A Single-Amino-Acid Substitution in Herpes Simplex Virus 1 Envelope Glycoprotein B at a Site Required for Binding to the Paired Immunoglobulin-Like Type 2 Receptor α (PILRα) Abrogates PILRα-Dependent Viral Entry and Reduces Pathogenesis

Paired immunoglobulin-like type 2 receptor α (PILRα) is a herpes simplex virus 1 (HSV-1) entry receptor that associates with O-glycans on HSV-1 envelope glycoprotein B (gB). Two threonine residues (Thr-53 and Thr-480) in gB, which are required for the addition of the principal gB O-glycans, are essential for binding to soluble PILRα. However, the role of the two threonines in PILRα-dependent viral entry remains to be elucidated. Therefore, we constructed a recombinant HSV-1 carrying an alanine replacement of gB Thr-53 alone (gB-T53A) or of both gB Thr-53 and Thr-480 (gB-T53/480A) and demonstrated that these mutations abrogated viral entry in CHO cells expressing PILRα. In contrast, the mutations had no effect on viral entry in CHO cells expressing known host cell receptors for HSV-1 gD, viral entry in HL60 cells expressing myelin-associated glycoprotein (MAG) (another HSV-1 gB receptor), viral attachment to heparan sulfate, and viral replication in PILRα-negative cells. These results support the hypothesis that gB Thr-53 and Thr-480 as well as gB O-glycosylation, probably at these sites, are critical for PILRα-dependent viral entry. Interestingly, following corneal inoculation in mice, the gB-T53A and gB-T53/480A mutations significantly reduced viral replication in the cornea, the development of herpes stroma keratitis, and neuroinvasiveness. The abilities of HSV-1 to enter cells in a PILRα-dependent manner and to acquire specific carbohydrates on gB are therefore linked to an increase in viral replication and virulence in the experimental murine model.Herpes simplex virus 1 (HSV-1) entry into host cells depends on interactions between cell surface receptors and HSV-1 virion envelope glycoproteins (39). Five of the 12 HSV-1 envelope glycoproteins that have been identified thus far (i.e., glycoprotein B [gB], gC, gD, gH, and gL) have roles in viral entry (39). Both gB and gC mediate virion attachment by interacting with cell surface glycosaminoglycan, primarily heparan sulfate (16, 17). Although not essential for entry, this step provides stable interactions between the virion and the cell that favor the next steps (39). These steps include gD binding to one of its identified receptors, i.e., herpesvirus entry mediator (HVEM), nectin-1, and specific sites on heparan sulfate 3-O-sulfated heparan sulfate (3-O-S-HS) generated by certain 3-O-sulfotransferases (3-O-STs) (14, 28, 38, 51). Subsequent fusion between the virion envelope and host cell membrane, which requires the cooperative function of gB, heterodimer gH/gL, gD, and a gD receptor, then produces nucleocapsid penetration into the cell (31, 46).In addition to the interaction of gD with a gD receptor, gB binding to a cellular receptor other than heparan sulfate has been suggested to mediate viral entry, based on the observation that a soluble form of gB binds to heparan sulfate-deficient cells and blocks HSV-1 infection of some cell lines (3). Consistent with this observation, we have reported that paired immunoglobulin-like type 2 receptor α (PILRα) associates with gB and functions as an HSV-1 entry receptor (36). Viral entry via PILRα appears to be conserved among alphaherpesviruses, but there is a PILRα preference based on the observation that PILRα is able to mediate the entry of pseudorabies virus, a porcine alphaherpesvirus, but not of HSV-2 (1). Importantly, HSV-1 infection of human primary monocytes expressing both HVEM and PILRα was blocked by either an anti-PILRα or anti-HVEM antibody, suggesting that cellular receptors for both gD and gB are required for HSV-1 infection (36). However, CHO-K1 cells, which are resistant to HSV-1 infection, can become susceptible to HSV-1 entry and HSV-1-induced cell fusion after the overexpression of either a gD receptor, such as nectin-1, or PILRα (14, 36). It was thought that CHO-K1 cells express endogenously low levels of gB and gD receptors that allow the single overexpression of either a gB or gD receptor to support detectable levels of HSV-1 entry and HSV-1-induced cell fusion (36). More recently, myelin-associated glycoprotein (MAG), which has homology to PILRα, was also reported to serve as the gB receptor for HSV-1 and varicella-zoster virus (40). However, the importance of PILRα- or MAG-dependent viral entry in HSV-1 infection and pathogenesis in vivo remains to be elucidated.PILRα is one of the paired receptor families, in which one receptor has inhibitory functions and the other mediates activation functions, and is expressed mainly in immune system cells (13, 29). In addition, PILRα was previously reported to be expressed in certain types of cells in neural tissues (36). We previously identified one of the PILRα ligands as CD99 (37). Interestingly, PILRα recognition of CD99 is dependent on the addition of sialylated O-linked sugar chains at particular CD99 threonines (50). Similarly, we recently demonstrated that a specific sialylated O-glycan(s) on gB is critical for PILRα binding, based on observations that neuraminidase, which removes sialic acid, and benzyl-α-GalNAc treatment, which blocks O-glycan synthesis, inhibited gB binding to a soluble PILRα (49). More importantly, one (Thr-53) or both (Thr-53 and Thr-480) putative O-glycosylation sites identified by bioinformatics analysis are required for the binding of gB to soluble PILRα, and the replacement of both Thr-53 and Thr-480 with alanine significantly inhibited the addition of O-glycans to gB (49). These observations suggest that Thr-53 and Thr-480 in gB are O-glycosylated, and these sites, and probably the addition of specific carbohydrates to them, are required for the interaction of gB with PILRα. However, it remains uncertain whether gB Thr-53 and Thr-480, and probably the gB O-glycosylation of these sites, are required for PILRα-dependent viral entry in natural infections.In the present study, we have shown that the alanine replacement of gB Thr-53 (gB-T53A) alone or of both gB Thr-53 and Thr-480 (gB-T53/480A) significantly inhibited cell-cell fusion in CHO cells expressing PILRα, gB, gD, gH, and gL, whereas the mutations had no effect on cell-cell fusion in CHO cells expressing nectin-1, gB, gD, gH, and gL. Furthermore, we constructed recombinant HSV-1 carrying the gB-T53A and gB-T53/480A mutations and found that these mutations abrogated PILRα-dependent viral entry but had no effect on viral entry via known receptors for HSV-1 gD and MAG, viral attachment to heparan sulfate, and viral replication in PILRα-negative cells. We also tested these recombinant viruses in mice and present data showing that the mutations in gB significantly reduced viral replication, the development of herpes stromal keratitis (HSK), and neuroinvasiveness.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

Mutations in the N termini of herpes simplex virus type 1 and 2 gDs alter functional interactions with the entry/fusion receptors HVEM,nectin-2, and 3-O-sulfated heparan sulfate but not with nectin-1

