Is nectin-1 the “master” receptor for deadly herpes B virus infection?

The family Herpesviridae is a large, diverse family of double-stranded enveloped DNA viruses. Herpes B virus (BV) and Cercopithecine herpesvirus 2 (CeHV-2) are simian herpesviruses. Like herpes simplex virus 1 (HSV-1), they belong to the α-herpesvirus subfamily. Interest in BV infections results from the observation that zoonotic infections often result in death of humans, whereas infection of macaques, the natural host of BV, results in disease similar to that observed on HSV infections of humans. We recently reported in cell-cell fusion assays in which nectin-1, a HSV-1 gD receptor, mediated fusion of cells expressing glycoproteins from both BV and CeHV-2. However, HVEM, another HSV-1 gD receptor, did not mediate fusion by BV and CeHV-2 glycoproteins. Paired immunoglobulin-like type 2 receptor α (PILRα), an HSV-1 gB fusion receptor, did not mediate fusion with BV or CeHV-2 glycoproteins. These results were further confirmed by BV infection. Our results may indicate that differential receptor usage by BV in humans when compared with macaques may have pathological consequences. Understanding human and simian receptor usage for BV and HSV may provide clues to understand the pathogenesis of these viruses, as well as related viruses, in their natural host as well zoonotic infections. This broader understanding may result in the development of novel therapeutics to control deadly BV infections as well as herpesvirus infections in general. Herpes B virus (BV, officially named as Macacine herpesvirus 1, formerly Herpesvirus simiae, monkey B virus, or Cercopithecine herpesvirus 1) and Cercopithecine herpesvirus 2 (CeHV-2, formerly simian agent 8) are primate herpesviruses belonging to the α-herpesvirus subfamily and are closely related to herpes simplex virus 1 (HSV-1) and HSV-2. HSV causes recurrent mucocutaneous lesions on the mouth, face, or genitalia and in rare cases can cause meningitis or encephalitis. BV naturally infects macaques whereas zoonotic infections of foreign hosts, such as humans, can result in encephalitis, encephalomyelitis and death. BV infection has high mortality in humans (greater than 50% in documented infections) and as such is recognized as a deadly virus for humans requiring biosafety level 4. Similarly to the high level of pathogenicity of BV in humans, HSV infection of marmosets can also be fatal. CeHV-2 is a pathogen of baboons and is not known to cause disease in primates outside the natural hosts. Since the receptors for HSV are well described, we chose to explore the receptor usage of the BV and CeHV-2. HSV entry into target cells requires a minimum four virus encoded glycoproteins—glycoproteins B (gB), D (gD), H (gH) and L (gL). These glycoproteins interact with a variety of cellular receptors to facilitate virus entry. Best described are the cellular receptors for gB and gD which we chose to study. The interaction of gD with the gD receptors has been extensively investigated. The binding of gD to herpesvirus entry mediator (HVEM) (Montgomery et al., Cell 1996), nectin-1 (Geraghty et al., Science 1998), nectin-2 (Warner et al., Virology 1998) and modified heparan sulfate (Shukla et al., Cell 1999) triggers fusion of the virion envelope with a cellular membrane. Paired immunoglobulin-like type 2 receptor α (PILRα) is a receptor that binds to gB and also mediates entry and fusion (Satoh et al., Cell 2008) even though it shows much less fusion activity than the gD receptors nectin-1 and HVEM. Two other gB receptors, with less known in regard to the significance in virus infection, are myelin-associated glycoprotein (MAG) (Suenaga et al., Proc Natl Acad Sci U S A 2010), and non-muscle myosin heavy chain IIA (NMHC-IIA) (Arii et al., Nature 2010). HVEM is a member of the tumor necrosis factor receptor family. Nectin-1 and nectin-2 are cell adhesion molecules in the immunoglobulin superfamily and are widely expressed by a variety of cell types, including epithelial cells and neurons. Specific sites in heparan sulfate generated by certain 3-O-sulfotransferases (3-O-S HS) can also serve as a gD-binding entry receptor. PILRα is expressed on cells of the immune system. MAG is a cell-surface molecule belonging to immunoglobulin superfamily, is usually expressed in neural tissues. NMHC-IIA not only functions in the cytoplasm but also functions on the cell surface with gB upon viral entry. NMHC-IIA is expressed in a broad range of cell lines, tissues and cell types in vivo. To begin our studies, we generated expression constructs for the homologous glycoproteins (gB, gD, gH and gL) necessary for HSV-1 fusion from BV and CeHV-2. We then performed cell-cell fusion assays to determine which cellular and viral proteins were required for cell-cell fusion. The results indicated that nectin-1 is the primary receptor that mediates fusion for BV and CeHV-2, and that HVEM and PILRα do not function for BV and CeHV-2. To verify our cell-cell fusion results, we used cells expressing human nectin-1, HVEM or PILRα and determined if they could be infected with BV. The results confirmed that BV only utilizes nectin-1 and not HVEM or PILRα. To confirm that BV and CeHV-2 do not utilize human HVEM as an entry receptor because of differences between human and simian HVEM, we performed cell-cell fusion assays with simian HVEM. In these experiments, we found that simian HVEM could mediate fusion with cells expressing HSV-1 glycoproteins however; simian HVEM did not mediate fusion of cells expressing glycoproteins from BV and CeHV-2. Thus, as found in earlier studies and reports from other investigators, our findings are compatible with the idea that nectin-1 is a pan α-herpesvirus entry receptor. Of particular interest from our recent studies is the idea that differential receptor usage may be important for pathogenesis in infected hosts. Compatible with this idea, studies in mice have shown that nectin-1 is the primary receptor responsible for the infection of the vaginal epithelium with HSV-2. Interestingly, in experimental vaginal infections, nectin-1 was not the sole receptor capable of enabling spread of HSV infection from the vaginal epithelium to the PNS and CNS (Taylor et al., Cell Host Microbe 2007). However, expression of nectin-1 is necessary for HSV-2 infection via the intracranial route and for encephalitis while HVEM was not important (Kopp et al., Proc Natl Acad Sci U S A 2009). In contrast, in HSV-1 infection of the murine eye, we found that both HVEM and nectin-1 must be present for maximal HSV-1 infection further suggesting that receptor requirements for HSV depends on the route of infection and/or serotype (Karaba et al., J Virol 2011). The host immune response may also be important in pathogenesis. Previous studies have shown that engagement of HVEM by gD alters the immune response following murine vaginal infection. In these studies, there was a transient increase in mucosal chemokine and IL-6 levels when compared with infection with wild-type control virus in agreement with gD-HVEM interaction elicits an innate response (Yoon et al., PloS One 2011). Overall, our studies, as well as studies by others, indicate that choice of receptor utilized as well as the host may be important determinants in regard to the outcome of the infection. Virus receptor usage, replication and control by the host immune are likely fine-tuned to allow maximal virus replication without being detrimental to the host to allow virus spread to naive hosts. In contrast, when a virus infects a non-natural host, this balance may not exist resulting in severe pathogenesis as observed in BV infections of humans. By understanding the required interactions between host receptors and the relevant glycoproteins required for infection, novel therapeutics might be developed to treat zoonotic infections and prevent deadly infections or more broadly used in natural HSV infections that routinely occur within the human population.