Herpes Simplex Virus 1 Glycoprotein B from a Hyperfusogenic Virus Mediates Enhanced Cell-Cell Fusion.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4 °C and also entered cells more efficiently at 15 °C, relative to wild type HSV-1 strain KOS virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type KOS. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type KOS gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the KOS gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.

Membrane fusion activity of herpes simplex virus 1 glycoproteins from a hyperfusogenic virus.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4°C and also entered cells more efficiently at 15°C relative to wild type virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the wild type gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. Wild type gC is proposed to facilitate fusion and entry into epithelial cells by optimizing conformational changes in the fusion protein gB. ANG gC substitution or addition also had no effect on cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.

Host Cell Neddylation Facilitates Alphaherpesvirus Entry in a Virus-Specific and Cell-Dependent Manner.

Herpes simplex virus 1 (HSV-1) commandeers the host cell proteasome at several steps of its replication cycle, including entry. Here we demonstrate that HSV-2, pseudorabies virus (PRV), and bovine herpesvirus 1 (BoHV-1) entry are blocked by bortezomib, a proteasome inhibitor that is an FDA-approved cancer drug. Proteasome-dependent entry of HSV-1 is thought to be ubiquitin-independent. To interrogate further the proteasomal mechanism of entry, we determined the involvement of the ubiquitin-like molecule NEDD8 and the neddylation cascade in alphaherpesvirus entry and infection. MLN4924 is a small-molecule inhibitor of neddylation that binds directly to the NEDD8-activating enzyme. Cell treatment with MLN4924 inhibited plaque formation and infectivity by HSV-1, PRV, and BoHV-1 at noncytotoxic concentrations. Thus, the neddylation pathway is broadly important for alphaherpesvirus infection. However, the neddylation inhibitor had little effect on entry of the veterinary viruses but had a significant inhibitory effect on entry of HSV-1 and HSV-2 into seven different cell types. Washout experiments indicated that MLN4924's effect on viral entry was reversible. A time-of-addition assay suggested that the drug was acting on an early step in the entry process. Small interfering RNA (siRNA) knockdown of NEDD8 significantly inhibited HSV entry. In probing the neddylation-dependent step in entry, we found that MLN4924 dramatically blocked endocytic uptake of HSV from the plasma membrane by >90%. In contrast, the rate of HSV entry into cells that support direct fusion of HSV with the cell surface was unaffected by MLN4924. Interestingly, proteasome activity was less important for the endocytic internalization of HSV from the cell surface. The results suggest that the NEDD8 cascade is critical for the internalization step of HSV entry. IMPORTANCE Alphaherpesviruses are ubiquitous pathogens of humans and veterinary species that cause lifelong latent infections and significant morbidity and mortality. Host cell neddylation is important for cell homeostasis and for the infection of many viruses, including HSV-1, HSV-2, PRV, and BoHV-1. Inhibition of neddylation by a pharmacologic inhibitor or siRNA blocked HSV infection at the entry step. Specifically, the NEDD8 pathway was critically important for HSV-1 internalization from the cell surface by an endocytosis mechanism. The results expand our limited understanding of cellular processes that mediate HSV internalization. To our knowledge, this is the first demonstration of a function for the neddylation cascade in virus entry.

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.

Viral entry and the ubiquitin-proteasome system.

Viruses confiscate cellular components of the ubiquitin-proteasome system (UPS) to facilitate many aspects of the infectious cycle. The 26S proteasome is an ATP-dependent, multisubunit proteolytic machine present in all eukaryotic cells. The proteasome executes the controlled degradation of functional proteins, as well as the hydrolysis of aberrantly folded polypeptides. There is growing evidence for the role of the UPS in viral entry. The UPS assists in several steps of the initiation of infection, including endosomal escape of the entering virion, intracellular transport of incoming nucleocapsids and uncoating of the viral genome. Inhibitors of proteasome activity, including MG132, epoxomicin, lactacystin and bortezomib have been integral to developments in this area. Here, we review the mechanistic details of UPS involvement in the entry process of viruses from a multitude of families. The possibility of proteasome inhibitors as therapeutic antiviral agents is highlighted.

