Assembly and organization of glycoproteins B, C, D, and H in herpes simplex virus type 1 particles lacking individual glycoproteins: No evidence for the formation of a complex of these molecules

Glycoprotein B (gB), gC, gD, and gH:L of herpes simplex virus type 1 (HSV-1) are implicated in virus adsorption and penetration. gB, gD, and gH:L are essential for these processes, and their expression is necessary and sufficient to induce cell fusion. The current view is that these molecules act in concert as a functional complex, and cross-linking studies support this view (C. G. Handler, R. J. Eisenberg, and G. H. Cohen, J. Virol. 70:6067-6075, 1996). We examined the glycoprotein composition, with respect to gB, gC, gD, and gH, of mutant virions lacking individual glycoproteins and the sedimentation characteristics of glycoproteins extracted from these virions. The amounts of gB, gC, gD, or gH detected in virions did not alter when any one of these molecules was absent, and it therefore appears that they are incorporated into the virion independently of each other. The sedimentation characteristics of gB and gD from mutant virions were not different from those of wild-type virions. We confirmed that gB, gC, and gD could be cross-linked to each other on the virion surface but found that the absence of one glycoprotein did not alter the outcome of cross-linking reactions between the remaining molecules. The incorporation and arrangement of these glycoproteins in the virion envelope therefore appear to be independent of the individual molecular species. This is difficult to reconcile with the concept that gB, gC, gD, and gH:L are incorporated as a functional complex into the virion envelope.     

Oligomeric structure of glycoproteins in herpes simplex virus type 1.

A number of herpes simplex virus (HSV) glycoproteins are found in oligomeric states: glycoprotein E (gE)-gI and gH-gL form heterodimers, and both gB and gC have been detected as homodimers. We have further explored the organization of glycoproteins in the virion envelope by using both purified virions to quantitate glycoprotein amounts and proportions and chemical cross-linkers to detect oligomers. We purified gB, gC, gD, and gH from cells infected with HSV type 1 and used these as immunological standards. Glycoproteins present in sucrose gradient-purified preparations of two strains of HSV type 1, KOS and NS, were detected with antibodies to each of the purified proteins. From these data, glycoprotein molar ratios of 1:2:11:16 and 1:1:14:9 were calculated for gB/gC/gD/gH in KOS and NS, respectively. gL was also detected in virions, although we lacked a purified gL standard for quantitation. We then asked whether complexes of these glycoproteins could be identified, and if they existed as homo- or hetero-oligomers. Purified KOS was incubated at 4 degrees C with bis (sulfosuccinimidyl) suberate (BS3), an 11.4 A (1A = 0.1 mm) noncleavable, water-soluble cross-linker. Virus extracts were examined by Western blotting (immunoblotting), or immunoprecipitation followed by Western blotting, to assay for homo- and hetero-oligomers. Homodimers of gB, gC, and gD were detected, and hetero-oligomers containing gB cross-linked to gC, gC to gD, and gD to gB were also identified. gH and gL were detected as a hetero-oligomeric pair and could be cross-linked to gD or gC but not to gB. We conclude that these glycoproteins are capable of forming associations with one another. These studies suggest that glycoproteins are closely associated in virions and have the potential to function as oligomeric complexes.     

Soluble V domain of Nectin-1/HveC enables entry of herpes simplex virus type 1 (HSV-1) into HSV-resistant cells by binding to viral glycoprotein D

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1(123)), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1(123), approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1(123) did not associate with the cell surface. sNec1(123)-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.     

Herpes simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and blocks virus entry

Virion glycoproteins gB, gD, and gH/gL play essential roles for herpes simplex virus (HSV) entry. The function of gD is to interact with a cognate receptor, and soluble forms of gD block HSV entry by tying up cell surface receptors. Both gB and the nonessential gC interact with cell surface heparan sulfate proteoglycan (HSPG), promoting viral attachment. However, cells deficient in proteoglycan synthesis can still be infected by HSV. This suggests another function for gB. We found that a soluble truncated form of gB bound saturably to the surface of Vero, A431, HeLa, and BSC-1 cells, L-cells, and a mouse melanoma cell line expressing the gD receptor nectin-1. The HSPG analog heparin completely blocked attachment of the gC ectodomain to Vero cells. In contrast, heparin only partially blocked attachment of soluble gB, leaving 20% of the input gB still bound even at high concentrations of inhibitor. Moreover, heparin treatment removed soluble gC but not gB from the cell surface. These data suggest that a portion of gB binds to cells independently of HSPG. In addition, gB bound to two HSPG-deficient cell lines derived from L-cells. Gro2C cells are deficient in HSPG, and Sog9 cells are deficient in HSPG, as well as chondroitin sulfate proteoglycan (CSPG). To identify particular gB epitopes responsible for HSPG-independent binding, we used a panel of monoclonal antibodies (MAbs) to gB to block gB binding. Only those gB MAbs that neutralized virus blocked binding of soluble gB to the cells. HSV entry into Gro2C and Sog9 cells was reduced but still detectable relative to the parental L-cells, as previously reported. Importantly, entry into Gro2C cells was blocked by purified forms of either the gD or gB ectodomain. On a molar basis, the extent of inhibition by gB was similar to that seen with gD. Together, these results suggest that soluble gB binds specifically to the surface of different cell types independently of HSPG and CSPG and that by doing so, the protein inhibits entry. The results provide evidence for the existence of a cellular entry receptor for gB.     

