Claude Krummenacher

Herpes simplex virus glycoprotein D bound to the human receptor HveA.

Herpes simplex virus (HSV) infection requires binding of the viral envelope glycoprotein D (gD) to cell surface receptors. We report the X-ray structures of a soluble, truncated ectodomain of gD both alone and in complex with the ectodomain of its cellular receptor HveA. Two bound anions suggest possible binding sites for another gD receptor, a 3-O-sulfonated heparan sulfate. Unexpectedly, the structures reveal a V-like immunoglobulin (Ig) fold at the core of gD that is closely related to cellular adhesion molecules and flanked by large N- and C-terminal extensions. The receptor binding segment of gD, an N-terminal hairpin, appears conformationally flexible, suggesting that a conformational change accompanying binding might be part of the viral entry mechanism.

Herpes Virus Fusion and Entry: A Story with Many Characters

Herpesviridae comprise a large family of enveloped DNA viruses all of whom employ orthologs of the same three glycoproteins, gB, gH and gL. Additionally, herpesviruses often employ accessory proteins to bind receptors and/or bind the heterodimer gH/gL or even to determine cell tropism. Sorting out how these proteins function has been resolved to a large extent by structural biology coupled with supporting biochemical and biologic evidence. Together with the G protein of vesicular stomatitis virus, gB is a charter member of the Class III fusion proteins. Unlike VSV G, gB only functions when partnered with gH/gL. However, gH/gL does not resemble any known viral fusion protein and there is evidence that its function is to upregulate the fusogenic activity of gB. In the case of herpes simplex virus, gH/gL itself is upregulated into an active state by the conformational change that occurs when gD, the receptor binding protein, binds one of its receptors. In this review we focus primarily on prototypes of the three subfamilies of herpesviruses. We will present our model for how herpes simplex virus (HSV) regulates fusion in series of highly regulated steps. Our model highlights what is known and also provides a framework to address mechanistic questions about fusion by HSV and herpesviruses in general.

Structure of Herpes Simplex Virus Glycoprotein D Bound to the Human Receptor Nectin-1

Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting β-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.

Localization of a Binding Site for Herpes Simplex Virus Glycoprotein D on Herpesvirus Entry Mediator C by Using Antireceptor Monoclonal Antibodies

ABSTRACT The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.

Understanding HSV-1 entry glycoproteins.

Herpes Simplex Virus‐1 is a common infectious agent, but the precise detail of entry and infection of cells has only now begun to be clarified. Four viral surface glycoproteins (gB, gD, gH and gL) are required. This review summarises the known structure and function of each of these essential viral envelope glycoproteins, and explores what is known about their close cooperation with each other in mediating cellular membrane fusion. It is suggested that, following gD binding to one of its entry receptors, membrane fusion is mediated by gB and the heterodimer gH/gL. Significantly, these four entry glycoproteins also play a key role in the interaction between HSV and the host immune system. The glycoproteins serve an important role as targets of adaptive immunity. However, recent studies have demonstrated that the same proteins also play a key role in initiating the early innate immune response to HSV. Understanding the complex functions of these HSV proteins may be essential for successful development of vaccines for HSV. Copyright

Entry of Herpes Simplex Virus Type 1 into Primary Sensory Neurons In Vitro Is Mediated by Nectin-1/HveC

ABSTRACT Primary cultures of rat and mouse sensory neurons were used to study the entry of herpes simplex virus type 1 (HSV-1). Soluble, truncated nectin-1 but not HveA prevented viral entry. Antibodies against nectin-1 also blocked infection of rat neurons. These results indicate that nectin-1 is the primary receptor for HSV-1 infection of sensory neurons.

Localization of the gD-binding region of the human herpes simplex virus receptor, HveA.

ABSTRACT During virus entry, herpes simplex virus (HSV) glycoprotein D (gD) binds to one of several human cellular receptors. One of these, herpesvirus entry mediator A (HveA), is a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ectodomain contains four characteristic cysteine-rich pseudorepeat (CRP) elements. We previously showed that gD binds the ectodomain of HveA expressed as a truncated, soluble protein [HveA(200t)]. To localize the gD-binding domain of HveA, we expressed three additional soluble forms of HveA consisting of the first CRP [HveA(76t)], the second CRP [HveA(77–120t)], or the first and second CRPs [HveA(120t)]. Biosensor and enzyme-linked immunosorbent assay studies showed that gD bound to HveA(120t) and HveA(200t) with the same affinity. However, gD did not bind to HveA(76t) or HveA(77–120t). Furthermore, HveA(200t) and HveA(120t), but not HveA(76t) or HveA(77–120t), blocked herpes simplex virus (HSV) entry into CHO cells expressing HveA. We also generated six monoclonal antibodies (MAbs) against HveA(200t). MAbs CW1, -2, and -4 bound linear epitopes within the second CRP, while CW7 and -8 bound linear epitopes within the third or fourth CRPs. None of these MAbs blocked the binding of gD to HveA. In contrast, MAb CW3 recognized a discontinuous epitope within the first CRP of HveA, blocked the binding of gD to HveA, and exhibited a limited ability to block virus entry into cells expressing HveA, suggesting that the first domain of HveA contains at least a portion of the gD binding site. The inability of gD to bind HveA(76t) suggests that additional amino acid residues of the gD binding site may reside within the second CRP.

