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.     

Fusion between Perinuclear Virions and the Outer Nuclear Membrane Requires the Fusogenic Activity of Herpes Simplex Virus gB

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic “fusion loops.” These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.Herpesvirus glycoproteins gB and gH/gL participate in two separate membrane fusion events that occur during different stages of virus replication. First, during virus entry into cells, gB and gH/gL promote fusion between the virion envelope and either the plasma membrane or endosomes (reviewed in references 6, 21, 27, and 39). Second, herpes simplex virus (HSV) gB and gH (likely complexed to form a heterodimer with gL), and likely homologues in other herpesviruses, promote nuclear egress (12). Herpesvirus capsids are produced in the nucleus and cross the nuclear envelope (NE) by envelopment at the inner nuclear membrane (NM), producing perinuclear virions that then fuse with the outer NM (reviewed in references 35 and 36). There is evidence that HSV gB and gH/gL function in a redundant fashion in fusion between enveloped, perinuclear virus particles and the outer NM (12), whereas both gB and gH/gL are essential for entry fusion (8, 13, 38). Much more is known about the mechanisms involved in entry fusion than those involved in egress fusion, and many important questions remain in terms of how these two membrane fusion processes relate to each other.Entry of HSV into cells involves interactions between the viral receptor-binding protein gD and the gD receptors (16, 28, 30, 37). When gD binds to its receptors, there are conformational changes in gD which apparently activate gB and gH/gL, so that these glycoproteins promote fusion involving the virion envelope and cellular membranes (21, 32). By using split green fluorescent protein fusion proteins, also denoted bimolecular complementation, two groups showed that gD binding to gD ligands triggers interactions between gB and gH/gL and that this is accompanied by cell-cell fusion (1, 2). There is also evidence that gB and gH/gL contribute to different stages of membrane fusion. When gH/gL is expressed with gD, there is hemifusion (mixing of the outer leaflets of membranes) of adjacent cells, and this partial fusion is apparently mediated by gH/gL (41). However, full fusion (mixing of both inner and outer leaflets) occurs only when gB is coexpressed with gD and gH/gL (41). Also supporting a role for gH in membrane fusion, peptides based on heptad repeats in gH can disrupt model membranes (14, 15, 17). HSV gB is a class III fusion protein, structurally similar to vesicular stomatitis virus G protein, with a three-stranded coil-coil barrel in the central region of the molecule reminiscent of class I fusion proteins, e.g., influenza virus hemagglutinin (22). Therefore, herpesvirus gB and gH/gL differ substantially from the fusion proteins expressed by all other well-studied viruses because both gB and gH/gL participate directly in membrane fusion, apparently functioning in different aspects of entry fusion.HSV gB and other viral class III fusion proteins differ from class I fusion proteins that have N-terminal, hydrophobic fusion peptides because class III fusion proteins possess internal bipartite “fusion loops” composed of both hydrophobic and hydrophilic residues (3, 22). In the solved structure of the HSV gB ectodomain, which might represent a postfusion form of the protein, the fusion loops are located near the base of the molecule, adjacent to the virion envelope (22). Mutant forms of gB with single amino acid substitutions in these fusion loops displayed diminished cell-cell fusion activity when transfected into cells with gD and gH/gL (20). Cell-cell fusion approximates the fusion that occurs during entry, defining the minimal fusion machinery, although there are differences between entry and cell-cell fusion (10). Moreover, full-length gB molecules with fusion loop mutations failed to complement gB null HSV (19). Recently, it was demonstrated that the HSV gB extracellular domain can interact with liposomes in vitro and that this binding depends upon gB''s fusion loops (19).Herpesvirus capsids are assembled in the nucleus and acquire an envelope by budding through the inner NM. For a short time, enveloped virus particles are found in the space between the inner and outer NMs (perinuclear space), but then the envelopes of these particles fuse with the outer NM, releasing capsids into the cytoplasm (reviewed in references 35 and 36). Cytoplasmic capsids acquire a second envelope by budding into the trans-Golgi network, and this secondary envelopment involves redundant or additive functions of gE/gI and gD, i.e., either of these glycoproteins will suffice (11). The second step of the nuclear egress pathway involving membrane fusion between the envelope of perinuclear particles and the outer NM requires HSV glycoproteins gB and gH/gL (12). HSV double mutants lacking both gB and gH accumulate enveloped virus particles in the perinuclear space and in herniations, i.e., membrane vesicles that bulge into the nucleoplasm and derive from the inner NM (12). These observations, coupled with the evidence that gB and gH/gL are fusion proteins, suggested that gB and gH/gL promote the fusion between virus particles and the outer NM. However, there is one important difference between nuclear egress fusion and entry fusion. Virus mutants lacking either gB or gH are unable to enter cells, but such mutants have fewer defects in nuclear egress than double mutants lacking both gB and gH (12). Thus, as with secondary envelopment that involves gD and gE/gI, glycoproteins gB and gH/gL act in a redundant or additive fashion to mediate the fusion between the envelope of perinuclear virus particles and the outer NM. It is also important to note that there appear to be other mechanisms by which HSV particles can exit the perinuclear space. For example, although a substantial number of gB gH null double mutants accumulated in herniations (increased by ∼10-fold), some virions were seen on cell surfaces, although their numbers were reduced by ∼2.5- to 5-fold compared with those of wild-type HSV (12, 46).HSV entry fusion is triggered by gD binding to one of its ligands. However, it is not clear what triggers fusion of the envelope of perinuclear particles with the outer NM. gD, gB, gH, gM, gK, and other viral membrane proteins are all present in NMs and in perinuclear virus particles (4, 12, 25, 40, 42, 44). It seems unlikely that there are substantial quantities of known gD receptors in NMs, although this has not been carefully examined and there may well be unidentified gD receptors present in NMs. However, if fusion at NMs is not activated by gD binding to gD receptors, there must be other mechanisms to trigger this fusion. There is evidence that HSV gK negatively regulates fusion at the NE because (i) overexpression of gK causes enveloped virus particles to accumulate in the perinuclear space (25) and (ii) gK is primarily localized to the endoplasmic reticulum and NM and is not substantially found in extracellular virions (26, 34). Another potential regulatory mechanism for fusion at the outer NM involves phosphorylation of the cytoplasmic domain of gB by the HSV kinase US3 (46). An HSV recombinant lacking gH and expressing a mutant gB with a substitution, T887A, affecting an amino acid in the gB cytoplasmic domain displayed reduced US3-dependent phosphorylation and accumulated enveloped virus particles in herniations (46). This mutation in gB did not alter HSV entry into cells (31, 46). Together, these results suggest that HSV fusion with the outer NM differs from entry fusion in some, but likely not all, important mechanistic details.Given that both gB and gH/gL are well established as fusion proteins for virus entry, we hypothesized that these glycoproteins directly mediate the membrane fusion that occurs between the envelope of perinuclear virus particles and the outer NM (12, 46). However, there are other possibilities. For example, it is conceivable that loss of both gB and gH alters the structure of the envelope of perinuclear HSV virions so that other HSV glycoproteins (that directly promote fusion) are affected. To address this issue and extend our understanding of how gB functions in nuclear egress fusion, we constructed HSV recombinants that express mutant forms of gB with substitutions in the fusion loops. These viruses also lacked gH, making nuclear egress totally dependent on a functional form of gB. By propagating these recombinants using gH-expressing cells, we could produce virus particles including gH and the mutant gB molecules. These HSV recombinants expressing gH as well as gB fusion loops, W174R, W174Y, Y179K, and A261D, were all unable to enter cells. However, two recombinants, expressing W174Y and Y179K, exhibited some cell-to-cell spread while the other two, expressing W174R and A261D, did not spread beyond single infected cells. All four recombinants infected into cells lacking gH exhibited defects in nuclear egress. These results provide strong support for the hypothesis that gB acts directly to mediate the fusion of the virion envelope with the outer NM during HSV egress.     

Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

Pseudorabies virus (PRV) Us9 is a small, tail-anchored (TA) membrane protein that is essential for axonal sorting of viral structural proteins and is highly conserved among other members of the alphaherpesvirus subfamily. We cloned the Us9 homologs from two human pathogens, varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1), as well as two veterinary pathogens, equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1), and fused them to enhanced green fluorescent protein to examine their subcellular localization and membrane topology. Akin to PRV Us9, all of the Us9 homologs localized to the trans-Golgi network and had a type II membrane topology (typical of TA proteins). Furthermore, we examined whether any of the Us9 homologs could compensate for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection in a compartmented chamber system. EHV-1 and BHV-1 Us9 were able to fully compensate for the loss of PRV Us9, whereas VZV and HSV-1 Us9 proteins were unable to functionally replace PRV Us9 when they were expressed in a PRV background.Alphaherpesviruses are classified by their variable host range, short reproductive cycle, and ability to establish latency in the peripheral nervous system (PNS) (36, 37). Commonly studied pathogens of this subfamily include herpes simplex virus (HSV) and varicella-zoster virus (VZV), as well as the veterinary pathogens pseudorabies virus (PRV), equine herpesvirus (EHV), and bovine herpesvirus (BHV). Initial infection begins with the virus entering the host mucosal surfaces and spreading between cells of the mucosal epithelium. Invariably, virus enters the PNS through the infection of peripheral nerves that innervate this region. The virus establishes a latent infection in PNS neurons that can be reactivated and that persists for the life of the host (36). In most natural infections, virus replication in the PNS never spreads to the central nervous system (CNS). However, on rare occasions, invasion of the CNS does occur, resulting in devastating encephalitis (46). Trafficking of virus particles from infected epithelial cells into the axon and subsequent transport to neuronal cell bodies is known as retrograde spread of infection. Trafficking of virus particles that are assembled in the neuronal cell body and subsequently sorted into axons for transport to epithelial cells at the initial site of infection (upon reactivation from latency) is known as anterograde spread of infection.Though the natural host of PRV is swine, the virus infects a wide variety of animals, including rodents, cats, dogs, rabbits, cattle, and chicken embryos, but not higher primates (1, 30, 47). In contrast to the well-contained spread of PRV within its natural host, infection of other mammals is usually lethal. Instead of stopping in the PNS, infection continues on to second-order and third-order neurons in the CNS (reviewed in reference 35). This facet of PRV infection makes it a useful tracer of neuronal connections (18). Work in our lab has identified three PRV proteins, Us9 and the gE/gI heterodimer, which are critical for efficient anterograde spread of infection in vivo (i.e., spread from presynaptic to postsynaptic neurons) (6, 45). The molecular mechanism by which these proteins function has been further elucidated in vitro using primary neuronal cultures of superior cervical ganglion (SCG) harvested from embryonic rat pups. PRV Us9 and, to a lesser extent, gE/gI are required for efficient axonal targeting of viral structural proteins, a necessary step for subsequent anterograde, transneuronal spread (10, 11, 27, 28, 42).PRV Us9 is a type II, tail-anchored (TA) membrane protein that is highly enriched in lipid raft microdomains and resides predominantly in or near the trans-Golgi network (TGN) inside infected cells (5-7, 27). It has homologs in most of the alphaherpesviruses, including VZV (16), HSV-1 (22), HSV-2 (17), EHV-1 (21, 40), EHV-4 (41), BHV-1 (25), and BHV-5 (14). Though several studies have examined individually the Us9 proteins encoded by VZV (16), HSV-1 (4, 22, 34, 39), BHV-1 (13), and BHV-5 (14), several gaps in our understanding of Us9 biology remain, namely, whether all of the PRV Us9 homologs are type II membrane proteins, if the proteins localize to similar subcellular compartments within different cell types, and if they can functionally substitute for the loss of PRV Us9 in axonal sorting and anterograde spread of infection. The aim of this study is to examine PRV Us9 in parallel assays with its homologs from VZV, HSV-1, EHV-1, and BHV-1 to identify potential similarities and differences between these highly conserved alphaherpesvirus proteins.     

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.     

Herpes Simplex Virus Type 1 Glycoprotein E Mediates Retrograde Spread from Epithelial Cells to Neurites

In animal models of infection, glycoprotein E (gE) is required for efficient herpes simplex virus type 1 (HSV-1) spread from the inoculation site to the cell bodies of innervating neurons (retrograde direction). Retrograde spread in vivo is a multistep process, in that HSV-1 first spreads between epithelial cells at the inoculation site, then infects neurites, and finally travels by retrograde axonal transport to the neuron cell body. To better understand the role of gE in retrograde spread, we used a compartmentalized neuron culture system, in which neurons were infected in the presence or absence of epithelial cells. We found that gE-deleted HSV-1 (NS-gEnull) retained retrograde axonal transport activity when added directly to neurites, in contrast to the retrograde spread defect of this virus in animals. To better mimic the in vivo milieu, we overlaid neurites with epithelial cells prior to infection. In this modified system, virus infects epithelial cells and then spreads to neurites, revealing a 100-fold retrograde spread defect for NS-gEnull. We measured the retrograde spread defect of NS-gEnull from a variety of epithelial cell lines and found that the magnitude of the spread defect from epithelial cells to neurons correlated with epithelial cell plaque size defect, indicating that gE plays a similar role in both types of spread. Therefore, gE-mediated spread between epithelial cells and neurites likely explains the retrograde spread defect of gE-deleted HSV-1 in vivo.Herpes simplex virus type 1 (HSV-1) is an alphaherpesvirus that characteristically infects skin and mucosal surfaces before spreading to sensory neurons, where it establishes a lifelong persistent infection. The virus periodically returns to the periphery via sensory axons and causes recurrent lesions as well as asymptomatic shedding. This life cycle requires viral transport along axons in two directions: toward the neuron cell body (retrograde direction) and away from the neuron cell body (anterograde direction).Many studies of alphaherpesvirus neuronal spread have focused on pseudorabies virus (PRV), a virus whose natural host is the pig. Three PRV proteins, glycoprotein E (gE), gI, and Us9, have been shown to mediate anterograde neuronal spread both in animal models of infection and in cultured neurons. However, these three proteins are dispensable for retrograde spread (3, 8, 11, 12, 31, 46). In contrast, numerous animal models of infection have shown that HSV-1 gE is required for retrograde spread from the inoculation site to the cell bodies of innervating neurons (4, 9, 44, 56). In the murine flank model, wild-type (WT) virus replicates in the skin and then infects sensory neurons and spreads in a retrograde direction to the dorsal root ganglia (DRG). In this model, gE-deleted HSV-1 replicates in the skin but is not detected in the DRG (9, 44). This phenotype differs from gE-deleted PRV, which is able to reach the DRG at WT levels (8). Thus, unlike PRV, gE-deleted HSV-1 viruses have a retrograde spread defect in vivo.HSV-1 gE is a 552-amino-acid type I membrane protein found in the virion membrane as well as in the trans-Golgi and plasma membranes of infected cells (1). gE forms a heterodimer with another viral glycoprotein, gI. The gE/gI complex is important for HSV-1 immune evasion through its Fc receptor activity. gE/gI binds to the Fc domain of antibodies directed against other viral proteins, sequestering these antibodies and blocking antibody effector functions (27, 32, 40). Additionally, gE/gI promotes spread between epithelial cells. Viruses lacking either gE or gI form characteristically small plaques in cell culture and small inoculation site lesions in mice (4, 9, 18, 40, 58). In animal models, gE and gI also mediate viral spread in both anterograde and retrograde directions (4, 19, 44, 56).In order to better understand the role of gE in HSV-1 retrograde neuronal spread, we employed a compartmentalized neuron culture system that has been used to study directional neuronal spread of PRV and West Nile virus (12, 14, 45). In the Campenot chamber system, neurites are contained in a compartment that is separate from their corresponding cell bodies. Therefore, spread in an exclusively retrograde direction can be measured by infecting neurites and detecting spread to neuron cell bodies.HSV-1 replication requires retrograde transport of incoming viral genomes to the nucleus. In neurites, fusion between viral and cellular membranes occurs at the plasma membrane (43, 48). Upon membrane fusion, the capsid and a subset of tegument proteins (the inner tegument) dissociate from glycoproteins and outer tegument proteins, which remain at the plasma membrane (28, 38). Unenveloped capsids and the associated inner tegument proteins are then transported in the retrograde direction to the nucleus (7, 48, 49).For both neurons and epithelial cells, retrograde transport is dependent upon microtubules, ATP, the retrograde microtubule motor dynein, and the dynein cofactor dynactin (22, 34, 49, 52). Several viral proteins interact with components of the dynein motor complex (23, 39, 60). However, none of these proteins suggest a completely satisfactory mechanism by which viral retrograde transport occurs, either because they are not components of the complex that is transported to the nucleus (UL34, UL9, VP11/12) or because capsids lacking that protein retain retrograde transport activity (VP26) (2, 17, 21, 28, 37). This implies that additional viral proteins are involved in retrograde trafficking.We sought to better characterize the role of gE in retrograde spread and found that gE is dispensable for retrograde axonal transport; however, it promotes HSV-1 spread from epithelial cells to neurites. This epithelial cell-to-neuron spread defect provides a plausible explanation for the retrograde spread defect of gE-deleted HSV-1 in animal models of infection.     

Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B

Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.Herpes simplex virus (HSV) is an important human pathogen, causing significant morbidity and mortality worldwide. HSV enters host cells by fusion of the viral envelope with either an endosomal membrane (38) or the plasma membrane (63). The entry pathway taken is thought to be determined by both virus (17, 45) and host cell (4, 17, 35, 39, 45) factors. Based on experiments with lysosomotropic agents, which elevate the normally low pH of endosomes, acidic pH has been implicated in the endocytic entry of HSV into several cell types, including human epithelial cells (37). Low pH has also recently been implicated in cell infection by several other human and veterinary herpesviruses (1, 21, 26, 47). The mechanistic role of endosomal pH in herpesvirus entry into cells is not known.Herpesviruses are a paradigm for membrane fusion mediated by a complex of several glycoproteins. We have proposed that HSV likely encodes machinery to mediate both pH-dependent and pH-independent membrane fusion reactions. Envelope glycoproteins glycoprotein B (gB) and gD and the heterodimer gH-gL are required for both pH-independent and pH-dependent entry pathways (11, 22, 30, 39, 46). Interaction of gD with one of its cognate receptors is an essential trigger for membrane fusion and entry (13, 52), regardless of the cellular pathway. However, engagement of a gD receptor is not sufficient for fusion, and at least one additional unknown trigger involving gB or gH-gL is likely necessary. gB is conserved among all herpesviruses, and in all cases studied to date, it plays roles in viral entry, including receptor binding and membrane fusion. The crystal structure of an ectodomain fragment of HSV type 1 (HSV-1) gB is an elongated, rod-like structure containing hydrophobic internal fusion loops (28). This structure bears striking architectural homology to the low pH, postfusion form of G glycoprotein from vesicular stomatitis virus (VSV-G) (43). Both the gB and G structures have features of class I and class II fusion proteins and are thus designated class III proteins (57).During entry of the majority of virus families, low pH acts directly on glycoproteins to induce membrane fusion (60). In some cases, the low pH trigger is not sufficient, or it plays an indirect role. For example, host cell proteases, such as cathepsins D and L, require intravesicular low pH to cleave Ebola virus and severe acute respiratory syndrome (SARS) glycoproteins to trigger fusion (14, 51).We investigated the role of low pH in the molecular mechanism of herpesviral entry. The results suggest that mildly acidic pH, similar to that found within endosomes, triggers a conformational change in gB. We propose that, together with other cellular cues such as receptor interaction, intracellular low pH can play a direct activating role in HSV membrane fusion and entry.     

The UL31 and UL34 Gene Products of Herpes Simplex Virus 1 Are Required for Optimal Localization of Viral Glycoproteins D and M to the Inner Nuclear Membranes of Infected Cells

U_L31 and U_L34 of herpes simplex virus type 1 form a complex necessary for nucleocapsid budding at the inner nuclear membrane (INM). Previous examination by immunogold electron microscopy and electron tomography showed that pU_L31, pU_L34, and glycoproteins D and M are recruited to perinuclear virions and densely staining regions of the INM where nucleocapsids bud into the perinuclear space. We now show by quantitative immunogold electron microscopy coupled with analysis of variance that gD-specific immunoreactivity is significantly reduced at both the INM and outer nuclear membrane (ONM) of cells infected with a U_L34 null virus. While the amount of gM associated with the nuclear membrane (NM) was only slightly (P = 0.027) reduced in cells infected with the U_L34 null virus, enrichment of gM in the INM at the expense of that in the ONM was greatly dependent on U_L34 (P < 0.0001). pU_L34 also interacted directly or indirectly with immature forms of gD (species expected to reside in the endoplasmic reticulum or nuclear membrane) in lysates of infected cells and with the cytosolic tail of gD fused to glutathione S-transferase in rabbit reticulocyte lysates, suggesting a role for the pU_L34/gD interaction in recruiting gD to the NM. The effects of U_L34 on gD and gM localization were not a consequence of decreased total expression of gD and gM, as determined by flow cytometry. Separately, pU_L31 was dispensable for targeting gD and gM to the two leaflets of the NM but was required for (i) the proper INM-versus-ONM ratio of gD and gM in infected cells and (ii) the presence of electron-dense regions in the INM, representing nucleocapsid budding sites. We conclude that in addition to their roles in nucleocapsid envelopment and lamina alteration, U_L31 and U_L34 play separate but related roles in recruiting appropriate components to nucleocapsid budding sites at the INM.Herpesvirus virions comprise a nucleocapsid containing genomic viral DNA, a proteinaceous tegument layer surrounding the nucleocapsid, and a virion envelope surrounding the tegument. The envelope of extracellular herpes simplex virus (HSV) virions contains glycoproteins gB, gC, gD, gE, gI, gG, gH, gK, gL, and gM (23, 51).As viewed by electron microscopy, nascent virions form as the nucleocapsid buds through densely staining regions of the nuclear membrane (NM) (21, 41). Electron tomograms of HSV perinuclear virions compared to those of extracellular virions infer that the former contain glycoproteins of considerably less glycosylation and a relatively sparse tegument layer compared to their counterparts in mature extracellular virions (6). The lower levels of glycosylation in HSV perinuclear virions are consistent with the fact that the lumen of the perinuclear space is continuous with that of the endoplasmic reticulum. Thus, the polysaccharide moieties of virion glycoproteins become fully processed as virions access Golgi enzymes during their egress to the extracellular space. Although the full proteome of the nascent perinuclear virion is unknown, immunogold studies have shown that they contain at least pU_L31, pU_L34, pUS3, gB, gC, gD, gH, gM, and the VP16 and pU_L11 tegument proteins in addition to the proteins that comprise the viral capsid (4, 5, 15, 25, 37, 40, 47, 50, 55).The U_L31 and U_L34 gene products of HSV-1 (pU_L31 and pU_L34, respectively) form a complex that localizes at the inner and outer NMs (INM and ONM, respectively) of infected cells (40). Both proteins are essential for nucleocapsid envelopment at the INM and become incorporated into nascent virions when nucleocapsids bud through the INM into the perinuclear space (39, 40, 42). The proteins and their essential role in nucleocapsid envelopment are conserved in all herpesvirus subfamilies (14, 20, 32, 45). pU_L31 of HSV-1 is a mostly hydrophobic phosphoprotein that is held in close approximation to the nucleoplasmic face of the INM by interaction with pU_L34, an integral membrane protein of type II orientation (33, 40, 46, 56). The first 248 amino acids of pU_L34 are predicted to reside in the nucleoplasm or cytoplasm, depending on whether the protein localizes in the INM or ONM, respectively. This is followed by an approximately 22-amino acid transmembrane domain with up to 5 amino acids residing in the perinuclear space or lumen of the endoplasmic reticulum.In the most prominent model of herpesvirion egress, the envelope of the perinuclear virion fuses with the ONM, releasing the deenveloped nucleocapsid into the cytoplasm, where it subsequently buds into cytoplasmic membranous organelles such as the Golgi or trans-Golgi network (34, 49). This model is supported by the observation that pU_L31 and pU_L34 are located in the perinuclear virion but not extracellular virions (18, 40). Thus, these proteins are lost from the virion upon fusion of the virion envelope with the ONM. Also supporting this egress model is the observation that deletion of both gB and gH causes virions to accumulate aberrantly in the perinuclear space (15). The involvement of gH and gB is potentially satisfying because these proteins comprise essential components of the machinery that mediates fusion of the virion envelope with the plasma or endosomal membranes during the initiation of infection (9, 12, 16, 44, 52). Moreover, expression of a combination of gB, gD, gH, and gL is sufficient to mediate fusion of cell membranes, whereas coexpression with gM or gK inhibits this fusion (3, 8, 11). Although the mechanism of fusion is unclear, gD is known to bind viral receptors on cell surfaces, and the structure of gB indicates features reminiscent of other viral fusion proteins (24, 35, 48). gD has been shown to interact with gB and gH at least transiently, suggesting that these interactions may be important for the fusion reaction (1, 2). Thus, fusion between the nascent and mature virion envelopes with target membranes may share mechanistic similarities.On the other hand, it is likely that the two fusion events are mechanistically distinct because (i) single deletion of either gH or gB precludes viral entry and cell/cell fusion but does not cause nascent virions to accumulate in the perinuclear space (9, 16, 31, 43) and (ii) the activity of a viral kinase encoded by U_S3 is dispensable for entry but believed to promote fusion of the perinuclear virion and ONM (28, 40). Moreover, the lack of glycoproteins from the pseudorabies virus perinuclear virion suggests that fusion is mediated by an entirely different mechanism in this system (26).The current study focuses on how glycoproteins are incorporated into the nascent virion. We show that optimal recruitment of gD to both leaflets of the NM and gM to the INM requires pU_L34 and pU_L31. We also show that immature gD interacts with pU_L34, suggesting a mechanism by which pU_L34 might recruit gD to the NM.     