Multiple cell surface molecules (herpesvirus entry mediator [HVEM], nectin-1, nectin-2, and 3-O-sulfated heparan sulfate) can serve as entry receptors for herpes simplex virus type 1 (HSV-1) or HSV-2 and also as receptors for virus-induced cell fusion. Viral glycoprotein D (gD) is the ligand for these receptors. A previous study showed that HVEM makes contact with HSV-1 gD at regions within amino acids 7 to 15 and 24 to 32 at the N terminus of gD. In the present study, amino acid substitutions and deletions were introduced into the N termini of HSV-1 and HSV-2 gDs to determine the effects on interactions with all of the known human and mouse entry/fusion receptors, including mouse HVEM, for which data on HSV entry or cell fusion were not previously reported. A cell fusion assay was used to assess functional activity of the gD mutants with each entry/fusion receptor. Soluble gD:Fc hybrids carrying each mutation were tested for the ability to bind to cells expressing the entry/fusion receptors. We found that deletions overlapping either or both of the HVEM contact regions, in either HSV-1 or HSV-2 gD, severely reduced cell fusion and binding activity with all of the human and mouse receptors except nectin-1. Amino acid substitutions described previously for HSV-1 (L25P, Q27P, and Q27R) were individually introduced into HSV-2 gD and, for both serotypes, were found to be without effect on cell fusion and the binding activity for nectin-1. Each of these three substitutions in HSV-1 gD enhanced fusion with cells expressing human nectin-2 (ordinarily low for wild-type HSV-1 gD), but the same substitutions in HSV-2 gD were without effect on the already high level of cell fusion observed with the wild-type protein. The Q27P or Q27R substitution in either HSV-1 and HSV-2 gD, but not the L25P substitution, significantly reduced cell fusion and binding activity for both human and mouse HVEM. Each of the three substitutions in HSV-1 gD, as well as the deletions mentioned above, reduced fusion with cells bearing 3-O-sulfated heparan sulfate. Thus, the N terminus of HSV-1 or HSV-2 gD is not necessary for functional interactions with nectin-1 but is necessary for all of the other receptors tested here. The sequence of the N terminus determines whether nectin-2 or 3-O-sulfated heparan sulfate, as well as HVEM, can serve as entry/fusion receptors.     

Differential Effects on Cell Fusion Activity of Mutations in Herpes Simplex Virus 1 Glycoprotein B (gB) Dependent on Whether a gD Receptor or a gB Receptor Is Overexpressed

Glycoprotein B (gB) of herpes simplex virus (HSV) is one of four glycoproteins essential for viral entry and cell fusion. Recently, paired immunoglobulin-like type 2 receptor (PILRα) was identified as a receptor for HSV type 1 (HSV-1) gB. Both PILRα and a gD receptor were shown to participate in HSV-1 entry into certain cell types. The purpose of this study was to determine whether insertional mutations in gB had differential effects on its function with PILRα and the gD receptor, nectin-1. Previously described gB mutants and additional newly characterized mutants were used in this study. We found that insertional mutations near the N terminus and C terminus of gB and especially in the central region of the ectodomain reduced cell fusion activity when PILRα was overexpressed much more than when nectin-1 was overexpressed. Most of the insertions reduced the binding of gB to PILRα, for at least some forms of gB, but this reduction did not necessarily correlate with the selective reduction in cell fusion activity with PILRα. These results suggest that the regions targeted by the relevant mutations are critical for functional activity with PILRα. They also suggest that, although both the binding of gB to a gB receptor and the binding of gD to a gD receptor may be required for HSV-induced cell fusion, the two receptor-binding activities may have unequal weights in triggering fusogenic activity, depending on the ratios of gB and gD receptors or other factors.Manifestations of disease caused by herpes simplex virus (HSV) include recurrent mucocutaneous lesions in the mouth or on the face or genitalia and, more rarely, meningitis or encephalitis. The infection of host cells occurs by the fusion of the virion envelope with a cell membrane to deliver the nucleocapsid containing the viral genome into the host cell. This entry process and virus-induced cell fusion require glycoprotein B (gB), along with gD, gH, and gL. The membrane-fusing activity of HSV depends in part on the binding of gD to one of its receptors, herpesvirus entry mediator (HVEM), nectin-1, nectin-2, or 3-O-sulfated heparan sulfate (18). HVEM is a member of the tumor necrosis factor receptor family and is expressed by cells of the immune system, as well as many other cell types, such as epithelial, stromal, and endothelial cells (23). Nectin-1 and nectin-2 are cell adhesion molecules belonging to the immunoglobulin superfamily and are widely expressed by a variety of cell types, including epithelial cells and neurons (20). Specific sites in heparan sulfate generated by particular 3-O-sulfotransferases can serve as gD-binding entry receptors (17). This binding of gD to a receptor is associated with conformational changes in gD that are thought to enable gD to interact with gB and/or the heterodimer gH-gL to trigger fusogenic activity (8, 12). Both gB and gH have properties of fusogenic viral proteins (1, 7). Although evidence has been presented that gD and gH-gL are sufficient for hemifusion and that gB, in addition, is required for fusion pore formation (19), the specific roles each plays in HSV-induced membrane fusion have not been fully defined.gB was recently discovered to bind to paired immunoglobulin-like type 2 receptor (PILRα) in an interaction that can mediate viral entry and cell fusion, provided that gD also binds to one of its receptors (14). For cells such as CD14⁺ monocytes, antibodies specific for either HVEM or PILRα were shown to block HSV entry. Also, entry requires the presence of both gD and gB in the virion. Although the overexpression of either a gD receptor or a gB receptor can enhance the susceptibility of cells to HSV entry and HSV-induced cell fusion, there are very few, if any, cell types that do not express at least low levels of endogenous receptors. Thus, the possibility exists that these endogenous receptors are cooperating with the introduced receptors to render the cells susceptible to HSV-induced membrane fusion.PILRα belongs to the paired-receptor families, which consist of activating and inhibitory receptors (4, 11, 19). They are conserved among mammals (24). The inhibitory form PILRα has an immunoreceptor tyrosine-based inhibition motif in its cytoplasmic domain and transduces inhibitory signals (4). On the other hand, the activating form PILRβ associates with the immunoreceptor tyrosine-based activation motif-bearing DAP12 adaptor molecule and delivers activating signals (16). Both PILRα and PILRβ are expressed on cells of the immune system, especially monocytes, dendritic cells, and NK cells (4, 11, 19), and also in neurons (14). CD99 has been identified as a natural ligand for both PILRα and PILRβ (16). The binding of either PILRα or PILRβ to CD99 depends on the presence of sialyated O-linked glycans on CD99 (22).In addition to binding to PILRα, gB can bind to heparin and heparan sulfate and may contribute, along with gC, to the binding of HSV to cell surface heparan sulfate (17). Also, gB and gC can bind to DC-SIGN, which serves as a binding receptor for the infection of dendritic cells (2). An X-ray structure of the HSV-1 gB ectodomain reveals a homotrimeric conformation with structural homology to vesicular stomatitis virus (VSV) G glycoprotein, the single glycoprotein responsible for the entry of VSV. Both HSV-1 gB and VSV G glycoprotein have features of class 1 and class 2 viral fusion proteins and have been designated class 3 fusion proteins (7, 14, 15). The heparan sulfate-binding determinant of gB has been localized to a lysine-rich domain in the N terminus and shown to be dispensable for viral entry (9). It lies within a region that is probably disordered and was not included in the defined coordinates of the X-ray structure. The binding of DC-SIGN to gB probably depends on high-mannose N-glycans of gB (6).In a previous study (10), 81 insertion mutants of HSV-1 gB were characterized to assess the effects of the insertions on protein processing and function in cell fusion with gD receptors, in relation to structural domains of gB identified in an X-ray structure (7). Only 27 mutants were found to be processed into mature glycosylated forms and transported to the cell surface. Only 11 of these retained fusion activity toward target cells expressing nectin-1 or HVEM. For the present study, we used 25 previously described gB insertion mutants shown to be expressed on cell surfaces and also identified an additional 10 such mutants.The present study was designed to determine whether the effects of insertions in gB on cell fusion activity would be dependent on whether a gD receptor (nectin-1) or a gB receptor (PILRα) was overexpressed in target cells that also expressed unidentified weak endogenous receptors. In addition, we assessed the abilities of the gB mutants to bind to PILRα. Our results showed that some insertions inhibited cell fusion activity when PILRα was overexpressed significantly more than when nectin-1 was overexpressed, but without necessarily preventing the binding of PILRα to gB, at least to some stable oligomeric forms of gB. The results indicate that, although both a gB receptor and a gD receptor may be required for cell fusion activity, the two receptor-binding activities have unequal weights in triggering fusogenic activity, depending on the ratios of gB and gD receptors or other factors.     

