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.

Low-pH Endocytic Entry of the Porcine Alphaherpesvirus Pseudorabies Virus.

The alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, a disease of great economic and welfare importance in swine. Other alphaherpesviruses, including herpes simplex virus (HSV), utilize low-pH-mediated endocytosis to enter a subset of cell types. We investigated whether PRV used this entry pathway in multiple laboratory model cell lines. Inhibition of receptor-mediated endocytosis by treatment with hypertonic medium prevented PRV entry. PRV entry into several cell lines, including porcine kidney (PK15) cells and African green monkey kidney (Vero) cells, was inhibited by noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of PRV virions to pH 5.0 in the absence of host cell membranes reduced entry into PK15 and Vero cells by >80%. Together, these findings suggest that endocytosis followed by fusion with host membranes triggered by low endosomal pH is an important route of entry for PRV.IMPORTANCE PRV is a pathogen of great economic and animal welfare importance in many parts of the world. PRV causes neurological, respiratory, and reproductive disorders, often resulting in mortality of young and immunocompromised animals. Mortality, decreased production, and trade restrictions result in significant financial losses for the agricultural industry. Understanding the molecular mechanisms utilized by PRV to enter host cells is an important step in identifying novel strategies to prevent infection and spread. A thorough understanding of these mechanisms will contribute to a broader understanding of alphaherpesvirus entry. Here, we demonstrate PRV entry into multiple model cell lines via a low-pH endocytosis pathway. Together, these results provide a framework for elucidating the early events of the PRV replicative cycle.

Pathogenesis of Human Gammaherpesviruses: Recent Advances.

PURPOSE OF THIS REVIEW: Human gammaherpesviruses have complex lifecycles that drive their pathogenesis. KSHV and EBV are the etiological agents of multiple cancers worldwide. There is no FDA-approved vaccine for either KSHV or EBV. This review will describe recent progress in understanding EBV and KSHV lifecycles during infection. RECENT FINDINGS: Determining how latency is established, particularly how non-coding RNAs influence latent and lytic infection, is a rapidly growing area of investigation into how gammaherpesviruses successfully persist in the human population. Many factors have been identified as restrictors of reactivation from latency, especially innate immune antagonism. Finally, new host proteins that play a role in lytic replication have been identified. SUMMARY: In this review we discuss recent findings over the last 5 years on both host and viral factors that are involved in EBV and KSHV pathogenesis.

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.

Herpes simplex virus Membrane Fusion.

Herpes simplex virus mediates multiple distinct fusion events during infection. HSV entry is initiated by fusion of the viral envelope with either the limiting membrane of a host cell endocytic compartment or the plasma membrane. In the infected cell during viral assembly, immature, enveloped HSV particles in the perinuclear space fuse with the outer nuclear membrane in a process termed de-envelopment. A cell infected with some strains of HSV with defined mutations spread to neighboring cells by a fusion event called syncytium formation. Two experimental methods, the transient cell-cell fusion approach and fusion from without, are useful surrogate assays of HSV fusion. These five fusion processes are considered in terms of their requirements, mechanism, and regulation. The execution and modulation of these events require distinct yet often overlapping sets of viral proteins and host cell factors. The core machinery of HSV gB, gD, and the heterodimer gH/gL is required for most if not all of the HSV fusion mechanisms.

Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry.

Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions.IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a class III fusion protein, undergoes reversible conformational changes in response to low-pH exposure. Here, we show that low-pH inactivation of HSV is irreversible and due to a defect in virion fusion activity. We identified an irreversible change in the fusion domain of gB following multiple sequential low-pH exposures or following prolonged low-pH treatment. This change appears to be separable from the alteration in gB quaternary structure. Together, the results are consistent with a model by which low pH can have an activating or inactivating effect on HSV depending on the presence of a target membrane.