Virus-specific glycoproteins associated with the nuclear fraction of herpes simplex virus type 1-infected cells.

Monospecific antisera to herpes simplex virus type 1 (HSV-1) glycoproteins gB, gC, and gD were used to identify the HSV-1-specific glycoproteins associated with the nuclear fraction as compared with those associated with cytoplasmic fraction, whole-cell lysates, and purified virions. The results indicate that a predominance of HSV glycoprotein precursors pgC(105), pgB(110), and pgD(52) is associated with the nuclear fraction. Treatment of the nuclear fraction with the enzyme endo-beta-N-acetylglucosaminidase H indicated that the lower-molecular-weight glycoproteins are sensitive to this endoglycosidase. These results suggest that in the nuclear fraction of HSV-1-infected cells virus-specific glycoproteins gB, gC, and gD are predominately in the high-mannose precursor form; however, detectable amounts of the fully glycosylated forms of gC and gD were also found.     

Cellular and viral requirements for rapid endocytic entry of herpes simplex virus

It was recently demonstrated that herpes simplex virus (HSV) successfully infects Chinese hamster ovary (CHO) cells expressing glycoprotein D (gD) receptors and HeLa cells by an endocytic mechanism (A. V. Nicola, A. M. McEvoy, and S. E. Straus, J. Virol. 77:5324-5332, 2003). Here we define cellular and viral requirements of this pathway. Uptake of intact, enveloped HSV from the cell surface into endocytic vesicles was rapid (t(1/2) of 8 to 9 min) and independent of the known cell surface gD receptors. Following uptake from the surface, recovery of intracellular, infectious virions increased steadily up to 20 min postinfection (p.i.), which corresponds to accumulation of enveloped virus in intracellular compartments. There was a sharp decline in recovery by 30 min p.i., suggesting loss of the virus envelope as a result of capsid penetration from endocytic organelles into the cytosol. In the absence of gD receptors, endocytosed virions did not successfully penetrate into the cytosol but were instead transported to lysosomes for degradation. Inhibitors of phosphatidylinositol (PI) 3-kinase, such as wortmannin, blocked transport of incoming HSV to the nuclear periphery and virus-induced gene expression but had no effect on virus binding or uptake. This suggests a role for PI 3-kinase activity in trafficking of HSV through the cytosol. Viruses that lack viral glycoproteins gB, gD, or gH-gL were defective in transport to the nucleus and had reduced infectivity. Thus, similar to entry via direct penetration at the cell surface, HSV entry into cells by wortmannin-sensitive endocytosis is efficient, involves rapid cellular uptake of viral particles, and requires gB, gD, and gH-gL.     

Herpes simplex virus: receptors and ligands for cell entry

Entry of herpes simplex virus (HSV) into cells depends upon multiple cell surface receptors and multiple proteins on the surface of the virion. The cell surface receptors include heparan sulphate chains on cell surface proteoglycans, a member of the tumor necrosis factor (TNF) receptor family and two members of the immunoglobulin superfamily related to the poliovirus receptor. The HSV ligands for these receptors are the envelope glycoproteins gB and gC for heparan sulphate and gD for the protein receptors and specific sites in heparan sulphate generated by certain 3-O-sulfotransferases. HSV gC also binds to the C3b component of complement and can block complement-mediated neutralization of virus. The purposes of this review are to summarize available information about these cell surface receptors and the viral ligands, gC and gD, and to discuss roles of these viral glycoproteins in immune evasion and cellular responses as well as in viral entry.     

Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry

Herpes simplex virus (HSV) entry requires the interaction of glycoprotein D (gD) with a cellular receptor such as herpesvirus entry mediator (HVEM or HveA) or nectin-1 (HveC). However, the fusion mechanism is still not understood. Since cholesterol-enriched cell membrane lipid rafts are involved in the entry of other enveloped viruses such as human immunodeficiency virus and Ebola virus, we tested whether HSV entry proceeds similarly. Vero cells and cells expressing either HVEM or nectin-1 were treated with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin or nystatin and then exposed to virus. In all cases, virus entry was inhibited in a dose-dependent manner, and the inhibitory effect was fully reversible by replenishment of cholesterol. To examine the association of HVEM and nectin-1 with lipid rafts, we analyzed whether they partitioned into nonionic detergent-insoluble glycolipid-enriched membranes (DIG). There was no constitutive association of either receptor with DIG. Binding of soluble gD or virus to cells did not result in association of nectin-1 with the raft-containing fractions. However, during infection, a fraction of gB but not gC, gD, or gH associated with DIG. Similarly, when cells were incubated with truncated soluble glycoproteins, soluble gB but not gC was found associated with DIG. Together, these data favor a model in which HSV uses gB to rapidly mobilize lipid rafts that may serve as a platform for entry and cell signaling. It also suggests that gB may interact with a cellular molecule associated with lipid rafts.     

Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is via interference with the interaction between gD in the virus and HVEM on the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding.     

Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. We studied the ability of these proteins to bind to mammalian cells, to bind to immobilized heparin, to block HSV type 1 (HSV-1) attachment to cells, and to inhibit plaque formation by HSV-1. Each of these gC proteins bound to conformation-dependent monoclonal antibodies and to human complement component C3b, indicating that they maintained the same conformation of gC proteins expressed in mammalian cells. Biotinylated gC1(457t) and gC2(426t) each bind to several cell lines. Binding was inhibited by an excess of unlabeled gC but not by gD, indicating specificity. The attachment of gC to cells involves primarily heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding by 50% but had no effect on gD binding. Moreover, binding of gC to two heparan sulfate-deficient L-cell lines, gro2C and sog9, both of which are mostly resistant to HSV infection, was markedly reduced. Purified gD1 (306t), however, bound equally well to the two mutant cell lines. In contrast, saturating amounts of gC1(457t) interfered with HSV-1 attachment to cells but failed to block plaque formation, suggesting a role for gC in attachment but not penetration. A mutant form of gC lacking residues 33 to 123, gC1(delta 33-123t), expressed in the baculovirus system, bound significantly less well to cells than did gC1(457t) and competed poorly with biotinylated gC1(457t) for binding. These results suggest that residues 33 to 123 are important for gC attachment to cells. In contrast, both the mutant and wild-type forms of gC bound to immobilized heparin, indicating that binding of these proteins to the cell surface involves more than a simple interaction with heparin. To determine that the contribution of the N-terminal region of gC is important for HSV attachment, we compared several properties of a mutant HSV-1 which contains gC lacking amino acids 33 to 123 to those of its parental virus, which contains full-length gC. The mutant bound less well to cells than the parental virus but exhibited normal growth properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blocking antibody access to neutralizing domains on glycoproteins involved in entry as a novel mechanism of immune evasion by herpes simplex virus type 1 glycoproteins C and E

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins.     

Copatching and lipid raft association of different viral glycoproteins expressed on the surfaces of pseudorabies virus-infected cells

Pseudorabies virus (PRV) is a swine alphaherpesvirus that is closely related to human herpes simplex virus (HSV). Both PRV and HSV express a variety of viral envelope glycoproteins in the plasma membranes of infected cells. Here we show that at least four major PRV glycoproteins (gB, gC, gD, and gE) in the plasma membrane of infected swine kidney cells and monocytes seem to be linked, since monospecific antibody-induced patching of any one of these proteins results in copatching of the others. Further, for all four PRV glycoproteins, monospecific antibody-induced patches were enriched in GM1, a typical marker of lipid raft microdomains, but were excluded for transferrin receptor, a nonraft marker, suggesting that these viral proteins may associate with lipid rafts. However, only gB and, to a lesser extent, gE were found in lipid raft fractions by using detergent floatation assays, indicating that gC and gD do not show strong lipid raft association. Addition of methyl-beta-cyclodextrin (MCD), a cholesterol-depleting agent that is commonly used to disrupt lipid rafts, only slightly reduced copatching efficiency between the different viral proteins, indicating that other factors, perhaps tegument-glycoprotein interactions, may be important for the observed copatching events. On the other hand, MCD strongly reduced polarization of the antibody-induced viral glycoprotein patches to a cap structure, a gE-dependent process that has been described for specific PRV- and HSV-infected cells. Therefore, we hypothesize that efficient gE-mediated capping of antibody-antigen patches may require the lipid raft-associated signal transduction machinery.     