Site-specific proteolytic cleavage of the amino terminus of herpes simplex virus glycoprotein K on virion particles inhibits virus entry

Herpes simplex virus 1 (HSV-1) glycoprotein K (gK) is expressed on virions and functions in entry, inasmuch as HSV-1(KOS) virions devoid of gK enter cells substantially slower than is the case for the parental KOS virus (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). Deletion of the amino-terminal 68-amino-acid (aa) portion of gK caused a reduction in efficiency and kinetics of virus entry similar to that of the gK-null virus in comparison to the HSV-1(F) parental virus. The UL20 membrane protein and gK were readily detected on double-gradient-purified virion preparations. Immuno-electron microscopy confirmed the presence of gK and UL20 on purified virions. Coimmunoprecipitation experiments using purified virions revealed that gK interacted with UL20, as has been shown in virus-infected cells (T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, J. Virol. 82:6310-6323, 2008). Scanning of the HSV-1(F) viral genome revealed the presence of a single putative tobacco etch virus (TEV) protease site within gD, while additional TEV predicted sites were found within the UL5 (helicase-primase helicase subunit), UL23 (thymidine kinase), UL25 (DNA packaging tegument protein), and UL52 (helicase-primase primase subunit) proteins. The recombinant virus gDΔTEV was engineered to eliminate the single predicted gD TEV protease site without appreciably affecting its replication characteristics. The mutant virus gK-V5-TEV was subsequently constructed by insertion of a gene sequence encoding a V5 epitope tag in frame with the TEV protease site immediately after gK amino acid 68. The gK-V5-TEV, R-gK-V5-TEV (revertant virus), and gDΔTEV viruses exhibited similar plaque morphologies and replication characteristics. Treatment of the gK-V5-TEV virions with TEV protease caused approximately 32 to 34% reduction of virus entry, while treatment of gDΔTEV virions caused slightly increased virus entry. These results provide direct evidence that the gK and UL20 proteins, which are genetically and functionally linked to gB-mediated virus-induced cell fusion, are structural components of virions and function in virus entry. Site-specific cleavage of viral glycoproteins on mature and fully infectious virions utilizing unique protease sites may serve as a generalizable method of uncoupling the roles of viral glycoproteins in virus entry and virion assembly.     

Herpes B virus utilizes human nectin-1 but not HVEM or PILRα for cell-cell fusion and virus entry

To investigate the requirements of herpesvirus entry and fusion, the four homologous glycoproteins necessary for herpes simplex virus (HSV) fusion were cloned from herpes B virus (BV) (or macacine herpesvirus 1, previously known as cercopithecine herpesvirus 1) and cercopithecine herpesvirus 2 (CeHV-2), both related simian simplexviruses belonging to the alphaherpesvirus subfamily. Western blots and cell-based enzyme-linked immunosorbent assay (ELISA) showed that glycoproteins gB, gD, and gH/gL were expressed in whole-cell lysates and on the cell surface. Cell-cell fusion assays indicated that nectin-1, an HSV-1 gD receptor, mediated fusion of cells expressing glycoproteins from both BV and CeHV-2. However, herpesvirus entry mediator (HVEM), another HSV-1 gD receptor, did not facilitate BV- and CeHV-2-induced cell-cell fusion. Paired immunoglobulin-like type 2 receptor alpha (PILRα), an HSV-1 gB fusion receptor, did not mediate fusion of cells expressing glycoproteins from either simian virus. Productive infection with BV was possible only with nectin-1-expressing cells, indicating that nectin-1 mediated entry while HVEM and PILRα did not function as entry receptors. These results indicate that these alphaherpesviruses have differing preferences for entry receptors. The usage of the HSV-1 gD receptor nectin-1 may explain interspecies transfer of the viruses, and altered receptor usage may result in altered virulence, tropism, or pathogenesis in the new host. A heterotypic cell fusion assay resulting in productive fusion may provide insight into interactions that occur to trigger fusion. These findings may be of therapeutic significance for control of deadly BV infections.     

Herpes simplex virus type 1 gK is required for gB-mediated virus-induced cell fusion, while neither gB and gK nor gB and UL20p function redundantly in virion de-envelopment

Multiple amino acid changes within herpes simplex virus type 1 (HSV-1) gB and gK cause extensive virus-induced cell fusion and the formation of multinucleated cells (syncytia). Early reports established that syncytial mutations in gK could not cause cell-to-cell fusion in the absence of gB. To investigate the interdependence of gB, gK, and UL20p in virus-induced cell fusion and virion de-envelopment from perinuclear spaces as well as to compare the ultrastructural phenotypes of the different mutant viruses in a syngeneic HSV-1 (F) genetic background, gB-null, gK-null, UL20-null, gB/gK double-null, and gB/UL20 double-null viruses were constructed with the HSV-1 (F) bacterial artificial chromosome pYEBac102. The gK/gB double-null virus YEbacDeltagBDeltagK was used to isolate the recombinant viruses gBsyn3DeltagK and gBamb1511DeltagK, which lack the gK gene and carry the gBsyn3 or gBamb1511 syncytial mutation, respectively. Both viruses formed small nonsyncytial plaques on noncomplementing Vero cells and large syncytial plaques on gK-complementing cells, indicating that gK expression was necessary for gBsyn3- and gBamb1511-induced cell fusion. Lack of virus-induced cell fusion was not due to defects in virion egress, since recombinant viruses specifying the gBsyn3 or gKsyn20 mutation in the UL19/UL20 double-null genetic background caused extensive cell fusion on UL20-complementing cells. As expected, the gB-null virus failed to produce infectious virus, but enveloped virion particles egressed efficiently out of infected cells. The gK-null and UL20-null viruses exhibited cytoplasmic defects in virion morphogenesis like those of the corresponding HSV-1 (KOS) mutant viruses. Similarly, the gB/gK double-null and gB/UL20 double-null viruses accumulated capsids in the cytoplasm, indicating that gB, gK, and UL20p do not function redundantly in membrane fusion during virion de-envelopment at the outer nuclear lamellae.     