Effects of Phosphorylation of Herpes Simplex Virus 1 Envelope Glycoprotein B by Us3 Kinase In Vivo and In Vitro

We recently reported that the herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) (A. Kato, J. Arii, I. Shiratori, H. Akashi, H. Arase, and Y. Kawaguchi, J. Virol. 83:250-261, 2009; T. Wisner, C. C. Wright, A. Kato, Y. Kawaguchi, F. Mou, J. D. Baines, R. J. Roller and D. C. Johnson, J. Virol. 83:3115-3126, 2009). In the studies reported here, we examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo and present data showing that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis and periocular skin disease in mice. The same effects have been reported for mice infected with a recombinant HSV-1 carrying a kinase-inactive mutant of Us3. These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically reacted with phosphorylated gB Thr-887 and used this antibody to show that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly on the cell surface of infected cells.The herpes simplex virus 1 (HSV-1) Us3 gene encodes a serine/threonine protein kinase with an amino acid sequence that is conserved in the subfamily Alphaherpesvirinae (9, 20, 29). The Us3 kinase phosphorylation target site has been reported to be similar to that of protein kinase A (PKA), a cellular cyclic AMP-dependent protein kinase (3, 12). Us3 catalytic activity plays important roles in viral replication and pathogenesis in vivo, based on studies showing that recombinant Us3 null mutant viruses and recombinant viruses encoding catalytically inactive Us3 have significantly reduced virulence, pathogenicity, and replication in mouse models (21, 34). In contrast, Us3 is not essential for growth in tissue culture cells (29). Thus, recombinant Us3 mutants grow as well as wild-type virus in Vero cells and have modestly impaired growth in a specific cell line such as HEp-2 cells (32, 33). The catalytic activity of Us3 is, in part, regulated by autophosphorylation of its serine at position 147 (Ser-147), and regulation of Us3 activity by autophosphorylation of Ser-147 appears to play a critical role in HSV-1 replication in vivo and in HSV-1 pathogenesis (34). Numerous studies have elucidated the potential downstream effects of Us3, including blocking apoptosis (18, 26-28), promoting nuclear egress of progeny nucleocapsids through the nuclear membrane (24, 32, 33), redistributing and phosphorylating nuclear membrane-associated viral nuclear egress factors UL31 and UL34 (13, 24, 30, 31) and cellular proteins including lamin A/C and emerin (16, 22, 23), controlling infected cell morphology (12, 27), and downregulating cell surface expression of viral envelope glycoprotein B (gB) (11).Two substrates that mediate some of the Us3 functions described above have been identified. First, it has been shown that Us3 phosphorylates Thr-887 in the cytoplasmic tail of gB, which appears to downregulate cell surface expression of gB (11). This conclusion is based on the observation that a T887A mutation in gB (gB-T887A) markedly upregulated cell surface expression of gB in infected cells: this upregulation was also observed with a recombinant virus encoding a Us3 kinase-inactive mutant, whereas a phosphomimetic substitution for gB Thr-887 restored wild-type cell surface expression of gB (11). Us3 phosphorylation of gB Thr-887 has also been proposed to be involved in regulation of fusion of the nascent progeny virion envelope with the cell''s outer nuclear membrane, based on the observation that virions accumulated aberrantly in the perinuclear space in cells infected with a mutant virus carrying the gB-T887A substitution mutation and lacking the capacity to produce gH (42). Second, it has been shown that Us3 may phosphorylate some or all of the six serines in the UL31 N-terminal region (24). Such phosphorylation might regulate proper localization of UL31 and UL34 at the nuclear membrane, nuclear egress of nucleocapsids, and viral growth in cell cultures since the Us3 kinase-inactive mutant phenotype for nuclear egress (i.e., mislocalization of UL31 and UL34 at the nuclear membrane, aberrant accumulation of virions within herniations of the nuclear membrane, and decreased viral growth in cell cultures) is also produced by replacement of the six serines in the UL31 N-terminal region with alanines while phosphomimetic substitutions of the six serines restored the wild-type phenotype (24).Thus, the molecular mechanisms for some of the downstream effects of Us3 phosphorylation have been gradually elucidated. However, it remains to be shown whether the Us3 functions reported to date are in fact involved in viral replication and pathogenicity in vivo. In the present study, we focused on Us3 phosphorylation of gB Thr-887 and examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo. These studies have shown that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis (HSK) and periocular skin disease in mice, as reported for infection of mice with a recombinant virus carrying a Us3 kinase-inactive mutant (34). These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically recognized phosphorylated gB Thr-887 and used this antibody to directly study the functional consequences of Us3 phosphorylation of gB Thr-887 in infected cells. We also present data showing that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly gB localization on the cell surface of infected cells.     

Mutagenesis of Varicella-Zoster Virus Glycoprotein B: Putative Fusion Loop Residues Are Essential for Viral Replication,and the Furin Cleavage Motif Contributes to Pathogenesis in Skin Tissue In Vivo