Herpes Simplex Virus Entry by a Nonconventional Endocytic Pathway.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and mortality in humans worldwide. HSV-1 enters epithelial cells via an endocytosis mechanism that is low-pH dependent. However, the precise intracellular pathway has not been identified, including the compartment where fusion occurs. In this study, we utilized a combination of molecular and pharmacological approaches to better characterize HSV entry by endocytosis. HSV-1 entry was unaltered in both cells treated with small interfering RNA (siRNA) to Rab5 or Rab7 and cells expressing dominant negative forms of these GTPases, suggesting entry is independent of the conventional endo-lysosomal network. The fungal metabolite brefeldin A (BFA) and the quinoline compound Golgicide A (GCA) inhibited HSV-1 entry via beta-galactosidase reporter assay and impaired incoming virus transport to the nuclear periphery, suggesting a role for trans-Golgi network (TGN) functions and retrograde transport in HSV entry. Silencing of Rab9 or Rab11 GTPases, which are involved in the retrograde transport pathway, resulted in only a slight reduction in HSV infection. Together, these results suggest that HSV enters host cells by an intracellular route independent of the lysosome-terminal endocytic pathway.IMPORTANCE Herpes simplex virus 1 (HSV-1), the prototype alphaherpesvirus, is ubiquitous in the human population and causes lifelong infection that can be fatal in neonatal and immunocompromised individuals. HSV enters many cell types by endocytosis, including epithelial cells, the site of primary infection in the host. The intracellular itinerary for HSV entry remains unclear. We probed the potential involvement of several Rab GTPases in HSV-1 entry and suggest that endocytic entry of HSV-1 is independent of the canonical lysosome-terminal pathway. A nontraditional endocytic route may be employed, such as one that intersects with the trans-Golgi network (TGN). These results may lead to novel targets for intervention.

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.

Conformational Change in Herpes Simplex Virus Entry Glycoproteins Detected by Dot Blot.

Conformational changes in viral membrane proteins drive membrane fusion, a critical step in virus entry and infection. Here we describe a simple and rapid virus blotting immunoassay to define conformational changes with a panel of monoclonal antibodies to distinct sites across a viral glycoprotein. This dot blot technique has been utilized to define low pH-triggered changes in the prefusion form of the herpesviral fusogen gB. At pH of <6.2 there are specific changes in herpes simplex virus 1 gB domains I and V. This corresponds broadly to host cell endosomal pH. Many of the identified changes are at least partially reversible. This method can be adapted to document changes in viral proteins that are not fusion proteins, including those induced by alternate triggers such as receptor-binding or protease cleavage.

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.

Early Steps in Herpes Simplex Virus Infection Blocked by a Proteasome Inhibitor.

Viruses commandeer host cell 26S proteasome activity to promote viral entry, gene expression, replication, assembly, and egress. Proteasomal degradation activity is critical for herpes simplex virus (HSV) infection. The proteasome inhibitor bortezomib (also known as Velcade and PS-341) is a clinically effective antineoplastic drug that is FDA approved for treatment of hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Low nanomolar concentrations of bortezomib inhibited infection by HSV-1, HSV-2, and acyclovir-resistant strains. Inhibition coincided with minimal cytotoxicity. Bortezomib did not affect attachment of HSV to cells or inactivate the virus directly. Bortezomib acted early in HSV infection by perturbing two distinct proteasome-dependent steps that occur within the initial hours of infection: the transport of incoming viral nucleocapsids to the nucleus and the virus-induced disruption of host nuclear domain 10 (ND10) structures. The combination of bortezomib with acyclovir demonstrated synergistic inhibitory effects on HSV infection. Thus, bortezomib is a novel potential therapeutic for HSV with a defined mechanism of action.IMPORTANCE Viruses usurp host cell functions to advance their replicative agenda. HSV relies on cellular proteasome activity for successful infection. Proteasome inhibitors, such as MG132, block HSV infection at multiple stages of the infectious cycle. Targeting host cell processes for antiviral intervention is an unconventional approach that might limit antiviral resistance. Here we demonstrated that the proteasome inhibitor bortezomib, which is a clinically effective cancer drug, has the in vitro features of a promising anti-HSV therapeutic. Bortezomib inhibited HSV infection during the first hours of infection at nanomolar concentrations that were minimally cytotoxic. The mechanism of bortezomib's inhibition of early HSV infection was to halt nucleocapsid transport to the nucleus and to stabilize the ND10 cellular defense complex. Bortezomib and acyclovir acted synergistically to inhibit HSV infection. Overall, we present evidence for the repurposing of bortezomib as a novel antiherpesviral agent and describe specific mechanisms of action.