Role for nectin-1 in herpes simplex virus 1 entry and spread in human retinal pigment epithelial cells

Herpes simplex virus 1 (HSV-1) demonstrates a unique ability to infect a variety of host cell types. Retinal pigment epithelial (RPE) cells form the outermost layer of the retina and provide a potential target for viral invasion and permanent vision impairment. Here we examine the initial cellular and molecular mechanisms that facilitate HSV-1 invasion of human RPE cells. High-resolution confocal microscopy demonstrated initial interaction of green fluorescent protein (GFP)-tagged virions with filopodia-like structures present on cell surfaces. Unidirectional movement of the virions on filopodia to the cell body was detected by live cell imaging of RPE cells, which demonstrated susceptibility to pH-dependent HSV-1 entry and replication. Use of RT-PCR indicated expression of nectin-1, herpes virus entry mediator (HVEM) and 3-O-sulfotransferase-3 (as a surrogate marker for 3-O-sulfated heparan sulfate). HVEM and nectin-1 expression was subsequently verified by flow cytometry. Nectin-1 expression in murine retinal tissue was also demonstrated by immunohistochemistry. Antibodies against nectin-1, but not HVEM, were able to block HSV-1 infection. Similar blocking effects were seen with a small interfering RNA construct specifically directed against nectin-1, which also blocked RPE cell fusion with HSV-1 glycoprotein-expressing Chinese hamster ovary (CHO-K1) cells. Anti-nectin-1 antibodies and F-actin depolymerizers were also successful in blocking the cytoskeletal changes that occur upon HSV-1 entry into cells. Our findings shed new light on the cellular and molecular mechanisms that help the virus to enter the cells of the inner eye.     

Herpes simplex virus glycoprotein K,but not its syncytial allele,inhibits cell-cell fusion mediated by the four fusogenic glycoproteins,gD, gB,gH, and gL

A Myc epitope was inserted at residue 283 of herpes simplex virus type 1 (HSV-1) glycoprotein K (gK), a position previously shown not to interfere with gK activity. The Myc-tagged gK localized predominantly to the endoplasmic reticulum, both in uninfected and in HSV-infected cells. gK, coexpressed with the four HSV fusogenic glycoproteins, gD, gB, gH, and gL, inhibited cell-cell fusion. The effect was partially dose dependent and was observed both in baby hamster kidney (BHK) and in Vero cells, indicating that the antifusion activity of gK may be cell line independent. The antifusion activity of gK did not require viral proteins other than the four fusogenic glycoproteins. A syncytial (syn) allele of gK (syn-gK) carrying the A40V substitution present in HSV-1(MP) did not block fusion to the extent seen with the wild-type (wt) gK, indicating that the syn mutation ablated, at least in part, the antifusogenic activity of wt gK. We conclude that gK is part of the mechanism whereby HSV negatively regulates its own fusion activity. Its effect accounts for the notion that cells infected with wt HSV do not fuse with adjacent, uninfected cells into multinucleated giant cells or syncytia. gK may also function to preclude fusion between virion envelope and the virion-encasing vesicles during virus transport to the extracellular compartment, thus preventing nucleocapsid de-envelopment in the cytoplasm.     

Binding of Herpes Simplex Virus Glycoprotein B (gB) to Paired Immunoglobulin-Like Type 2 Receptor α Depends on Specific Sialylated O-Linked Glycans on gB