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation Δ⁴⁹¹RSRR⁴⁹⁴ and point mutation ⁴⁹¹GSGG⁴⁹⁴ in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the Δ⁴⁹¹RSRR⁴⁹⁴ or ⁴⁹¹GSGG⁴⁹⁴ mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴ or pOka-gB⁴⁹¹GSGG⁴⁹⁴ virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.Varicella-zoster virus (VZV), an alphaherpesvirus, causes chicken pox (varicella) as a primary infection and shingles (zoster) upon reactivation from infected ganglia in humans (reviewed in reference 16). Although not yet investigated in VZV, herpesvirus entry requires fusion of the virus envelope with cell membranes governed by viral glycoprotein B (gB) and gH/gL, which are conserved across the family Herpesviridae (12, 27, 57). gB is the most conserved glycoprotein, with its function as a fusion protein well documented for several of the herpesviruses (10, 19, 38, 48, 51, 52).Open reading frame 31 (ORF31) codes for the 931 amino acids of VZV gB (18, 37). The successive N- and O-linked glycosylation plus the sialation and sulfation of VZV gB yields a mature protein with a molecular mass of approximately 140 kDa (45). Upon maturation, gB is cleaved, presumably by cellular proteases, into two polypeptides of 66 and 68 kDa. Intracellular trafficking of gB was shown to be dependent upon amino acid motifs in the cytoplasmic domain (24-26). In transfection studies, gB was transported to the cellular surface where it was endocytosed and localized to the trans-Golgi, where envelopment of viral particles is thought to occur.The structures of gB in the two human alphaherpesviruses, VZV and herpes simplex virus type 1 (HSV-1), are likely to be very similar as they have 49% amino acid identity (reviewed in reference 16). The ectodomain of HSV-1 gB was shown to form a spike that consisted of trimers with the structural homology to gG of vesicular stomatitis virus (28). Heldwein et al. (28) proposed that HSV-1 gB is a class II fusion protein based on homology to VSV G. The herpesvirus gB monomer was divided into five domains, I to V. Domain I consisted of a continuous amino acid sequence that folded into a pleckstrin homology-like domain, while domain II was comprised of two discontinuous segments, which also had a pleckstrin homology-like domain. A loop region exposed to the exterior of gB connected domain II with domain III. Domain III was comprised of three discontinuous segments and connected to the external loop by a long α helix that ended in a central coiled coil. Domain IV crowned gB and was connected to domain V, which stretched from the top to the bottom of the gB monomer, forming the core of the trimer making contacts with the two other subunits. The structural homology and lack of furin cleavage suggest that the herpesvirus gB and VSV G proteins have undergone convergent evolution.Although not proven experimentally, VZV gB is likely to be cleaved by the subtilisin-like proprotein convertase furin as the glycoprotein has a furin recognition motif [R]-X-[KR]-R-|-X (where | indicates the position at which the polypeptide is cleaved) (29). The [R]-X-[KR]-R-|-X motif is conserved in gBs for all of the herpesvirus families (5, 9, 21, 36, 40, 53, 63, 64). This site has been shown to be dispensable for the replication of human cytomegalovirus (HCMV), bovine herpesvirus type 1 (BHV-1), and pseudorabies virus (PRV) in vitro (32, 49, 58). Furin site mutants for BHV-1 and PRV show an altered phenotype in vitro, but effects were not examined in vivo. HSV-1 gB is not cleaved and lacks the [R]-X-[KR]-R-|-X motif at the canonical site, which is of interest because HSV-1 is genetically the most closely related human herpesvirus to VZV.Domain I of HSV gB showed structural conservation of putative fusion loops similar to those found in domain IV of the VSV G protein (28). Despite the lack of conserved amino acids within these loops, the hydrophobicity of the residues appears to be conserved for the Herpesviridae (4). Substitution of hydrophobic residues in Epstein-Barr virus gB and linker insertion mutagenesis close to the putative fusion loops of HSV-1 gB abrogated fusion based on in vitro transfection studies (4, 22, 34). However, the effect of substitutions in these putative fusion loops on viral replication has not been characterized. Since the development of fusion assays for VZV has proven elusive, the effect of substitutions in the putative fusion loop using viral mutagenesis to make recombinant viruses provides an alternative approach for identifying functional residues in VZV gB.In contrast to HSV-1, VZV is a human-restricted pathogen (reviewed in reference 16). To study the pathogenesis of VZV in vivo, well-established human xenograft models have been developed using SCID mice (6, 7, 13, 14, 41, 44, 54, 65). Lesions formed by VZV in the skin are similar to those seen in human subjects following primary infection (15, 43). The relevance of the model was demonstrated by studies with the varicella vaccine virus (vOka) that exhibited decreased growth in skin xenografts in vivo but does not cause disease in the healthy human host. In contrast, the vaccine virus and its parent strain, parental Oka (pOka), have indistinguishable replication kinetics in vitro (15, 43).The present study was designed to investigate the effects of structure-based targeted mutations in VZV gB on viral replication in cultured cells and in human skin xenografts in the SCIDhu mouse model. This was performed in the context of infectious virus recovered using the self-excisable bacterial artificial chromosome (BAC) containing the genome of a clinical isolate, Oka (62). The roles of the conserved residues W180 (gB-W180G) and Y185 (gB-Y185G) in the putative fusion loop were evaluated using glycine substitution, and the role of the furin recognition motif (⁴⁹¹RSRR⁴⁹⁴) was assessed by a complete deletion of the furin motif (gBΔ⁴⁹¹RSRR⁴⁹⁴) or a substitution of the arginine residues with glycine (gB⁴⁹¹GSGG⁴⁹⁴) to conserve the carbon backbone.     

Reevaluating Herpes Simplex Virus Hemifusion

Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.Membrane fusion is an essential step during the entry process of enveloped viruses, such as herpes simplex virus (HSV), into target cells. The general pathway by which enveloped viruses fuse with target membranes through the action of fusion proteins is fairly well understood. Viral fusion proteins use the free energy liberated during their own protein conformational changes to draw the two membranes—viral and target—together. Fusion is thought to proceed through a “hemifusion” intermediate, in which the proximal leaflets of the two bilayers have merged but a viral pore has not yet formed and viral contents have not yet mixed with the cell cytoplasm (10, 38). Fusion proteins then drive the completion of fusion, which includes fusion pore formation, pore enlargement, and complete content mixing.HSV, an enveloped neurotropic virus, requires four glycoproteins—glycoprotein B (gB), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL)—to execute fusion (9, 57, 60). gB, gD, and gH are membrane bound; gL is a soluble protein which complexes with gH to form a heterodimer (gH/gL). HSV-1 gH is not trafficked to the cell or virion surface in the absence of gL (32, 52). The requirement of four entry glycoproteins sets HSV apart from other enveloped viruses, most of which induce fusion through the activity of a single fusion protein. Although the specific mode of HSV entry is cell type dependent—fusion with neurons and Vero cells occurs at the plasma membrane at neutral pH; fusion with HeLa and CHO cells involves pH-dependent endocytosis, and fusion with C10 cells involves pH-independent endocytosis (42, 45)—all routes of entry require gD, gB, and gH/gL. Furthermore, although some discrepancies between virus-cell and cell-cell fusion have been observed (8, 44, 55, 58), both generally require the actions of gD, gB, and gH/gL.Much work has gone toward the understanding of how the required HSV entry glycoproteins work together to accomplish fusion, and many questions remain. After viral attachment, mediated by glycoprotein C and/or gB (54), the first step in HSV fusion is thought to be gD binding a host cell receptor (either herpesvirus entry mediator [HVEM], nectin-1, nectin-2, or heparan sulfate modified by specific 3-O-sulfotransferases) (56). The gD-receptor interaction induces a conformational change in gD (39) that is thought to trigger gD-gB and/or gD-gH/gL interactions that are required for the progression of fusion (1-4, 13, 18, 23, 49).gB and gH/gL are considered the core fusion machinery of most herpesviruses. The HSV-1 gB structure revealed surprising structural homology to the postfusion structures of two known viral fusion proteins (31, 35, 51). This structural homology indicates that despite not being sufficient for HSV fusion, gB is likely a fusion protein. Although the gB cytoplasmic tail (CT) is not included in the solved structure, it acts as a regulator of fusion, as CT truncations can cause either hyperfusion or fusion-null phenotypes (5, 17). The gB CT has been proposed to bind stably to lipid membranes and negatively regulate membrane fusion (12). Another proposed regulator of gB function is gH/gL. Despite conflicting accounts of whether gD and a gD receptor are required for the interaction of gH/gL and gB (1, 3, 4), a recent study indicates that gH/gL and gB interact prior to fusion and that gB may interact with target membranes prior to an interaction with gH/gL (2). The gB-gH/gL interaction seems to be required for the progression of fusion.Compared to the other required HSV entry glycoproteins, the role of gH/gL during fusion remains enigmatic. Mutational studies have revealed several regions of the gH ectodomain, transmembrane domain (TM), and CT that are required for its function (19, 25, 26, 30, 33). gH/gL of another herpesvirus, Epstein-Barr virus (EBV), have been shown to bind integrins during epithelial cell fusion, and soluble forms of HSV gH/gL have been shown to bind cells and inhibit viral entry in vitro (24, 46). However, the role of gH/gL binding to target cells in regard to the fusion process remains to be determined.There are some lines of evidence that suggest that gH/gL is a fusion protein. The gH/gL complexes of VZV and CMV have been reported to independently execute some level of cell-cell fusion (14, 37). HSV-1 gH/gL has been reported to independently mediate membrane fusion during nuclear egress (15). In silico analyses and studies of synthetic HSV gH peptides have proposed that gH has fusogenic properties (20, 21, 25-28). Finally, of most importance to the work we report here, gH/gL has been shown to be sufficient for induction of hemifusion in the presence of gD and a gD receptor, further promoting the premise that gH/gL is a fusion protein (59). However, the recently solved crystal structure of HSV-2 gH/gL revealed a tight complex of gH/gL in a “boot-like” structure, which bears no structural homology to any known fusion proteins (11). The HSV-2 gH/gL structure and research demonstrating that gH/gL and gB interactions are critical to fusion (2) have together prompted a new model of HSV fusion in which gH/gL is required to either negatively or positively regulate the activity of gB through direct binding.We wanted to investigate the ability of a previously reported gH CT mutant, 824L, to execute hemifusion. 824L gH contains a five-residue insertion at gH residue 824, just C-terminal of the TM domain. 824L is expressed on cell surfaces and incorporated into virions at levels indistinguishable from those of wild-type gH by either cell-based ELISA or immunoblotting, yet it is nonfunctional (33). We relied on a fusion assay capable of detecting hemifusion, developed by Subramanian et al. (59), which we modified to include an additional control for hemifusion or nonenlarging pore formation, glycosylphosphatidylinositol (GPI)-linked hemagglutinin (GPI-HA). GPI-HA is a variant of the influenza virus fusion protein, HA, that is known to stall the fusion process before enlarging fusion pores are formed.We were surprised to find that in our hands, gD, a gD receptor, and gH/gL were insufficient for the induction of hemifusion or lipid mixing in both cell-based and virus-based fusion assays. We found that gD, gB, and gH/gL are all required to observe lipid mixing. Further, we found that gB, gD, gL, and 824L gH are insufficient for lipid mixing. Our findings support the emerging view, based on gH/gL structure, that the gH/gL complex does not function as a fusion protein and does not insert into target membranes to initiate the process of fusion through a hemifusion intermediate. Our findings also further demonstrate that mutations in the CT of gH can have a dramatic effect on the ability of gH/gL to function in fusion.     

Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD

The gD, gB, and gH/gL glycoprotein quartet constitutes the basic apparatus for herpes simplex virus (HSV) entry into the cell and fusion. gD serves as a receptor binding glycoprotein and trigger of fusion. The conserved gB and gH/gL execute fusion. Central to understanding HSV entry/fusion has become the dissection of how the four glycoproteins engage in cross talk. While the independent interactions of gD with gB and gD with gH/gL have been documented, less is known of the interaction of gB with gH/gL. So far, this interaction has been detected only in the presence of gD by means of a split green fluorescent protein complementation assay. Here, we show that gB interacts with gH/gL in the absence of gD. The gB-gH/gL complex was best detected with a form of gB in which the endocytosis and phosphorylation motif have been deleted; this form of gB persists in the membranes of the exocytic pathway and is not endocytosed. The gB-gH/gL interaction was detected both in whole transfected cells by means of a split yellow fluorescent protein complementation assay and, biochemically, by a pull-down assay. Results with a panel of chimeric forms of gB, in which portions of the glycoprotein bracketed by consecutive cysteines were replaced with the corresponding portions from human herpesvirus 8 gB, favor the view that gB carries multiple sites for interaction with gH/gL, and one of these sites is located in the pleckstrin-like domain 1 carrying the bipartite fusion loop.Entry of herpes simplex virus (HSV) into the cell requires a multipartite apparatus made of a quartet of viral glycoproteins, gD, gB, and the heterodimer gH/gL, and a multistep process that culminates in the fusion of the virion envelope with cell membranes (5, 6, 10, 25, 36, 41). gD serves as the receptor-binding glycoprotein, able to interact with alternative receptors, nectin1, herpesvirus entry mediator (HVEM) and, in some cells, modified heparan sulfate (9, 13, 30, 39). It can also be engineered to accept heterologous ligands able to interact with selected receptors present on tumor cells and thus represents a tool to redirect HSV tropism (21, 28, 29, 42). The heterodimer gH/gL and gB execute fusion and constitute the conserved fusion apparatus across the Herpesviridae family. gB structure in the postfusion conformation shows a trimer with a central coiled coil (19). gH shows elements typical of type 1 fusion glycoproteins, in particular, helices able to interact with membranes, and two heptad repeats potentially able to form a coiled coil (12, 15-18). The discovery that a soluble form of gD enables entry of gD-null virions revealed that gD serves the additional function of triggering fusion and led to the view that the major roles of gD are to sense that virus has reached a receptor-positive cell and to signal to gB and gH/gL that fusion is to be executed (8). Biochemical and structural analyses showed that the C-terminal region of the gD ectodomain, containing the profusion domain required for fusion but not for receptor binding, can undergo major conformational changes (11, 24). Specifically, it binds the gD core and masks or hinders the receptor binding sites, conferring upon the molecule a closed, auto-inhibited conformation (24). Alternatively, it may unfold, conferring upon gD an open conformation. It was proposed that the C terminus of gD unfolds from gD core at receptor binding and recruits gH/gL and gB to a quaternary complex. A key feature of the model was that complexes among the glycoprotein quartet were not preformed, but, rather, they would assemble at the onset of or at fusion execution.Central to understanding HSV entry/fusion has become the dissection of the interactions that occur among the members of the glycoprotein quartet and their significance to the process. A first evidence of a gD-gH/gL interaction was provided in coimmunoprecipitation studies (35). Interactions between gD and gH/gL and between gD and gB were subsequently detected by split green fluorescence protein (GFP) complementation assays, implying that gD can recruit gB and gH/gL independently of one another, a result that argues against a stepwise recruitment of the glycoproteins to gD. In agreement with the proposed model, the interaction between gH/gL and gB was detected in the presence of transfected or soluble gD (1, 2). However, further studies highlighted levels of complexity not foreseen in the initial model. Thus, pull-down analyses showed that the interaction sites in gD with gB and with gH/gL lie in part outside the C-terminal portion of the gD ectodomain, that resting virions contain small amounts of gD in complex with gB and with gH/gL prior to encountering cells, and that de novo gD-gB complexes were not detected at virus entry into the cell (14).A major objective of current studies was to analyze the interaction of gB with gH/gL. We documented the interaction by two independent assays, i.e., by a complementation assay of split yellow fluorescent protein Venus (herein indicated as YFP) (31) in whole cells and, biochemically, by a pull-down assay. The latter was applied recently in our laboratory and is based on the ability of One-Strep-tagged proteins (e.g., gH) to specifically absorb to Strep-Tactin resin and thus retain any protein in complex (14). To preliminarily search for gB regions critical for the interaction with gH/gL, we engineered chimeric forms of HSV-1 and human herpesvirus 8 (HHV-8) gB in which the cysteines were preserved. While none of the chimeras was completely defective in the interaction, the interactions in the chimeras carrying substitutions in the pleckstrin-like domain 1—the domain that carries the bipartite fusion loops—were hampered. Altogether, the results underscore the ability of gB to interact with gH/gL in the absence of gD and favor the view that sites in gB for interaction with gH/gL involve multiple contacts, one of which is located in the domain that carries the fusion loops.     

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.Entry of herpes simplex virus (HSV) into cells requires four viral glycoproteins, gB, gD, gH, and gL, plus one of several cell receptors, either herpesvirus entry mediator (HVEM), nectin-1, or 3-OST (45). Crystal structures and other studies have documented that receptor binding triggers conformational changes to gD that trigger the downstream events leading to fusion (10, 11, 18, 26, 28, 52). Moreover, when HSV receptor-bearing cells are transfected with expression plasmids for glycoproteins gB, gD, gH, and gL, the cells fuse to form multinucleated giant cells or syncytia (39, 48). However, the precise series of events that take place after receptor binding have not yet been fully elucidated. What we do know is that both gB and a heterodimer of gH/gL constitute the core fusion machinery that is conserved and required for the fusion step of entry of all herpesviruses (18, 26, 30, 46, 49).Thus far, we know the crystal structure of one form of the gB ectodomain of HSV type 1 (HSV-1) (19). This protein has the characteristics of a fusion protein and is a charter member of the class III group of viral fusion proteins (4). Others in this class include Epstein-Barr virus gB, vesicular stomatitis virus (VSV) G, and baculovirus gp64 (5, 22, 41). Like VSV G and gp64, gB has two putative fusion loops at the base of each protomer of the crystallized trimer. Single-amino-acid mutations in many of the hydrophobic residues of the putative fusion loops of gB ablate its ability to function in cell-cell fusion assays (16, 17). Moreover, these mutants are unable to complement the entry of a gB-null virus (16). Finally, the ectodomains of these mutants, unlike wild-type protein, failed to coassociate with liposomes, indicating that the putative fusion loops do insert into membranes (16, 17). Recently, it was shown that several of these mutants are also defective for fusion events involved in virus egress (51). Together, these studies provide compelling evidence that HSV gB functions as a fusion protein and that the fusion loops are critical for this function. However, unlike VSV G and baculovirus gp64, gB does not function on its own in entry but, rather, requires the participation of gH/gL. In the absence of crystallographic data for gH/gL, it is not yet clear what role it plays in herpesvirus fusion. In a previous study, we used bimolecular complementation (BiMC) to examine protein-protein interactions that occur among the viral glycoproteins during fusion (1). A similar study was carried out by Avitabile et al. (2). The BiMC assay is based on the observation that N- and C-terminal fragments of green fluorescent protein (GFP) (and derivatives such as enhanced yellow fluorescent protein [EYFP]) do not spontaneously reconstitute a functional fluorophore (20, 29, 40). However, the codons for each half can be appended to the genes for two interacting proteins (23, 24). When these are cotransfected, an interaction between the two proteins of interest brings the two halves of the fluorophore in close enough contact to restore fluorescence.When HSV receptor-bearing cells, such as B78H1 cells that are engineered to express nectin-1, are transfected with plasmids that express gB, gD, gH, and gL, they undergo cell-cell fusion (13, 15, 27, 31, 48). When gD is omitted, no fusion occurs. We found that fusion of these transfected cells could be triggered by addition of a soluble form of gD (the gD ectodomain). We then used this approach to examine interactions between gB and gH/gL during cell fusion (1). Therefore, we tagged gB with the C-terminal half of EYFP and gH with the N-terminal half. When plasmids bearing these forms were cotransfected into C10 cells along with a plasmid for untagged gL, no fusion occurred, but importantly, no BiMC occurred. However, when we added gD306, cells began to fuse within 10 min, and all of the syncytia that formed exhibited bright EYFP fluorescence indicative of BiMC. We concluded that gD triggers both fusion and a physical interaction between gB and gH/gL. However, these experiments did not separate these two events, so we were unable to determine if the interaction preceded fusion or merely was a by-product of it.The purpose of this study was to determine if the gB-gH/gL interaction is essential for fusion and if it occurs prior to fusion. We focused on gB because its structure is known and we have a panel of well-characterized monoclonal antibodies (MAbs) to gB. Our approach was to determine which of these MAbs, if any, could block fusion and also block the interaction with gH/gL. We also examined the effect of mutations to the fusion loops of gB on its interaction with gH/gL. We previously mapped these MAbs to four functional regions (FR) of gB, three of which were resolved in the crystal structure (6, 19). Of these, FR1 contains the fusion loops, FR2 is in the center of the gB structure with no known function, and FR3 is at in the crown of the protein and may be involved in binding to cells (7). Our rationale was that if the interaction between gB and gH/gL is important for fusion, then it should not be blocked by nonneutralizing anti-gB MAbs. At the same time, we thought that some neutralizing MAbs might not only block fusion but also block BiMC. We found that neutralizing MAbs to FR1 and FR2 inhibited both BiMC and fusion. In contrast, we found that neutralizing MAbs that map to FR3 blocked fusion but failed to block the interaction between gB and gH/gL, thereby dissociating the two events. Finally, we found that gB mutants with changes in the fusion loops that were fusion negative were also unable to bind to gH/gL. The latter results suggest that insertion of gB into the target membrane precedes its interaction with gH/gL.     

Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Papillomavirus capsids are composed of 72 pentamers reinforced through inter- and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.Human papillomaviruses (HPVs) exclusively infect cutaneous or mucosal epithelial tissues (14, 15, 30). HPV types that infect the mucosal epithelia can lead to the development of benign or malignant neoplasms, thus allowing for their categorization into low-risk or high-risk HPV types, respectively (14, 15, 30). A small subset of the more than 200 HPV types now identified are the causative agents of over 75% of all cervical cancers. HPV16 is the most prevalent type worldwide, found in ca. 50 to 62% of squamous cell carcinomas (14, 50).HPV16 virions contain a single, circular double-stranded DNA genome of ∼8 kb which associates with histones to form a chromatin-like structure. This minichromosome is packaged within a nonenveloped, icosahedral capsid composed of the major capsid protein L1 and the minor capsid protein L2. Similar to polyomaviruses, 72 capsomeres of L1 are geometrically arranged on a T=7 icosahedral lattice (2, 9, 17, 19, 36, 42). Recent cryoelectron microscopy images of HPV16 pseudovirions (PsV) suggest that L2 is arranged near the inner conical hollow of each L1 pentamer, although it is not known whether each L1 pentamer is occupied with a single L2 protein (5, 42).Due to technical constraints in the production of native HPV virions in organotypic culture, assembly studies of HPV particles have largely been restricted to the utilization of in vitro-derived particles such as virus-like particles (VLPs), PsV, and quasivirions (QV) (6, 12, 25, 40, 43). Recent research suggests that HPV and bovine papillomavirus PsV can undergo a redox-dependent conformational change that takes place over the course of many hours. This conformational change is characterized by resistance to proteolysis and chemical reduction and the appearance of a more orderly capsid structure via transmission electron microscopy (TEM) (7, 20).We present evidence that native virions, in the context of the complete papillomavirus life cycle, utilize a tissue-spanning redox gradient that facilitates multiple redox-dependent assembly and maturation events over the course of many days. We show that stability and specific infectivity of 20-day virions increases over 10-day virions, 20-day virions are more susceptible to neutralization than 10-day virions, and both viral DNA encapsidation and infectivity of HPV-infected tissues are redox dependent in that they can be manipulated via the treatment of organotypic tissues with oxidized glutathione (GSSG), which is concentration and temporally dependent.     

A Double Mutation in Glycoprotein gB Compensates for Ineffective gD-Dependent Initiation of Herpes Simplex Virus Type 1 Infection

Herpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling.Entry of herpes simplex virus type 1 (HSV-1) into susceptible cells involves the coordinated activities of at least five viral envelope glycoproteins (9, 18, 33, 40). Virions initially bind to glycosaminoglycan (GAG) moieties of cell surface proteoglycans through glycoproteins B and C (gB and gC, respectively) (32, 51), facilitating the interaction of gD with one of its specific receptors, herpesvirus entry mediator (HVEM, or HveA), nectin-1 (HveC), or 3-O-sulfated heparan sulfate (24, 45, 50). Receptor binding is believed to result in a conformational change in gD, which in turn activates the fusion mechanism mediated by gB and the gH/gL heterodimer; fusion merges the virus envelope with the cell surface or endosomal membrane, resulting in capsid release into the cytoplasm (11, 23, 30, 37, 44, 47, 48). Prior to receptor binding, the N-terminal region of the gD ectodomain is folded back over the immunoglobulin (Ig)-like core domain in a position to engage the C-terminal effector region (pro-fusion domain), thereby keeping the effector domain in an inactive state (23, 37). Receptor binding disrupts this engagement and liberates the effector domain to activate gB and/or gH/gL. The crystal structure of the gB ectodomain shows unexpected homology to the postfusion form of glycoprotein G from vesicular stomatitis virus (VSV G), a well-characterized fusion protein (30), providing strong evidence that gB plays a major role in membrane fusion. In addition, gH displays structural hallmarks of fusion proteins (26, 27), and gB and gH each have fusogenic activity, as indicated by the finding that either alone is sufficient for membrane fusion during nuclear egress (20). However, gB and gH/gL are both required for complete fusion during virus entry, although gB is dispensable for hemifusion, an intermediate state (53).Results from biochemical and bimolecular-complementation assays have shown that gD binds individually to both gB and gH/gL, regardless of the presence of gD receptors (4, 5, 25), while complexes of gB and gH/gL assemble only in the presence of receptor-bound gD (4, 5). These observations suggested that receptor-dependent gD activation brings gB and gH/gL together for execution of the fusion event. However, based on new evidence that gB and gH/gL can also interact in the absence of gD, an alternative model has been proposed in which activated gD signals to preformed gB-gH/gL complexes (6). While these models are not mutually exclusive, the functional significance of the detected complexes remains to be firmly established (15). However, there is broad consensus that the gD-receptor interaction triggers the initiation of fusion by direct interaction with either or both gB and gH/gL, indicating that the quality of the gD-receptor interaction is key to the efficiency of HSV infection.Viruses have an intrinsic ability to evolve and adapt to changes in the environment, including the acquisition of an extended host range which can lead to epidemic infections (56). We previously described gain-of-function derivatives of a gD mutant virus, K26-gD:R222N/F223I, that was impaired in its ability to use nectin-1 as an entry receptor (54). Repeated passage of this virus through cells that express nectin-1 as the sole gD receptor yielded phenotypic revertants that had regained the ability to use nectin-1 for infection. This phenotype resulted from reversion or forward mutations at the parental mutant positions or from substitutions elsewhere in gD that likely affect the integrity of the discontinuous interface with nectin-1. Since these types of experiments can reveal novel factors or interactions that are important for virus entry, we performed a similar study at higher stringency in an attempt to avoid simple reversion mutations. The strategy was to use our previous gD:R222N/F223I mutant virus that is defective for entry via nectin-1 and ask if this virus could adapt to host cells expressing a mutant form of nectin-1 whose binding to wild-type gD is severely impaired. A specific goal of this effort was to find mutations in gD or other envelope glycoproteins that could enhance infection through atypical receptors, including cell-type-specific receptors that can be engaged by retargeted HSV vectors.Here we report the identification of a hyperactive gB double mutation, gB:D285N/A549T, referred to herein as gB:N/T, that allows virus entry in the absence of authentic gD receptors, enhances virus entry through unconventional receptors, including a targeted receptor, and appears to act by sensitizing gB to activation by gD, directly or indirectly via gH/gL, and increasing the rate of virus entry into different host cells. Our observations demonstrate that hyperactive gB can compensate for ineffective gD-receptor interactions in the process of HSV entry into cells.     

