Generation of Herpesvirus Entry Mediator (HVEM)-Restricted Herpes Simplex Virus Type 1 Mutant Viruses: Resistance of HVEM-Expressing Cells and Identification of Mutations That Rescue Nectin-1 Recognition

Abstract

Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.Entry of herpes simplex virus type 1 (HSV-1) into cells requires the envelope glycoprotein D (gD), gB, gH, and gL (5, 13, 21, 27). After virion adsorption to cell surface proteoglycans, gD binds to one of its receptors, herpesvirus entry mediator (HVEM, or HveA), nectin-1 (HveC), or 3-O-sulfated heparan sulfate (3-O-S HS) (19,32,43). Receptor binding results in a conformational change in gD, which is believed to activate either or both gB and gH as mediators of fusion between the viral envelope and the plasma membrane or an endosomal membrane (7,18,20,26,31,34,35). Fusion leads to nucleocapsid release into the cytoplasm for subsequent viral genome delivery to the nucleus. Since productive HSV-1 infection is set in motion by the gD-receptor interaction, cellular susceptibility to HSV-1 is initially determined by the presence of one or more functional gD receptors on the cell surface.In addition to supporting HSV-1 entry, gD receptors also support HSV-1 cell-to-cell spread (8,41). However, it is not clear whether they act by the same mechanism in entry and spread. For example, gD mutants that allow gD-receptor-independent virus spread but not receptor-independent entry have been isolated (39). Likewise, gD is required for entry of the related alphaherpesvirus pseudorabies virus (PRV) but not for PRV lateral spread (2,36,37,40). Two additional glycoproteins, gE an...

Novel Mutations in gB and gH Circumvent the Requirement for Known gD Receptors in Herpes Simplex Virus 1 Entry and Cell-to-Cell Spread

“…Alternatively, either or both N753K and A778V could change the positioning of this surface relative to the flap in such a manner that complete disengagement of these two structures requires only a weak signal, likely from gD. This second scenario is the most attractive, as it would offer an explanation for the gD dependence of gH:KV-enhanced spread between cells that lack canonical gD receptors but express minor receptors capable of limited gD activation, such as nectin-3 (36). As discussed below, our molecular dynamics simulations suggested another possibility.…”

“…We performed infectious center assays (17,36) to determine whether the new gB and gH mutations affected cell-to-cell spread. Vero cells were infected with wild-type KOS or mutant viruses at an MOI of 10 to achieve 100% infection.…”

“…This approach identified new mutations in gD as well as revertants (36). In an attempt to isolate a broader spectrum of adaptive mutations, including mutations in other essential glycoproteins, we further disabled the gD-nectin-1 interaction by (i) creating mutations in both binding partners or (ii) introducing further changes in nectin-1 without mutations in gD.…”

“…Infectious center assays were performed as described previously (36). Briefly, donor cells (Vero or VD60) were infected at an MOI of 10 at 37°C for 2 h followed by an acidic wash.…”

A Herpes Simplex Virus 2 (HSV-2) gD Mutant Impaired for Neural Tropism Is Superior to an HSV-2 gD Subunit Vaccine To Protect Animals from Challenge with HSV-2

“…The HVEM and nectin-1 binding domains of HSV gD are located in different regions of the glycoprotein: the HVEM binding domain is at the N terminus (26)(27)(28), and the nectin-1 domain is in the middle of HSV gD (28)(29)(30). Mutations in one domain can impair the ability of HSV gD to use one receptor but not the other receptor (28,31,32). Accordingly, we constructed an HSV-2 gD mutant virus, HSV2-gD27, in which the nectin-1 binding domain was mutated and the virus was impaired for infecting neuronal cells in vitro and in vivo (33).…”