Paired immunoglobulin-like type 2 receptor α (PILRα) is an inhibitory receptor expressed on both hematopoietic and nonhematopoietic cells. Its binding to a cellular ligand, CD99, depends on the presence of sialylated O-linked glycans on CD99. Glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1) binds to PILRα, and this association is involved in HSV-1 infection. Here, we found that the presence of sialylated O-glycans on gB is required for gB to associate with PILRα. Furthermore, we identified two threonine residues on gB that are essential for the addition of the principal O-glycans acquired by gB and that are also essential for the binding of PILRα to gB.Four envelope glycoproteins, gB, gD, gH, and gL, are required for herpes simplex virus type 1 (HSV-1) to enter into host cells. Paired immunoglobulin (Ig)-like type 2 receptor α (PILRα) binds to gB and functions as an entry receptor during HSV-1 infection in concert with an interaction between gD and gD receptors (10). An X-ray structure of gB suggested that it is a class III fusogenic glycoprotein with internal fusion loops (6), and evidence that these loops can associate with lipid membranes was presented recently (5). The interaction between PILRα and gB might help the fusion loops of gB to associate with cellular membranes. However, it has remained unclear how PILRα associates with gB. PILRα also binds to CD99, which is expressed mainly on T-cell subsets (12). Specific O-glycan structures on CD99 are required for recognition of CD99 by PILRα (15). Here, we addressed whether O-glycans on gB are involved in the association between PILRα and gB. One approach was to use benzyl-α-GalNAc, which specifically blocks the extension of O-glycans through its ability to compete with GalNAc-O-Ser/Thr, a substrate for β1-3-galactosyl-transferases, which generate core 1 structures of O-glycans (8). 293T cells transfected with gB (HSV-1 strain KOS) were treated with benzyl-α-GalNAc (Sigma) at 37°C for 48 h and were then stained with PILRα-Ig (15) or anti-gB monoclonal antibody ([MAb] clone 1105; Rumbaugh-Goodwin Institute) (Fig. ​(Fig.1A).1A). Recognition of gB by PILRα was abrogated almost completely by the treatment of gB transfectants with benzyl-α-GalNAc, whereas cell surface expression of gB was not affected. Because benzyl-α-GalNAc functions competitively, the weak binding of PILRα-Ig to benzyl-α-GalNAc-treated gB transfectants might have been due to an insufficient effect of benzyl-α-GalNAc on O-glycans. Benzyl-α-GalNAc did not affect the viability of cells (data not shown). Similarly, Western blot analysis showed that recognition of gB by PILRα-Ig was reduced by treatment with benzyl-α-GalNAc in a dose-dependent manner (Fig. ​(Fig.1B).1B). The molecular weight of gB expressed on cells treated with benzyl-α-GalNAc was slightly lower than that of gB on untreated cells. Thus, the presence of O-glycans on gB is critical for the interaction between PILRα and gB, as it is for the interaction between PILRα and CD99 (15).Open in a separate windowFIG. 1.Requirement of sialylated O-glycans on gB for the interaction with PILRα. (A) 293T cells transfected with gB were treated with benzyl-α-GalNAc (10 mM) and were then stained with PILRα-Ig or anti-gB MAb (solid line). As a control, the transfectants were stained with control Ig or control MAb (dotted line). Histograms show fluorescence intensity measured in arbitrary units on a log scale (x axis) and relative cell number on a linear scale (y axis). (B) Total cell lysates of mock (M)- or gB-transfected 293T cells treated with benzyl-α-GalNAc at the indicated concentrations were separated by SDS-PAGE, followed by blotting with anti-gB antiserum (R74; see reference 2) or PILRα-Ig. (C) gB (left)- or gD (right)-transfected 293T cells were incubated in the presence or absence of sialidase (0.01 U/ml) for 3 h and were stained with PILRα-Ig (solid line), nectin-Ig (solid line), or control Ig (dotted line). Expression of gB or gD was analyzed by using anti-gB MAb (solid line), anti-gD MAb (solid line), or control MAb (dotted line). Histograms show fluorescence intensity measured in arbitrary units on a log scale (x axis) and relative cell number on a linear scale (y axis).HSV gB is sialylated, and sialic acids on virions play an essential role in HSV-1 infection (14). Interestingly, sialic acids on O-glycans are required for recognition of CD99 by PILRα (13, 15). Therefore, we analyzed the involvement of sialic acids on gB in the interaction with PILRα. gB-transfected 293T cells treated with neuraminidase (Vibrio cholera; Roche) at 37°C for 3 h were not recognized by PILRα-Ig, whereas nontreated cells were recognized by PILRα-Ig (Fig. ​(Fig.1C).1C). Neuraminidase treatment did not affect the binding of nectin-Ig to gD transfectants or the cell surface expression of gB and gD.Four to 10% of the amino acids of PILRα are identical to Siglec (sialic acid-binding Ig-like lectin) family proteins, which recognize sialic acids on glycans (15). An arginine residue that is essential for sialic acid recognition by Siglecs is conserved in PILRα. Indeed, PILRα-Ig with this arginine residue mutated did not recognize gB or CD99 (data not shown). These results suggest that sialic acids on gB are involved in the recognition of gB by PILRα, as they are in the recognition of CD99 by PILRα. Along with the result that O glycosylation on gB is important for association with PILRα, sialylated O-glycans on gB are involved in the interaction with PILRα.We analyzed the glycosylation sites on gB that are responsible for recognition by PILRα. Although the NetOGlyc 3.1 algorithm (www.cbs.dtu.dk/services/NetOGlyc/) is useful in predicting potential O glycosylation sites, prediction of O glycosylation sites is still imprecise. The NetOGlyc 3.1 algorithm predicted seven threonine or serine residues (threonines at 37, 44, 53, 64, 67, and 480 and serine at 487) to be potential O glycosylation sites. Of note, five threonines were located near the N terminus. In order to analyze whether this N-terminal region is involved in recognition by PILRα, we constructed a gB chimeric molecule (gB30-115) possessing a BM-40 signal sequence (amino acid residues 1 to 40), a Flag-tag, an N-terminal gB fragment from its signal peptide cleavage site (amino acid residues 30 to 115) containing the five possible O glycosylation sites, and a transmembrane region of mouse PILRα (amino acid residues 196 to 256; GenBank accession number, {"type":"entrez-nucleotide","attrs":{"text":"NM_153510","term_id":"256355021","term_text":"NM_153510"}}NM_153510) to serve as an anchor to cellular membranes. This short N-terminal fragment of gB expressed on the cell surface was stained with both anti-Flag MAb and a PILRα-Ig fusion protein similar to wild-type (WT) gB (Fig. ​(Fig.2).2). In order to identify the amino acid residues of gB that are involved in association with PILRα, we generated a series of mutations of the N-terminal gB fragment. The gB fragment, in which all possible O glycosylation sites were mutated to alanine (gB30-115m), was not recognized by PILRα-Ig, whereas cell surface expression was not affected by these mutations. A revertant that has a threonine at amino acid residue 53 (A53T gB30-115m) was recognized by PILRα-Ig. In contrast, a WT N-terminal gB fragment in which only threonine 53 (T53) was mutated to alanine (T53A gB30-115) was not recognized by PILRα-Ig. Furthermore, the binding of PILRα-Ig to the A53T gB30-115m revertant was abrogated by sialidase or benzyl-α-GalNAc treatment (data not shown). Therefore, T53 is the only threonine within residues 30 to 115 of gB whose O glycosylation is required for the association of gB with PILRα.Open in a separate windowFIG. 2.Mutational analyses of O glycosylation sites in the N terminus domain of gB. Flag-tagged N terminus fragments of gB (amino acid residues 30 to 115) containing five potential O glycosylation sites or point mutations of these possible O glycosylation sites were transfected into 293T cells. The transfectants were stained with control Ig (dotted line) or PILRα-Ig (solid line). Expression of the N terminus domain of gB was analyzed by staining with anti-Flag MAb (solid line) or control MAb (dotted line). Histograms show fluorescence intensity measured in arbitrary units on a log scale (x axis) and relative cell number on a linear scale (y axis).We generated full-length gB in which T53 was mutated to alanine (T53A gB). The single point mutation at T53 partially affected the recognition of full-length gB by PILRα-Ig, whereas cell surface expression of gB was not affected (Fig. ​(Fig.3A).3A). This finding suggests that T53 is a dominant O glycosylation site on gB, which is involved in interactions with PILRα, although additional potential O glycosylation sites other than those near the N terminus might also be involved. Interestingly, gB with a mutation at threonine 480 (T480) in addition to T53 (T53AT480A) was not recognized by PILRα, whereas, similar to T53A gB, gB with an additional mutation at serine 487 (T53AS487A) was recognized by PILRα-Ig. gB with a mutation at T480 alone (T480A) was recognized by PILRα, as was WT gB. None of these mutations affected the cell surface expression of gB. Similar results were obtained using several other cell lines, such as COS cells (data not shown). These data suggest that two O glycosylation sites of gB, T53 and T480, are involved in the association with PILRα.Open in a separate windowFIG. 3.Mutational analyses of O glycosylation sites of full-length gB. (A) 293T cells were transfected with various mutated gBs, and the transfectants were stained with control Ig (dotted line) or PILRα-Ig (solid line). Expression of gB was analyzed by staining with anti-gB MAb (solid line) or control MAb (dotted line). The histograms show fluorescence intensity measured in arbitrary units on a log scale (x axis) and relative cell number on a linear scale (y axis). (B) Membrane proteins prepared from COS-7 cells transfected with WT gB and mutated gBs were boiled or left unheated in SDS sample buffer in reducing or nonreducing conditions, respectively. Samples were separated by SDS-PAGE, followed by blotting with anti-gB MAb.Both T53 and T480 are located in a proline-rich region, which may be important for protein folding (16). It has been reported that functional gB forms oligomers (1, 3, 6). Therefore, we analyzed whether the point mutations of gB affected oligomer formation. The oligomeric structure of gB is resistant in sodium dodecyl sulfate (SDS) sample buffer but is denatured by boiling (9). As shown in Fig. ​Fig.3B,3B, there was no difference in SDS resistance between WT gB and the mutated gBs. This suggests that point mutations of the O glycosylation sites at T53 and T480 of gB did not greatly affect the physical characteristics of gB. Moreover, there was no difference in the molecular weight between WT and mutated gB or in cell surface expression. Because the molecular weight of gB is relatively high and gB has several N glycosylation sites, mutations of one or two O glycosylation sites alone did not affect the total molecular weight of gB. However, the molecular weight of gB expressed in benzyl-α-GalNAc-treated cells was slightly lower than that of gB expressed in nontreated cells (Fig. ​(Fig.1B).1B). Because benzyl-α-GalNAc treatment inhibits synthesis of all the O-glycans on gB, other O glycosylation sites on gB might exist. However, it is noteworthy that only O glycosylation sites at T53 and T480 are involved in association with PILRα.Although mutations at T53 and T480 of gB completely abrogated recognition by PILRα, there is no direct evidence to suggest that these two residues are O glycosylated. In order to analyze O glycosylation on gB, we employed a novel method to label O-linked glycans, using Click-iT O-GalNAz metabolic glycoprotein-labeling reagent (azido-GalNAc) (Invitrogen). Because O-linked glycans generally possess peptide-proximal GalNAc residues (7), we cultured cells transfected with WT gB or mutated gB for 3 days in the presence of azido-GalNAc, which is metabolically incorporated into O-linked glycoproteins (4). gBs were immunoprecipitated with anti-gB MAb, and the azido-GalNAc incorporated into gB was treated with phosphine-Flag, which specifically reacts with the azido-GalNAc (11), followed by detection with anti-Flag MAb by Western blotting. WT gB, T53A-mutated gB, and T480A-mutated gB were blotted with anti-Flag MAb, whereas T53AT480A gB was only weakly blotted with anti-Flag MAb (Fig. ​(Fig.4).4). In contrast, there was no significant difference in the total amount of gB expressed. This result suggests that T53 and T480 of gB are O glycosylated. However, weak detection of O-glycans on the T53AT480A gB suggest the presence of O glycosylation sites other than T53 and T480 on gB.Open in a separate windowFIG. 4.Analysis of O-glycans on WT and mutated gB. O-glycans on gB expressed on 293T cells were metabolically labeled with Ac₄GalNAz and were then immunoprecipitated with anti-gB MAb. The labeled O-glycans in immunoprecipitates were modified with phosphine-Flag and were then analyzed by Western blotting. The labeled O-glycans and total amount of gB were detected by anti-Flag or anti-gB MAb, respectively.PILRα specifically associates with HSV-1 gB (10), but not with other HSV-1 glycoproteins, although some other envelope proteins are known to be O glycosylated. Recently, it was shown that insertion mutations in gB could reduce the binding of gB to PILRα, suggesting that the conformation of gB is also involved in the interaction (2). Therefore, PILRα does not associate with glycans alone and seems to recognize both protein structure and O-glycans (13, 15), which may be a reason that PILRα specifically associates with gB. It is interesting that both T53 and T480 are involved in the interaction with PILRα, because these two residues are widely separated on the polypeptide chain. Because PILRα bound to gB by Western blotting, PILRα might recognize linear epitopes in gB; therefore, PILRα might bind to the two sites in gB independently. Alternatively, elements of higher-order structure retained in the unreduced samples examined by SDS-polyacrylamide gel electrophoresis (PAGE) (Fig. ​(Fig.1B)1B) could have been necessary for the binding of PILRα-Ig to the blots. Thus, the binding of PILRα might depend upon the close proximity of the O-glycans attached to T53 and T480 in the trimeric conformation of gB. Determination of the structure of gB associated with PILRα will facilitate understanding the mechanism of membrane fusion during HSV-1 infection.     