Phosphorylation of the UL31 Protein of Herpes Simplex Virus 1 by the US3-Encoded Kinase Regulates Localization of the Nuclear Envelopment Complex and Egress of Nucleocapsids

Herpes simplex virus 1 nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space to obtain a primary viral envelope. This process requires a protein complex at the INM composed of the U_L31 and U_L34 gene products. While it is clear that the viral kinase encoded by the U_S3 gene regulates the localization of pU_L31/pU_L34 within the INM, the molecular mechanism by which this is accomplished remains enigmatic. Here, we have determined the following. (i) The N terminus of pU_L31 is indispensable for the protein''s normal function and contains up to six serines that are phosphorylated by the U_S3 kinase during infection. (ii) Phosphorylation at these six serines was not essential for a productive infection but was required for optimal viral growth kinetics. (iii) In the presence of active U_S3 kinase, changing the serines to alanine caused the pU_L31/pU_L34 complex to aggregate at the nuclear rim and caused some virions to accumulate aberrantly in herniations of the nuclear membrane, much as in cells infected with a U_S3 kinase-dead mutant. (iv) The replacement of the six serines of pU_L31 with glutamic acid largely restored the smooth distribution of pU_L34/pU_L31 at the nuclear membrane and precluded the accumulation of virions in herniations whether or not U_S3 kinase was active but also precluded the optimal primary envelopment of nucleocapsids. These observations indicate that the phosphorylation of pU_L31 by pU_S3 represents an important regulatory event in the virion egress pathway that can account for much of pU_S3''s role in nuclear egress. The data also suggest that the dynamics of pU_L31 phosphorylation modulate both the primary envelopment and the subsequent fusion of the nascent virion envelope with the outer nuclear membrane.The U_L31 and U_L34 proteins of herpes simplex virus 1 (HSV-1) form a complex that accumulates at the inner nuclear membrane (INM) of infected cells (26, 27). This complex is essential for the budding of nucleocapsids through the INM into the perinuclear space (26, 28). pU_L34 is a type 2 integral membrane protein with a 247-amino-acid nucleoplasmic domain that binds pU_L31 and holds the latter in close approximation to the INM (16, 19, 26, 31, 36, 37). Both proteins become incorporated into nascent virions, indicating that they directly or indirectly interact with nucleocapsids during the budding event (27). Interestingly, the coexpression of the pseudorabies virus homologs of HSV pU_L31 and pU_L34 are sufficient to induce budding from the INM in the absence of other viral proteins (13).The most prominent model of nuclear egress proposes that the step following primary envelopment involves the fusion of the perinuclear virion envelope with the outer nuclear membrane (ONM), allowing subsequent steps in which the deenveloped capsid engages budding sites in the Golgi or trans-Golgi network (20, 32). The U_S3 protein is a promiscuous kinase that phosphorylates pU_L31, pU_L34, and several other viral and cellular components (1, 2, 5, 11, 15, 21-23, 25). In the absence of pU_S3 kinase activity, (i) virions accumulate within distensions of the perinuclear space that herniate into the nucleoplasm (14, 27, 29), (ii) the pU_L31/pU_L34 complex is mislocalized at the nuclear rim from a smooth pattern to discrete foci that accumulate adjacent to nuclear membrane herniations (12, 14, 27, 29), and (iii) the onset of infectious virus production is delayed (21, 29).Aberrant accumulations of perinuclear virions similar to those observed in cells infected with U_S3 kinase-dead viruses have been observed in cells infected with viruses lacking the capacity to produce glycoproteins H and B (gH and gB, respectively) (8). Because these proteins are required for fusion with the plasma membrane or endocytic vesicles during HSV entry (3, 4, 9, 10, 18, 30, 33), it has been proposed that the accumulation of perinuclear virions in the absence of gH and gB reflects a failure in the apparatus that normally mediates the fusion between the nascent virion envelope and the ONM (8). By extension of this hypothesis, pU_S3 might act to trigger or otherwise regulate this perinuclear fusion event.The substrate(s) of the pU_S3 kinase responsible for the altered localization of the pU_L31/pU_L34 complex and the aberrant accumulation of perinuclear virions were heretofore unknown. In one study to identify such a substrate, it was determined that precluding the phosphorylation of pU_L34 was not responsible for the nuclear egress defects induced by the absence of pU_S3 or its kinase activity (29). The current study was therefore undertaken to investigate the hypothesis that the pU_S3-mediated phosphorylation of pU_L31 is critical to regulate nuclear egress. The presented evidence indicates that aspects of the U_S3 kinase-dead phenotype, including the retention of virions in the perinuclear space, the mislocalization of the pU_L31/pU_L34 complex, and the delayed onset of virus replication, can be replicated by precluding pU_L31 phosphorylation in the presence or absence of pU_S3 kinase activity. The data also suggest that the dynamic phosphorylation of pU_L31 is important during the primary envelopment of nucleocapsids.     

Retrograde Axon Transport of Herpes Simplex Virus and Pseudorabies Virus: a Live-Cell Comparative Analysis

Upon entry, neuroinvasive herpesviruses traffic from axon terminals to the nuclei of neurons resident in peripheral ganglia, where the viral DNA is deposited. A detailed analysis of herpes simplex virus type 1 (HSV-1) transport dynamics in axons following entry is currently lacking. Here, time lapse fluorescence microscopy was used to compare the postentry viral transport of two neurotropic herpesviruses: HSV-1 and pseudorabies virus (PRV). HSV-1 capsid transport dynamics were indistinguishable from those of PRV and did not differ in neurons of human, mouse, or avian origin. Simultaneous tracking of capsids and tegument proteins demonstrated that the composition of actively transporting HSV-1 is remarkably similar to that of PRV. This quantitative assessment of HSV-1 axon transport following entry demonstrates that HSV-1 and PRV share a conserved mechanism for postentry retrograde transport in axons and provides the foundation for further studies of the retrograde transport process.Herpes simplex virus type 1 (HSV-1) and the veterinary herpesvirus pathogen pseudorabies virus (PRV) establish latent infections within the peripheral nervous systems (PNS) of their hosts. Neurotropic herpesviruses gain access to the PNS at nerve endings present in infected skin or mucosal tissue. Upon entry at the nerve terminal, viral particles are transported in axons toward the neuronal cell body to ultimately deposit the viral genome into the nucleus. This process is referred to as retrograde transport and is critical for the establishment of latency. Following reactivation, progeny viral particles travel anterogradely from the ganglia toward the nerve terminals, resulting in reinfection of the dermis or other innervated tissues. Reactivated infection can manifest in various forms, including asymptomatic virus shedding or mild focal lesions (herpes labialis), or less frequently in more-severe disease (herpes keratitis, encephalitis, and in the case of varicella-zoster virus, shingles).All herpesviruses consist of an icosahedral capsid that contains the viral genome surrounded by a layer of proteins known as the tegument, which is contained within a membrane envelope (33). HSV-1 and PRV capsids disassociate from the viral envelope (2, 13, 14, 22, 23, 25, 28, 30, 40) and several tegument proteins (13, 16, 21, 25) upon fusion-mediated entry into cells. However, following entry into epithelial cell lines, the VP1/2 and UL37 tegument proteins are detected in association with cytosolic capsids of PRV by immunogold electron microscopy (16) and colocalize with HSV-1 capsids at the nuclear membrane by immunofluorescence microscopy (8). In primary sensory neurons, VP1/2 and UL37 are observed to be cotransported with PRV capsids during retrograde transport by time lapse fluorescence microscopy (21), and the kinetics of axon transport have been assessed (39).Although HSV-1 and PRV share similarities in their neurotropism in vivo (reviewed in reference 12), studies of axon transport have indicated possible mechanistic differences relevant to the underlying cell biology of neural transmission (reviewed in reference 10). As a result, a live-cell analysis comparing PRV and HSV-1 is needed to determine if axon transport mechanisms are conserved between the two neuroinvasive herpesvirus genera: Simplexvirus (HSV-1) and Varicellovirus (PRV). In this study, the retrograde transport process that delivers capsids to the nuclei of sensory neurons was compared for HSV-1 (strains KOS and F) and PRV (strain Becker).