Function of herpes simplex virus type 1 gD mutants with different receptor-binding affinities in virus entry and fusion

We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(inverted Delta 34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(inverted Delta 34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(inverted Delta 243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(inverted Delta 243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.     

Characterization of a heparan sulfate octasaccharide that binds to herpes simplex virus type 1 glycoprotein D

Herpes simplex virus type 1 utilizes cell surface heparan sulfate as receptors to infect target cells. The unique heparan sulfate saccharide sequence offers the binding site for viral envelope proteins and plays critical roles in assisting viral infections. A specific 3-O-sulfated heparan sulfate is known to facilitate the entry of herpes simplex virus 1 into cells. The 3-O-sulfated heparan sulfate is generated by the heparan sulfate d-glucosaminyl-3-O-sulfotransferase isoform 3 (3-OST-3), and it provides binding sites for viral glycoprotein D (gD). Here, we report the purification and structural characterization of an oligosaccharide that binds to gD. The isolated gD-binding site is an octasaccharide, and has a binding affinity to gD around 18 microm, as determined by affinity coelectrophoresis. The octasaccharide was prepared and purified from a heparan sulfate oligosaccharide library that was modified by purified 3-OST-3 enzyme. The molecular mass of the isolated octasaccharide was determined using both nanoelectrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The results from the sequence analysis suggest that the structure of the octasaccharide is a heptasulfated octasaccharide. The proposed structure of the octasaccharide is DeltaUA-GlcNS-IdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH(2)3S6S. Given that the binding of 3-O-sulfated heparan sulfate to gD can mediate viral entry, our results provide structural information about heparan sulfate-assisted viral entry.     

The Mature Virion of Ectromelia Virus,a Pathogenic Poxvirus,Is Capable of Intrahepatic Spread and Can Serve as a Target for Delayed Therapy

Orthopoxviruses (OPVs), which include the agent of smallpox (variola virus), the zoonotic monkeypox virus, the vaccine and zoonotic species vaccinia virus, and the mouse pathogen ectromelia virus (ECTV), form two types of infectious viral particles: the mature virus (MV), which is cytosolic, and the enveloped virus (EV), which is extracellular. It is believed that MVs are required for viral entry into the host, while EVs are responsible for spread within the host. Following footpad infection of susceptible mice, ECTV spreads lymphohematogenously, entering the liver at 3 to 4 days postinfection (dpi). Afterwards, ECTV spreads intrahepatically, killing the host. We found that antibodies to an MV protein were highly effective at curing mice from ECTV infection when administered after the virus reached the liver. Moreover, a mutant ECTV that does not make EV was able to spread intrahepatically and kill immunodeficient mice. Together, these findings indicate that MVs are sufficient for the spread of ECTV within the liver and could have implications regarding the pathogenesis of other OPVs, the treatment of emerging OPV infections, as well as strategies for preparedness in case of accidental or intentional release of pathogenic OPVs.     

Structural basis of local, pH-dependent conformational changes in glycoprotein B from herpes simplex virus type 1

Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV.     

Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells

Purinergic Signalling - Wound healing is an important outcome of tissue damage and can be stimulated by adenosine released from cells during events such as tissue injury, ischaemia or tumour...     

Examination of the Kinetics of Herpes Simplex Virus Glycoprotein D Binding to the Herpesvirus Entry Mediator, Using Surface Plasmon Resonance

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (K_D) of 3.2 × 10⁻⁶ M and gD2(306t) had a K_D of 1.5 × 10⁻⁶ M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Δ290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (K_D = 3.3 × 10⁻⁸ M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Δ290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.     

In vivo role of nectin-1 in entry of herpes simplex virus type 1 (HSV-1) and HSV-2 through the vaginal mucosa

Herpes simplex virus type 2 (HSV-2) is transmitted through the genital mucosa during sexual encounters. In recent years, HSV-1 has also become commonly associated with primary genital herpes. The mechanism of viral entry of HSV-1 and HSV-2 in the female genital tract is unknown. In order to understand the molecular interactions required for HSV entry into the vaginal epithelium, we examined the expression of herpesvirus entry mediator nectin-1 in the vagina of human and mouse at different stages of their hormonal cycle. Nectin-1 was highly expressed in the epithelium of human vagina throughout the menstrual cycle, whereas the mouse vaginal epithelium expressed nectin-1 only during the stages of the estrous cycle in which mice are susceptible to vaginal HSV infection. Furthermore, the ability of nectin-1 to mediate viral entry following intravaginal inoculation was examined in a mouse model of genital herpes. Vaginal infection with either HSV-1 or HSV-2 was blocked by preincubation of the virus with soluble recombinant nectin-1. Viral entry through the vaginal mucosa was also inhibited by preincubation of HSV-2 with antibody against gD. Together, these results suggest the importance of nectin-1 in mediating viral entry for both HSV-1 and HSV-2 in the genital mucosa in female hosts.     

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.