Distinct cellular responses differentiating alcohol- and hepatitis C virus-induced liver cirrhosis

Background

Herpes simplex virus (HSV) can utilize multiple pathways to enter host cells. The factors that determine which route is taken are not clear. Chinese hamster ovary (CHO) cells that express glycoprotein D (gD)-binding receptors are model cells that support a pH-dependent, endocytic entry pathway for all HSV strains tested to date. Fusion-from-without (FFWO) is the induction of target cell fusion by addition of intact virions to cell monolayers in the absence of viral protein expression. The receptor requirements for HSV-induced FFWO are not known. We used the syncytial HSV-1 strain ANG path as a tool to evaluate the complex interplay between receptor usage, membrane fusion, and selection of entry pathway.

Results

Inhibitors of endocytosis and endosome acidification blocked ANG path entry into CHO cells expressing nectin-1 receptors, but not CHO-nectin-2 cells. Thus, under these conditions, nectin-2 mediates pH-independent entry at the plasma membrane. In addition, CHO-nectin-2 cells supported pH-dependent, endocytic entry of different strains of HSV-1, including rid1 and HFEM. The kinetics of ANG path entry was rapid (t_1/2 of 5–10 min) regardless of entry route. However, HSV-1 ANG path entry by fusion with the CHO-nectin-2 cell plasma membrane was more efficient and resulted in larger syncytia. ANG path virions added to the surface of CHO-nectin-2 cells, but not receptor-negative CHO cells or CHO-nectin-1 cells, induced rapid FFWO.

Conclusion

HSV-1 ANG path can enter CHO cells by either endocytic or non-endocytic pathways depending on whether nectin-1 or nectin-2 is present. In addition to these cellular receptors, one or more viral determinants is important for the selection of entry pathway. HSV-induced FFWO depends on the presence of an appropriate gD-receptor in the target membrane. Nectin-1 and nectin-2 target ANG path to divergent cellular pathways, and these receptors may have different roles in triggering viral membrane fusion.     

