are ubiquitous pathogens that cause lifelong latent infections and that are characterized by multiple entry pathways. We propose that herpes simplex virus (HSV) gC plays a selective role in modulating HSV entry, such as entry into epithelial cells, by a low-pH pathway. gC facilitates a conformational change of the main fusogen gB, a class III fusion protein. We propose a model whereby gC functions with gB, gD, and gH/gL to allow low-pH entry. In the absence of gC, HSV entry occurs at a lower pH, coincident with trafficking to a lower pH compartment where gB changes occur at more acidic pHs. This report identifies a new function for gC and provides novel insight into the complex mechanism of HSV entry and fusion.

Glycoprotein C of Herpes Simplex Virus 1 Shields Glycoprotein B from Antibody Neutralization.

Viruses have evolved strategies to avoid neutralization by the host antibody response. Herpes simplex virus (HSV) glycoprotein C (gC) functions in viral entry and binds to complement component C3b, inhibiting complement-mediated immunity. We investigated whether gC protects HSV from antibody neutralization. HSV-1 that lacks gC was more sensitive to complement-independent neutralization by a panel of gB monoclonal antibodies than a wild-type gC rescuant virus. The presence of gC decreased neutralization by 2- to 16-fold. The gB in the native envelope of HSV-1 had reduced reactivity with antibodies in comparison to gB from the gC-null virus, suggesting that gC hampers the recognition of gB epitopes in the viral particle. The protein composition of the gC-null virus, including the surface glycoproteins essential for entry, was equivalent to that of the wild type, suggesting that gC is directly responsible for the reduced antibody recognition and neutralization. The neutralizing activity of antibodies to gD and gH antibodies was also increased in HSV lacking gC. Together, the data suggest that HSV-1 gC protects the viral envelope glycoproteins essential for entry, including gB, by shielding them from neutralization as a potential mechanism of immune evasion.IMPORTANCE HSV-1 causes lifelong infection in the human population and can be fatal in neonatal and immunocompromised individuals. There is no vaccine or cure, in part due to the ability of HSV to escape the host immune response by various mechanisms. The HSV particle contains at least 15 envelope proteins, four of which are required for entry and replication. This work suggests a novel role for gC in shielding the HSV entry glycoproteins. gC may function to help HSV escape neutralization by antibodies.

Acidic pH Mediates Changes in Antigenic and Oligomeric Conformation of Herpes Simplex Virus gB and Is a Determinant of Cell-Specific Entry.

Herpes simplex virus (HSV) is an important human pathogen with a high worldwide seroprevalence. HSV enters epithelial cells, the primary site of infection, by a low-pH pathway. HSV glycoprotein B (gB) undergoes low pH-induced conformational changes, which are thought to drive membrane fusion. When neutralized back to physiological pH, these changes become reversible. Here, HSV-infected cells were subjected to short pulses of radiolabeling, followed by immunoprecipitation with a panel of gB monoclonal antibodies (MAbs), demonstrating that gB folds and oligomerizes rapidly and cotranslationally in the endoplasmic reticulum. Full-length gB from transfected cells underwent low-pH-triggered changes in oligomeric conformation in the absence of other viral proteins. MAbs to gB neutralized HSV entry into cells regardless of the pH dependence of the entry pathway, suggesting a conservation of gB function in distinct fusion mechanisms. The combination of heat and acidic pH triggered irreversible changes in the antigenic conformation of the gB fusion domain, while changes in the gB oligomer remained reversible. An elevated temperature alone was not sufficient to induce gB conformational change. Together, these results shed light on the conformation and function of the HSV-1 gB oligomer, which serves as part of the core fusion machinery during viral entry.IMPORTANCE Herpes simplex virus (HSV) causes infection of the mouth, skin, eyes, and genitals and establishes lifelong latency in humans. gB is conserved among all herpesviruses. HSV gB undergoes reversible conformational changes following exposure to acidic pH which are thought to mediate fusion and entry into epithelial cells. Here, we identified cotranslational folding and oligomerization of newly synthesized gB. A panel of antibodies to gB blocked both low-pH and pH-neutral entry of HSV, suggesting conserved conformational changes in gB regardless of cell entry route. Changes in HSV gB conformation were not triggered by increased temperature alone, in contrast to results with EBV gB. Acid pH-induced changes in the oligomeric conformation of gB are related but distinct from pH-triggered changes in gB antigenic conformation. These results highlight critical aspects of the class III fusion protein, gB, and inform strategies to block HSV infection at the level of fusion and entry.

Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene.

Herpes simplex virus 1 (HSV-1) ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture.

Contributions of herpes simplex virus 1 envelope proteins to entry by endocytosis.

Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the required HSV glycoproteins, gB, gD, and gH-gL, may be sufficient for entry regardless of entry route taken. This may be distinct from entry mechanisms employed by other human herpesviruses.