Characterization of cell-binding properties of bovine herpesvirus 1 glycoproteins B, C, and D: identification of a dual cell-binding function of gB.

Previous studies have suggested that the attachment of bovine herpesvirus 1 (BHV-1) to permissive cells is mediated by its major glycoproteins B (gB), C (gC), and D (gD). In order to gain further insight into the mechanism of the BHV-1 attachment process, we purified authentic gB, gC, and gD from BHV-1-infected cells and membrane anchor-truncated, soluble gB, gC, and gD from stably transfected cell lines by affinity chromatography and examined their cell-binding properties on Madin-Darby bovine kidney cells. All of the glycoproteins tested exhibited saturable binding to Madin-Darby bovine kidney cells. All of the glycoproteins tested exhibited saturable binding to Madin-Darby bovine kidney cells. Addition of exogenous heparin or treatment of cells with heparinase to remove cellular heparan sulfate (HS) prevented both gC and gB from binding to cells but had no effect on gD binding. An assessment of competition between gB, gC, and gD for cell binding revealed that gC was able to inhibit gB binding, whereas other combinations showed no effect. Cell-bound gC could be dissociated by heparin or heparinase treatment. The response of bound gB to heparin and heparinase treatments differed for the authentic and soluble forms; while soluble gB was susceptible to the treatment, a significant portion of cell-bound authentic gB was resistant to the treatment. Binding affinity analysis showed that soluble gB and both forms of gC and gD each had single binding kinetics with comparable dissociation constants (Kds), ranging from 1.5 x 10(-7) to 5.1 x 10(-7) M, whereas authentic gB exhibited dual binding kinetics with Kd1 = 5.2 x 10(-7) M and Kd2 = 4.1 x 10(-9) M. These results demonstrate that BHV-1 gC binds only to cellular HS, gD binds to a non-HS component, and gB initially binds to HS and then binds with high affinity to a non-HS receptor. Furthermore, we found that while authentic gB was able to inhibit viral plaque formation, soluble gB, which retains the HS-binding property but lacks the high-affinity binding property, was defective in this respect. These results suggest that the interaction between gB and its high-affinity receptor may play a critical role in the virus entry process.     

Pseudotyping of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein G enables mutant virus attachment and entry

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.     

Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity.

The purpose of this study was to identify the herpes simplex virus glycoprotein(s) that mediates the adsorption of virions to cells. Because heparan sulfate moieties of cell surface proteoglycans serve as the receptors for herpes simplex virus adsorption, we tested whether any of the viral glycoproteins could bind to heparin-Sepharose in affinity chromatography experiments. Two glycoproteins, gB and gC, bound to heparin-Sepharose and could be eluted with soluble heparin. In order to determine whether virions devoid of gC or gB were impaired for adsorption, we quantitated the binding of wild-type and mutant virions to cells. We found that at equivalent input concentrations of purified virions, significantly fewer gC-negative virions bound to cells than did wild-type or gB-negative virions. In addition, the gC-negative virions that bound to cells showed a significant delay in penetration compared with wild-type virus. The impairments in adsorption and penetration of the gC-negative virions can account for their reduced PFU/particle ratios, which were found to be about 5 to 10% that of wild-type virions, depending on the host cell. Although gC is dispensable for replication of herpes simplex virus in cell culture, it clearly facilitates virion adsorption and enhances infectivity by about a factor of 10.     

Partial resistance to gD-mediated interference conferred by mutations affecting herpes simplex virus type 1 gC and gK.

Cells expressing herpes simplex virus (HSV) gD can be resistant to HSV entry as a result of gD-mediated interference. HSV strains differ in sensitivity to this interference, which blocks viral penetration but not binding. Previous studies have shown that mutations or variations in virion-associated gD can confer resistance to gD-mediated interference. Here we show that HSV-1 mutants selected for enhanced ability to bind and penetrate in the presence of inhibitory concentrations of heparin were partially resistant to gD-mediated interference. The resistance was largely due to the presence of two mutations: one in gC (the major heparin-binding glycoprotein) resulting in the absence of gC expression and the other in gK resulting in a syncytial phenotype. The results imply that heparin selected for mutants with altered postbinding requirements for entry. Resistance to gD-mediated interference conferred by mutations affecting gC and gK has not been previously described.     

A critical review on antiviral peptides derived from viral glycoproteins and host receptors to decoy herpes simplex virus

The health of the human population has been continuously challenged by viral infections. Herpes simplex virus (HSV) is one of the common causes of illness and can lead to death in immunocompromised patients. Existing anti-HSV therapies are not completely successful in eliminating the infection due to anti-viral drug resistance, ineffectiveness against the latent virus and high toxicity over prolonged use. There is a need to update our knowledge of the current challenges faced in anti-HSV therapeutics and realize the necessity of developing alternative treatment approaches. Protein therapeutics are now being explored as a novel approach due to their high specificity and low toxicity. This review highlights the significance of HSV viral glycoproteins and host receptors in the pathogenesis of HSV infection. Proteins or peptides derived from HSV glycoproteins gC, gB, gD, gH and host cell receptors (HSPG, nectin and HVEM) that act as decoys to inhibit HSV attachment, entry, or fusion have been discussed. Few researchers have tried to improve the efficacy and stability of the identified peptides by modifying them using a peptidomimetic approach. With these efforts, we think developing an alternative treatment option for immunocompromised patients and drug-resistant organisms is not far off.     

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.     

Infectivity and glycoprotein processing of herpes simplex virus type 1 grown in a ricin-resistant cell line deficient in N-acetylglucosaminyl transferase I.

We report on the replication of herpes simplex virus type 1 (HSV-1) and viral glycoprotein processing in RicR14 cells, a mutant ricin-resistant cell line defective in N-acetylglucosaminyl transferase I activity. In these cells HSV-1(MP) and (F) replicated to yields very similar to those in parental BHK cells. The kinetics of HSV-1 adsorption in mutant and in parent cells was also essentially identical. Progeny virions from ricin-resistant and wild-type cells displayed comparable specific infectivities. However, in the mutant cells the efficiency of plating of progeny virus from both RicR14 and BHK cells was reduced. HSV-1(MP) failed to induce syncytia in RicR14 cells either in a plaque assay or after a high-multiplicity infection. Moreover, the fully glycosylated forms of glycoproteins (gB, gC, and gD) were totally absent, and only the partially glycosylated precursors (pgC, pgD. and a triplet in the gB-gA region) accumulated in HSV-1-infected ricin-resistant cells and in herpesvirions made in these cells. Consistent with these results analysis of pronase glycopeptides from cells labeled with [14C]glucosamine showed a strong decrease of sialylated complex-type oligosaccharides and a dramatic accumulation of the neutral mannose-rich chains. The latter chains predominate in partially glycosylated precursors, whereas the complex acidic chains predominate in the fully processed forms of HSV glycoproteins. These results taken together indicate that (i) host-cell N-acetylglucosaminyl transferase I participates in the processing of HSV glycoproteins; and (ii) infectivity of herpesvirions does not necessarily require the mature form of gB. The absence of HSV-1(MP)-induced fusion in RicR14 cells is discussed.     

Glycoproteins required for entry are not necessary for egress of pseudorabies virus

In the current perception of the herpesvirus replication cycle, two fusion processes are thought to occur during entry and nuclear egress. For penetration, glycoproteins gB and gH/gL have been shown to be essential, whereas a possible role of these glycoproteins in nuclear egress remains unclear. Viral envelope glycoproteins have been detected by immunolabeling in the nuclear membrane as well as in primary enveloped particles in several herpesviruses, indicating that they might be involved in the fusion process. Moreover, a herpes simplex virus type 1 mutant simultaneously lacking gB and gH was described to be deficient in nuclear egress (A. Farnsworth, T. W. Wisner, M. Webb, R. Roller, G. Cohen, R. Eisenberg, and D. C. Johnson, Proc. Natl. Acad. Sci. USA 104:10187-10192, 2007). To analyze the situation in the related alphaherpesvirus pseudorabies virus (PrV), mutants carrying single and double deletions of glycoproteins gB, gD, gH, and gL were constructed and characterized. We show here that the simultaneous deletion of gB and gD, gB and gH, gD and gH, or gH and gL has no detectable effect on PrV egress, implying that none of these glycoproteins either singly or in the tested combinations is required for nuclear egress. In addition, immunolabeling studies using different mono- or polyclonal sera raised against various PrV glycoproteins did not reveal the presence of viral glycoproteins in the inner nuclear membrane or in primary virions. Thus, our data strongly suggest that different fusion mechanisms are active during virus entry and egress.