Plasma membrane topology of syncytial domains of herpes simplex virus type 1 glycoprotein K (gK): the UL20 protein enables cell surface localization of gK but not gK-mediated cell-to-cell fusion

Most spontaneously occurring mutations that cause extensive herpes simplex virus type 1 (HSV-1)-induced cell fusion are single amino acid changes within glycoprotein K (gK). Despite the strong genetic association of gK with virus-induced cell fusion, its direct involvement in cellular membrane fusion has been controversial, largely due to previously unsuccessful efforts to detect gK expression on virion and cellular surfaces. Recently, we showed that gK is expressed on HSV-1 virions and functioned in virus entry (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). To determine whether gK is expressed on cellular surfaces, as well as its membrane topology, we generated the recombinant viruses gKV5DI, gKV5DII, gKV5DIII, and gKV5DIVcontaining insertions of the V5 antigenic epitope within each of four domains of gK predicted to localize either in the cytoplasmic side or in the extracytoplasmic side of cellular membranes. Immunohistochemical and confocal microscopy analyses of infected cells showed that both wild-type and syncytial forms of gK were expressed on cell surfaces. Analysis of the topology of the V5-tagged gK revealed that gK domains I and IV were located extracellularly, whereas domains II and III were localized intracellularly. Transiently expressed gK failed to localize in cellular plasma membranes. In contrast, infection of gK-transfected cells with the gK-null virus DeltagK enabled expression of gK on cell surfaces, as well as gK-mediated membrane fusion. Transient-coexpression experiments revealed that the UL20 protein enabled cell surface expression of gK, but not gK-mediated cell-to-cell fusion, indicating that additional viral proteins are required for expression of the gK syncytial phenotype.     

The herpes simplex virus type 1 UL20 protein modulates membrane fusion events during cytoplasmic virion morphogenesis and virus-induced cell fusion

The herpes simplex virus type 1 (HSV-1) UL20 protein is an important determinant for virion morphogenesis and virus-induced cell fusion. A precise deletion of the UL20 gene in the HSV-1 KOS strain was constructed without affecting the adjacent UL20.5 gene. The resultant KOS/UL20-null virus produced small plaques of 8 to 15 cells in Vero cells while it produced wild-type plaques on the complementing cell line G5. Electron microscopic examination of infected cells revealed that the KOS/UL20-null virions predominantly accumulated capsids in the cytoplasm while a small percentage of virions were found as enveloped virions within cytoplasmic vacuoles. Recently, it was shown that UL20 expression was necessary and sufficient for cell surface expression of gK (T. P. Foster, X. Alvarez, and K. G. Kousoulas, J. Virol. 77:499-510, 2003). Therefore, we investigated the effect of UL20 on virus-induced cell fusion caused by syncytial mutations in gB and gK by constructing recombinant viruses containing the gBsyn3 or gKsyn1 mutations in a UL20-null genetic background. Both recombinant viruses failed to cause virus-induced cell fusion in Vero cells while they readily caused fusion of UL20-null complementing G5 cells. Ultrastructural examination of UL20-null viruses carrying the gBsyn3 or gKsyn1 mutation revealed a similar distribution of virions as the KOS/UL20-null virus. However, cytoplasmic vacuoles contained aberrant virions having multiple capsids within a single envelope. These multicapsid virions may have been formed either by fusion of viral envelopes or by the concurrent reenvelopment of multiple capsids. These results suggest that the UL20 protein regulates membrane fusion phenomena involved in virion morphogenesis and virus-induced cell fusion.     

Syncytial mutations in the herpes simplex virus type 1 gK (UL53) gene occur in two distinct domains.

Syncytial (syn) mutants of herpes simplex virus cause cell fusion. Many syn mutations map to the syn1 locus, which has been identified with the gK (UL53) gene. In this work, the gK genes of eight syn mutants derived from the KOS strain were sequenced to identify residues and, possibly, domains important for the fusion activity of mutant gK. DNA sequencing showed that six mutants (syn30, syn31, syn32, syn102, syn103, and syn105) had single missense mutations in the gK gene. Two of these, syn31 and syn32, had identical mutations that caused the introduction of a potential site for N-linked glycosylation. syn31 gK was analyzed by in vitro translation and found to utilize the novel glycosylation site. Two other mutants, syn8 and syn33, had three mutations each, resulting in three amino acid substitutions in syn8 and two substitutions in syn33. Of the 10 gK syn mutant sequences known, 8 have mutations in the N-terminal domain of gK, suggesting that this domain, which is likely to be an ectodomain, is important for the function of the protein. The other two mutants, syn30 and syn103, have mutations near the C terminus of gK.     

The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.Herpes simplex virus type 1 (HSV-1) can enter into cells via the fusion of its viral envelope with cellular membranes. Also, the virus can spread from infected to uninfected cells by causing virus-induced cell fusion, allowing virions to enter into uninfected cells without being exposed to extracellular spaces. These membrane fusion phenomena are known to be mediated by viral glycoproteins and other viral proteins (reviewed in reference 36). Although wild-type viruses cause a limited amount of virus-induced cell fusion, certain mutations cause extensive virus-induced cell-to-cell fusion (syncytial, or syn, mutations). These syncytial mutations are located predominantly within the UL20 gene (5, 27, 28); the UL24 gene (25, 38); the UL27 gene, encoding glycoprotein gB (7, 15, 18, 32); and the UL53 gene, coding for gK (6, 11, 24, 34, 35, 37).The presence of syncytial mutations within different viral genes, as well as other accumulating evidence, suggests that virus-induced cell fusion is mediated by the concerted action and interactions of the viral glycoproteins gD, gB, and gH/gL as well as gK and the membrane protein UL20p. Specifically, recent studies have shown that gD interacts with both gB and gH/gL (1, 2, 21). However, gB and gH/gL can also interact with each other even in the absence of gD (3). In this membrane fusion model, the binding of gD to its cognate receptors, including nectin-1, herpesvirus entry mediator (HVEM), and other receptors (8, 19, 30, 39-42), is thought to trigger sequential conformational changes in gH/gL and gB causing the fusion of the viral envelope with cellular membranes during virus entry as well as fusion among cellular membranes (22, 23). The transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (31, 43), suggesting that these four viral glycoproteins are necessary and sufficient for membrane fusion. However, this transient fusion system does not accurately depict virus-induced cell fusion. Specifically, viral glycoprotein K (gK) and the UL20 membrane protein (UL20p) have been shown to be strictly required for virus-induced cell fusion (10, 27, 29). Moreover, syncytial mutations within gK (6, 11, 24, 34, 35, 37) or UL20 (5, 27, 28) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than the wild-type virus into susceptible cells (17). In contrast, the transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while the coexpression of wild-type gK with gB, gD, and gH/gL was reported previously to inhibit cell fusion in certain cell lines (4). To date, there is no direct evidence that either gK or UL20p interacts with gB, gD, gH, or gL.The X-ray structure of the ectodomain of HSV-1 gB has been determined and was predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB (23). Single-amino-acid changes within the carboxyl terminus of gB located intracellularly as well as the deletion of the terminal 28 amino acids (aa) of gB cause extensive virus-induced cell fusion, presumably because they alter the extracellular conformation of gB (15, 31, 43). We have previously shown that HSV-1 gK and UL20p functionally and physically interact and that these interactions are absolutely necessary for their coordinate intracellular transport, cell surface expression, and functions in the HSV-1 life cycle (13, 16). In contrast to gB, syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (12), while syncytial mutations of UL20 are located within the amino terminus of UL20p shown to be located intracellularly (27).Recently, we showed that the a peptide composed of the amino-terminal 82 amino acids of gK (gKa) can complement in trans for gB-mediated cell fusion caused by the deletion of the carboxyl-terminal 28 amino acids of gB, suggesting that the gKa peptide interacted with gB or other viral glycoproteins involved in virus-induced cell fusion (10). In this work, we demonstrate that UL20p and the amino terminus of gKa physically interact with gB in infected cells, while the gKa peptide is also capable of binding to the extracellular portion of gH, suggesting that gK/UL20p modulates virus-induced cell fusion via direct interactions with gB and gH.     