Role of Sphingomyelin in Alphaherpesvirus Entry.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes disease in cattle populations worldwide. Sphingomyelin (SM) is the most abundant sphingolipid in the mammalian cell membrane, where it preferentially associates with cholesterol to form lipid raft domains. SM is a substrate for the lysosome-resident enzyme acid sphingomyelinase, which plays a role in cell membrane repair following injury. Treatment of cells with noncytotoxic concentrations of Staphylococcus aureus-derived sphingomyelinase successfully reduced cell surface-exposed sphingomyelin but did not significantly inhibit BoHV-1 entry and infection, as measured by the beta-galactosidase reporter assay. Interestingly, entry of the porcine alphaherpesvirus pseudorabies virus (PRV) was inhibited by sphingomyelin-depletion of cells. Treatment of BoHV-1 particles with sphingomyelinase inhibited viral entry activity, suggesting that viral SM plays a role in BoHV-1 entry, while cellular SM does not. Treatment of cells with noncytotoxic concentrations of the functional inhibitors of host acid sphingomyelinase, imipramine and amitriptyline, which induce degradation of the cellular enzyme, did not significantly inhibit BoHV-1 entry. In contrast, inhibition of cellular acid sphingomyelinase inhibited PRV entry. Entry of the human alphaherpesvirus herpes simplex virus 1 (HSV-1) was independent of both host SM and acid sphingomyelinase, in a manner similar to BoHV-1. Together, the results suggest that among the alphaherpesviruses, there is variability in entry requirements for cellular sphingomyelin and acid sphingomyelinase activity.IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. Available vaccines limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of BoHV-1 are significant. In order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. We recently identified a low pH endocytosis pathway for BoHV-1. Here, we examine the role of cellular factors responsible for membrane integrity and repair in alphaherpesviral entry. This study allows comparisons of the BoHV-1 entry pathway with those of other alphaherpesviruses (pseudorabies virus [PRV] and herpes simplex virus 1 [HSV-1]). Lastly, this is the first report of sphingomyelin and lysosomal sphingomyelinase playing a role in the entry of a herpesvirus. The results may lead to the development of more effective prevention and treatment regimens.

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.

Bovine Herpesvirus 1 Entry by a Low-pH Endosomal Pathway.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that poses a significant challenge to health and welfare in the cattle industry. We investigated the cellular entry route utilized by BoHV-1. We report that BoHV-1 enters Madin Darby bovine kidney (MDBK) cells, bovine turbinate cells, and African green monkey kidney (Vero) cells via a low-pH-mediated endocytosis pathway. Treatment of MDBK cells with hypertonic medium, which inhibits receptor-mediated endocytosis, prevented infection as measured by a beta-galactosidase reporter assay. Treatment of cells with noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes, inhibited BoHV-1 entry in a concentration-dependent fashion. The kinetics of endocytic uptake of BoHV-1 from the cell surface was rapid (50% uptake by ∼5 min). Time-of-addition experiments indicated that the lysosomotropic agents acted at early times postinfection, consistent with entry. Inactivation of virions by pretreatment with mildly acidic pH is a hallmark characteristic of viruses that utilize a low-pH-activated entry pathway. When BoHV-1 particles were exposed to pH 5.0 in the absence of target membrane, infectivity was markedly reduced. Lastly, treatment of cells with the proteasome inhibitor MG132 inhibited BoHV-1 entry in a concentration-dependent manner. Together, these results support a model of BoHV-1 infection in which low endosomal pH is a critical host trigger for fusion of the viral envelope with an endocytic membrane and necessary for successful infection of the target cell.IMPORTANCE BoHV-1 is a ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. While there are vaccines on the market, they only limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of this virus are significant, and in order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. This research establishes the initial entry pathway of BoHV-1, which provides a foundation for future development of effective treatments and preventative vaccines. Additionally, it allows comparisons to the entry pathways of other alphaherpesviruses, such as HSV-1.

