We report on the functional cloning of a hitherto unknown member of the immunoglobulin (Ig) superfamily selected for its ability to confer susceptibility to herpes simplex virus (HSV) infection on a highly resistant cell line (J1.1-2 cells), derived by exposure of BHKtk− cells to a recombinant HSV-1 expressing tumor necrosis factor alpha (TNF-α). The sequence of herpesvirus Ig-like receptor (HIgR) predicts a transmembrane protein with an ectodomain consisting of three cysteine-bracketed domains, one V-like and two C-like. HIgR shares its ectodomain with and appears to be an alternative splice variant of the previously described protein PRR-1 (poliovirus receptor-related protein). Both HIgR and PRR-1 conferred on J1.1-2 cells susceptibility to HSV-1, HSV-2, and bovine herpesvirus 1. The viral ligand of HIgR and PRR-1 is glycoprotein D, a constituent of the virion envelope long known to mediate viral entry into cells through interaction with cellular receptor molecules. Recently, PRR-1, renamed HveC (herpesvirus entry mediator C), and the related PRR-2, renamed HveB, were reported to mediate the entry of HSV-1, HSV-2, and bovine herpesvirus 1, and the homologous poliovirus receptor was reported to mediate the entry of pseudorabies virus (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618–1620, 1998; M. S. Warner, R. J. Geraghty, W. M. Martinez, R. I. Montgomery, J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear, Virology 246:179–189, 1998). Here we further show that HIgR or PRR-1 proteins detected by using a monoclonal antibody to PRR-1 are widely distributed among human cell lines susceptible to HSV infection and commonly used for HSV studies. The monoclonal antibody neutralized virion infectivity in cells transfected with HIgR or PRR-1 cDNA, as well as in the human cell lines, indicating a direct interaction of virions with the receptor molecule, and preliminarily mapping this function to the ectodomain of HIgR and PRR-1. Northern blot analysis showed that HIgR or PRR-1 mRNAs were expressed in human tissues, with the highest expression being detected in nervous system samples. HIgR adds a novel member to the cluster of Ig superfamily members able to mediate the entry of alphaherpesviruses into cells. The wide distribution of HIgR or PRR-1 proteins among human cell lines susceptible to HSV infection, coupled with the neutralizing activity of the antibody in the same cells, provides direct demonstration of the actual use of this cluster of molecules as HSV-1 and HSV-2 entry receptors in human cell lines. The high level of expression in samples from nervous system makes the use of these proteins in human tissues very likely. This cluster of molecules may therefore be considered to constitute bona fide receptors for HSV-1 and HSV-2.

Infections with human herpesvirus 6 (HHV-6), a ß-herpesvirus of which two variant groups (A and B) are recognized, is very common, approaching 100% in seroprevalence. Primary infection with HHV-6B causes roseola infantum or exanthem subitum, a common childhood disease that resolves spontaneously. After primary infection, the virus replicates in the salivary glands and is shed in saliva, the recognized route of transmission for variant B strains; it remains latent in lymphocytes and monocytes and persists at low levels in cells and tissues. Not usually associated with disease in the immunocompetent, HHV-6 infection is a major cause of opportunistic viral infections in the immunosuppressed, typically AIDS patients and transplant recipients, in whom HHV-6 infection/reactivation may culminate in rejection of transplanted organs and death. Other opportunistic viruses, human cytomegalovirus and HHV-7, also infect or reactivate in persons at risk. Another disease whose pathogenesis may be correlated with HHV-6 is multiple sclerosis. Data in favor of and against the correlation are discussed.

A number of sentinels sense incoming herpes simplex virus (HSV) virions and initiate an immediate innate response. The first line of defense at the cell surface is TLR2 (Toll-like receptor 2), whose signature signaling activity leads to activation of the key transcription factor NF-κB. We report that the HSV pathogen-associated molecular patterns for TLR2 are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the members of the Herpesviridae family. Specifically, virions devoid singly of one of essential fusion glycoproteins (gD, gB, or gH null), able to attach to cells but defective in fusion/entry, were sufficient to elicit the first wave of NF-κB response to HSV. The most effective were the gD-null virions, positive for gH/gL and gB. A soluble form of gB, truncated upstream of the transmembrane sequence (gB _730t-st ), was produced in human cells and purified by means of a Strep tag. gH/gL and gB were each able to physically interact with TLR2 in coimmunoprecipitation assays, one independently of the other, yet gH _t-st /gL, but not gB _730t-st , elicited an NF-κB response. Thus, whereas both HSV gH/gL and gB are ligands to TLR2, only gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation.

We have made two stocks of a herpes simplex virus 1 mutant lacking intact U_S5 and U_S6 open reading frames encoding glycoproteins J (gJ) and D (gD), respectively. The stock designated gD^−/+, made in cells carrying U_S6 and expressing gD, was capable of productively infecting cells, whereas the stock designated gD^−/−, made in cells lacking viral DNA sequences, was known to attach but not initiate infection. We report the following. (i) Both stocks of virus induced apoptosis in SK-N-SH cells. Thus, annexin V binding to cell surfaces was detected as early as 8 h after infection. (ii) U_S5 or U_S6 cloned into the baculovirus under the human cytomegalovirus immediate-early promoter was expressed in SK-N-SH cells and blocked apoptosis in cells infected with either gD^−/+or gD^−/−virus, whereas glycoprotein B, infected cell protein 22, or the wild-type baculovirus did not block apoptosis. (iii) In SK-N-SH cells, internalized, partially degraded virus particles were detected at 30 min after exposure to gD^−/−virus but not at later intervals. (iv) Concurrent infection of cells with baculoviruses did not alter the failure of gD^−/−virus from expressing its genes or, conversely, the expression of viral genes by gD^−/+virus. These results underscore the capacity of herpes simplex virus to initiate the apoptotic cascade in the absence of de novo protein synthesis and indicate that both gD and gJ independently, and most likely at different stages in the reproductive cycle, play a key role in blocking the apoptotic cascade leading to cell death.

Entry of herpes simplex virus (HSV) 1 into cells requires the interaction of HSV gD with herpesvirus entry mediator or nectin1 receptors, and fusion with cell membrane mediated by the fusion glycoproteins gB, gH, and gL. We report that the gD ectodomain in soluble form (amino acids 1-305) was sufficient to rescue the infectivity of a gD-null HSV mutant, indicating that gD does not need to be anchored to the virion envelope to mediate entry. Entry mediated by soluble gD required, in addition to the receptor-binding sites contained within residues 1-250, a discrete downstream portion (amino acids 261-305), located proximal to the transmembrane segment in full-length gD. We named it as profusion domain. The pro-fusion domain was required for entry mediated by virion-bound gD, because its substitution with the corresponding region of CD8 failed to complement the infectivity of gD ^-/+ HSV. Furthermore, a receptor-negative gD (gD _Δ6-259 ) inhibited virus infectivity when coexpressed with wild-type gD; i.e., it acted as a dominant-negative gD mutant. The pro-fusion domain is proline-rich, which is characteristic of regions involved in protein-protein interactions. P291L-P292A substitutions diminished the gD capacity to complement gD ^-/+ HSV infectivity. We propose that gD forms a tripartite complex with its receptor and, by way of the proline-rich pro-fusion domain, with the fusion glycoproteins, or with one of them. The tripartite complex would serve to recruit/activate the fusion glycoproteins and bring them from a fusion-inactive to a fusion-active state, such that they execute fusion of the virion envelope with cell membrane.