Loss of protein kinase PKR expression in human HeLa cells complements the vaccinia virus E3L deletion mutant phenotype by restoration of viral protein synthesis

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (ΔE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of ΔE3L virus was increased by nearly 2 log₁₀ in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the ΔE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in ΔE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the α subunit of protein synthesis initiation factor 2 (eIF-2α) was elevated severalfold in ΔE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2α phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with ΔE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.     

Pseudorabies Virus Glycoprotein gK Is a Virion Structural Component Involved in Virus Release but Is Not Required for Entry

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560–5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase–gK fusion protein expressed in Escherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKβ) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB⁻ genome (PrV-gK^gB). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKβ was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.Herpesvirions are complex structures consisting of a nucleoprotein core, capsid, tegument, and envelope. They comprise at least 30 structural proteins (35). Pseudorabies virus (PrV), a member of the Alphaherpesvirinae, is an economically important animal pathogen, causing Aujeszky’s disease in swine. It is also highly pathogenic for most other mammals except higher primates, including humans (28, 45), and a wide range of cultured cells from different species support productive virus replication, reflecting the wide in vivo host range. Envelope glycoproteins play major roles in the early and late interactions between virion and host cell. They are required for virus entry and participate in release of free virions and viral spread by direct cell-to-cell transmission (27, 37). For PrV, 10 glycoproteins, designated gB, gC, gD, gE, gG, gH, gI, gL, gM, and gN, have been characterized (20, 27); these glycoproteins are involved in the attachment of virion to host cell (gC and gD), fusion of viral envelope and cellular cytoplasmic membrane (gB, gD, gH, and gL), spread from infected to noninfected cells (gB, gE, gH, gI, gL, and gM), and egress (gC, gE, and gI) (27, 37). Homologs of these glycoproteins are also present in other alphaherpesviruses (37). The gene coding for a potential 11th PrV glycoprotein, gK, has been described recently (3), but the protein and its function have not been identified.The product of the homologous UL53 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) is gK (13, 32). gK was detected in nuclear membranes and in membranes of the endoplasmic reticulum but was not observed in the plasma membrane (14). Also, it did not appear to be present in purified virion preparations (15). The latter result was surprising since earlier studies identified several mutations in HSV-1 gK resulting in syncytium-inducing phenotypes (7, 14), which indicates participation of gK in membrane fusion events during HSV-1 infection. Moreover, HSV-1 mutants in gK exhibited a delayed entry into noncomplementing cells, which is difficult to reconcile with absence of gK from virions (31). Mutants deficient for gK expression have been isolated and investigated by different groups (16, 17). Mutant F-gKβ carries a lacZ gene insertion in the HSV-1 strain F gK gene, which interrupts the ORF after codon 112 (16). In mutant ΔgK, derived from HSV-1 KOS, almost all of the UL53 gene was deleted (17). Both mutants formed small plaques on Vero cells, and virus yield was reduced to an extent which varied with the different confluencies of the infected cells, cell types, and mutants used for infection. However, both HSV-1 gK mutants showed a defect in efficient translocation of virions from the cytoplasm to the extracellular space, and only a few enveloped virions were present in the extracellular space after infection of Vero cells (16, 17). The authors therefore suggested that HSV-1 gK plays a role in virion transport during egress.Different routes of final envelopment and egress of alphaherpesvirions are discussed. It has been suggested that HSV-1 nucleocapsids acquire their envelope at the inner nuclear membrane and are transported as enveloped particles through the endoplasmic reticulum to the Golgi stacks, where glycoproteins are modified in situ during transport (5, 6, 19, 39), although other potential egress pathways cannot be excluded (4). In contrast, maturation of varicella-zoster virus and PrV involves primary envelopment at the nuclear membrane, followed by release of nucleocapsids into the cytoplasm and secondary envelopment in the trans-Golgi area (10, 12, 43). Final egress of virions appears to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. The possibility of different routes of virion egress is supported by studies of other proteins involved in egress, e.g., the UL20 proteins of HSV-1 and PrV and the PrV UL3.5 protein, which lacks a homolog in the HSV-1 genome (1, 8, 9). In UL20-negative HSV-1, virions accumulated in the perinuclear cisterna of Vero cells (1), while PrV UL20⁻ virions accumulated and were retained in cytoplasmic vesicles (9). PrV UL3.5 is important for budding of nucleocapsids into Golgi-derived vesicles during secondary envelopment (8). Thus, there appear to be profound differences in the egress pathways. Since HSV-1 gK was also implicated in egress, we were interested in identifying the PrV homolog and analyzing its function.     

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.     

Antibodies Are Required for Complete Vaccine-Induced Protection against Herpes Simplex Virus 2

Herpes simplex virus 2 (HSV-2) 0ΔNLS is a live HSV-2 ICP0 ^- mutant vaccine strain that is profoundly attenuated in vivo due to its interferon-hypersensitivity. Recipients of the HSV-2 0ΔNLS vaccine are resistant to high-dose HSV-2 challenge as evidenced by profound reductions in challenge virus spread, shedding, disease and mortality. In the current study, we investigated the requirements for HSV-2 0ΔNLS vaccine-induced protection. Studies using (UV)-inactivated HSV-2 0ΔNLS revealed that self-limited replication of the attenuated virus was required for effective protection from vaginal or ocular HSV-2 challenge. Diminished antibody responses in recipients of the UV-killed HSV-2 vaccine suggested that antibodies might be playing a critical role in early protection. This hypothesis was investigated in B-cell-deficient μMT mice. Vaccination with live HSV-2 0ΔNLS induced equivalent CD8⁺ T cell responses in wild-type and μMT mice. Vaccinated μMT mice shed ~40-fold more infectious HSV-2 at 24 hours post-challenge relative to vaccinated wild-type (B-cell⁺) mice, and most vaccinated μMT mice eventually succumbed to a slowly progressing HSV-2 challenge. Importantly, passive transfer of HSV-2 antiserum restored full protection to HSV-2 0ΔNLS-vaccinated μMT mice. The results demonstrate that B cells are required for complete vaccine-induced protection against HSV-2, and indicate that virus-specific antibodies are the dominant mediators of early vaccine-induced protection against HSV-2.