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.

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.

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.

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13Herpes simplex viruses (HSVs) cause significant morbidity and mortality in humans worldwide. 14 Herpesviruses mediate entry by a multi-component, virus-encoded machinery. Herpesviruses 15 enter cells by endosomal low pH and pH-neutral mechanisms in a cell-specific manner. HSV 16 mediates cell entry via envelope glycoproteins gB, gD, and the heterodimer gH/gL regardless of 17 pH or endocytosis requirements. HSV envelope proteins that function selectively in a given 18 entry pathway have been elusive. Here we demonstrate that gC regulates cell entry and infection 19 by a low pH pathway. Conformational changes in the core herpesviral fusogen gB are critical for 20 membrane fusion. The presence of gC conferred a higher pH threshold to acid-induced antigenic 21 changes in gB. Thus, gC may selectively facilitate low pH entry by regulating conformational 22 changes in the fusion protein gB. We propose that gC modulates the HSV fusion machinery 23 during entry into pathophysiologically relevant cells, such as human epidermal keratinocytes. 24 Keywords: herpesviruses, herpes simplex virus, viral entry, viral glycoproteins 25 26 Importance 27Herpesviruses are ubiquitous pathogens that cause lifelong latent infections and are characterized 28 by multiple entry pathways. We propose that herpes simplex virus (HSV) gC plays a selective 29 role in modulating HSV entry by a low pH pathway, such as into epithelial cells. gC facilitates 30 conformational change of the main fusogen gB, a class III fusion protein. We propose a model 31 whereby gC functions with gB, gD, and gH/gL to allow low pH entry. In the absence of gC, 32HSV entry occurs at a lower pH, coincident with trafficking to a lower pH compartment where 33 gB changes occur at more acidic pHs. This study identifies a new function for gC and provides 34 novel insight into the complex mechanism of HSV entry and fusion. 35 Glycoprotein B is conserved among herpesviruses and is a member of the class III fusion 50 protein family. Unlike other class III fusion proteins such as VSV G and baculovirus gp64, 51 herpesviral gB alone is not sufficient for fusion and requires additional viral proteins, most 52 commonly gH/gL (12). Activation and regulation of the fusion function of gB are incompletely 53 understood. The gH/gL complex is thought to positively regulate gB (13-15). HSV-1 gB 54 undergoes conformational changes during fusion and entry (16, 17). Low pH specifically induces 55 reversible changes in gB domains I and V, which comprise a functional region containing 56 hydrophobic fusion loops (16). Acid-triggered changes in specific gB epitopes correlate with 57 fusion activity: (i) HSV particles entering by endocytosis have reduced reactivity with gB 58 domain I antibody H126, and elevation of endosomal pH blocks this change (16), (ii) irreversible 59 acid-triggered changes in the H126 epitope coincide with irreversible acid-inactivation of HSV 60 fusion and entry (18), and (iii) a hyperfusogenic form of gB has reduced reactivity with domain I 61 and domain V antibodies, si...