Herpes Simplex Virus Glycoprotein D Interferes with Binding of Herpesvirus Entry Mediator to Its Ligands through Downregulation and Direct Competition

ABSTRACT To initiate membrane fusion and virus entry, herpes simplex virus (HSV) gD binds to a cellular receptor such as herpesvirus entry mediator (HVEM). HVEM is a tumor necrosis factor (TNF) receptor family member with four natural ligands that either stimulate (LIGHT and LTα) or inhibit (BTLA and CD160) T cell function. We hypothesized that the interaction of gD with HVEM affects the binding of natural ligands, thereby modulating the immune response during infection. Here, we investigated the effect that gD has on the interaction of HVEM with its natural ligands. First, HSV gD on virions or cells downregulates HVEM from the cell surface. Similarly, trans-interaction with BTLA or LIGHT also downregulates HVEM from the cell surface, suggesting that HSV may subvert a natural mechanism for regulating HVEM activity. Second, we showed that wild-type gD had the lowest affinity for HVEM compared with the four natural ligands. Moreover, gD directly competed for binding to HVEM with BTLA but not LTα or LIGHT, indicating the possibility that gD selectively controls HVEM signals. On the other hand, natural ligands influence the use of HVEM by HSV. For instance, soluble BTLA, LTα, and LIGHT inhibited the binding of wild-type gD to HVEM, and soluble BTLA and LTα blocked HSV infection of HVEM-expressing cells. Thus, gD is at the center of the interplay between HVEM and its ligands. It can interfere with HVEM function in two ways, by competing with the natural ligands and by downregulating HVEM from the cell surface.

Engineered Disulfide Bonds in Herpes Simplex Virus Type 1 gD Separate Receptor Binding from Fusion Initiation and Viral Entry

ABSTRACT Glycoprotein D (gD) is the receptor binding protein of herpes simplex virus (HSV) and binds to at least two distinct protein receptors, herpesvirus entry mediator (HVEM) and nectin-1. While both receptor binding regions are found within the first 234 amino acids, a crystal structure shows that the C terminus of the gD ectodomain normally occludes the receptor binding sites. Receptor binding must therefore displace the C terminus, and this conformational change is postulated to be required for inducing fusion via gB and gH/gL. When cysteine residues are introduced at positions 37 and 302 of gD, a disulfide bond is formed that stabilizes the C terminus and prevents binding to either receptor. We speculated that if disulfide bonds were engineered further upstream, receptor binding might be separated from the induction of fusion. To test this, we made five additional double cysteine mutants, each potentially introducing a disulfide bond between the ectodomain C terminus and the core of the gD ectodomain. The two mutants predicted to impose the greatest constraint were unable to bind receptors or mediate cell-cell fusion. However, the three mutants with the most flexible C terminus bound well to both HVEM and nectin-1. Two of these mutants were impaired in cell-cell fusion and null-virus complementation. Importantly, a third mutant in this group was nonfunctional in both assays. This mutant clearly separates the role of gD in triggering fusion from its role in receptor binding. Based upon the properties of the panel of mutants we conclude that fusion requires greater flexibility of the gD ectodomain C terminus than does receptor binding.

Entry of Herpesviruses into Cells: The Enigma Variations

The entry of herpesviruses into their target cells is complex at many levels. Virus entry proceeds by a succession of interactions between viral envelope glycoproteins and molecules on the cell membrane. The process is divided into distinct steps: attachment to the cell surface, interaction with a specific entry receptor, internalization of the particle (optional and cell specific), and membrane fusion. Several viral envelope glycoproteins are involved in one or several of these steps. The most conserved entry glycoproteins in the herpesvirus family (gB, gH/gL) are involved in membrane fusion. Around this functional core, herpesviruses have a variety of receptor binding glycoproteins, which interact with cell surface proteins often from different families. This interaction activates and controls the actual fusion machinery. Interactions with cellular receptors and between viral glycoproteins have to be tightly coordinated and regulated to guarantee successful entry. Although additional entry receptors for herpesviruses continue to be identified, the molecular interactions between viral glycoproteins remain mostly enigmatic. This chapter will review our current understanding of the molecular interactions that occur during herpesvirus entry from attachment to fusion. Particular emphasis will be placed on structure-based representation of receptor binding as a trigger of fusion during herpes simplex virus entry.