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 1 Envelope Cholesterol Facilitates Membrane Fusion.

Methyl beta-cyclodextrin (MßCD) treatment of herpes simplex virus 1 (HSV-1) reduced envelope cholesterol levels and inhibited viral entry and infectivity in several cell types, regardless of the dependence of entry on endocytosis or low pH. Viral protein composition was similar in MßCD-treated and untreated virions, and ultrastructural analysis by electron microscopy revealed that cholesterol removal did not grossly affect virion structure or integrity. Removal of envelope cholesterol greatly reduced virion fusion activity as measured by fusion-from-without, suggesting that virion cholesterol is critical for the step of membrane fusion. MßCD-treatment of HSV-1 did not reduce viral attachment to the cells nor endocytic uptake of HSV-1 from the cell surface. The pre-fusion form of gB present in the HSV-1 envelope undergoes conformational changes in response to mildly acidic pH. These gB changes occurred independently of envelope cholesterol. Removal of cholesterol compromised virion stability as measured by recovery of infectivity following cycles of freeze-thaw. Taken together, the data suggest that HSV-1 envelope cholesterol is important for viral entry and infectivity due to a critical role in membrane fusion.

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.

Cellular Cholesterol Facilitates the Postentry Replication Cycle of Herpes Simplex Virus 1.

Cholesterol is an essential component of cell membranes and is required for herpes simplex virus 1 (HSV-1) entry (1-3). Treatment of HSV-1-infected Vero cells with methyl beta-cyclodextrin from 2 to 9 h postentry reduced plaque numbers. Transport of incoming viral capsids to the nuclear periphery was unaffected by the cholesterol reduction, suggesting that cell cholesterol is important for the HSV-1 replicative cycle at a stage(s) beyond entry, after the arrival of capsids at the nucleus. The synthesis and release of infectious HSV-1 and cell-to-cell spread of infection were all impaired in cholesterol-reduced cells. Propagation of HSV-1 on DHCR24-/- fibroblasts, which lack the desmosterol-to-cholesterol conversion enzyme, resulted in the generation of infectious extracellular virions (HSVdes) that lack cholesterol and likely contain desmosterol. The specific infectivities (PFU per viral genome) of HSVchol and HSVdes were similar, suggesting cholesterol and desmosterol in the HSV envelope support similar levels of infectivity. However, infected DHCR24-/- fibroblasts released ∼1 log less infectious HSVdes and ∼1.5 log fewer particles than release of cholesterol-containing particles (HSVchol) from parental fibroblasts, suggesting that the hydrocarbon tail of cholesterol facilitates viral synthesis. Together, the results suggest multiple roles for cholesterol in the HSV-1 replicative cycle.IMPORTANCE HSV-1 infections are associated with a wide range of clinical manifestations that are of public health importance. Cholesterol is a key player in the complex interaction between viral and cellular factors that allows HSV-1 to enter host cells and establish infection. Previous reports have demonstrated a role for cellular cholesterol in the entry of HSV-1 into target cells. Here, we employed both chemical treatment and cells that were genetically defined to synthesize only desmosterol to demonstrate that cholesterol is important at stages following the initial entry and transport of viral capsids to the nucleus. Viral protein expression, encapsidation of the viral genome, and the release of mature virions were impacted by the reduction of cellular cholesterol. Cholesterol was also critical for cell-to-cell spread of infection. These findings provide new insights into the cholesterol dependence of HSV-1 replication.

Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion.

Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism.IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular factors necessary for virus-cell membrane fusion. We found that OvHV-2 glycoproteins B, H, and L are sufficient for, and viral glycoprotein Ov8 can significantly enhance, cell-cell membrane fusion.

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.

Exploration of bivalent ligands targeting putative mu opioid receptor and chemokine receptor CCR5 dimerization.

Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR-CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5-MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5-MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation.