B Virus (Macacine herpesvirus 1) Glycoprotein D Is Functional but Dispensable for Virus Entry into Macaque and Human Skin Cells

Abstract

Glycoprotein D (gD) plays an essential role in cell entry of many simplexviruses. B virus (Macacine herpesvirus 1) is closely related to herpes simplex virus 1 (HSV-1) and encodes gD, which shares more than 70% amino acid similarity with HSV-1 gD. Previously, we have demonstrated that B virus gD polyclonal antibodies were unable to neutralize B virus infectivity on epithelial cell lines, suggesting gD is not required for B virus entry into these cells. In the present study, we confirmed this finding by producing a B virus mutant, BV-⌬gDZ, in which the gD gene was replaced with a lacZ expression cassette. Recombinant plaques were selected on complementing VD60 cells expressing HSV-1 gD. Virions lacking gD were produced in Vero cells infected with BV⌬gDZ. In contrast to HSV-1, B virus lacking gD was able to infect and form plaques on noncomplementing cell lines, including Vero, HEp-2, LLC-MK2, primary human and macaque dermal fibroblasts, and U373 human glioblastoma cells. The gD-negative BV-⌬gDZ also failed to enter entry-resistant murine B78H1 cells bearing a single gD receptor, human nectin-1, but gained the ability to enter when phenotypically supplemented with HSV-1 gD. Cell attachment and penetration rates, as well as the replication characteristics of BV-⌬gDZ in Vero cells, were almost identical to those of wild-type (wt) B virus. These observations indicate that B virus can utilize gD-independent cell entry and transmission mechanisms, in addition to generally used gD-dependent mechanisms. A lphaherpesviruses share a strategy to enter host cells (1-3). Initial cell attachment of free virions is mediated by glycoprotein C (gC) and/or gB binding to cell surface heparan sulfate (4). This interaction facilitates specific binding of gD to one of several cellular receptors. To date, five gD receptors have been identified, including herpesvirus entry mediator (HVEM, or HveA), nectin-1 (HveC), nectin-2 (HveB), poliovirus receptor (PVR, or HveD), and 3-O-sulfated heparin sulfate (5-8). Receptor binding induces a conformational change in gD and subsequent transition into an active state. Activated gD then induces gB and gH-gL conformational changes, which trigger fusion between viral and cellular membranes (9). A key role of gD homologs in cell entry was established for all known alphaherpesviruses expressing the protein, including herpes simplex virus 1 (HSV-1), pseudorabies virus (PRV), bovine herpesvirus 1 (BHV-1), and equine herpesvirus 1 (EHV-1). Investigations of deletion mutants of these viruses showed that gD is essential for virus penetration into target cells (10)(11)(12)(13)(14). Numerous studies showing complete inhibition of virus cell entry by monoclonal gD antibodies, soluble recombinant gD protein, or soluble gD receptors further confirmed the crucial role of gD in in vitro infectivity of alphaherpesviruses (15)(16)(17)(18). Experiments demonstrating that vaginal infection of experimental animals with HSV-1 and HSV-2 could be prevented by pretreatment of a virus inoculum with gD-specific anti...

B Virus (Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies

“…HSV-1 strain KOS (ATCC VR-1493), HSV-2 strain G (ATCC VR-734), B virus laboratory strain E2490 (a kind gift from the late R. N. Hull, Eli Lilly, Indianapolis, IN), a B virus gD deletion mutant (BV-⌬gDZ) containing the ␤-galactosidase (␤-Gal) gene under the control of a human cytomegalovirus promoter (13), and 19 B virus clinical isolates (obtained from the National B Virus Resource Laboratory, Atlanta, GA) were propagated in Vero cells in maintenance medium with DMEM containing 2% heat-inactivated FBS (2% DMEM). Virus stocks were titrated by plaque assay on Vero cells and were stored in aliquots at Ϫ80°C.…”

“…Postmortem examinations reveal focal neuronal lesions occasionally seen in parietal neurons, but far more often in the brainstem and cervical spinal cord, which are primary sites of virus recovery (5-11). The molecular basis for the differences in neurovirulence between HSV and B virus in humans remains a mystery despite the fact that specific molecular differences between these two viruses have been identified (12)(13)(14)(15)(16)(17)(18)(19).B virus is genetically and immunologically closely related to HSV, and some aspects of cell entry and cell-to-cell transmission of B virus and HSV are conserved (14,(20)(21)(22)(23). The specific interactions of glycoprotein D (gD) with cognate cellular receptors, viz., herpesvirus entry mediator (HVEM), nectin-1, and nectin-2, as well as one of the several isoforms of 3-O-sulfated heparan …”

“…Postmortem examinations reveal focal neuronal lesions occasionally seen in parietal neurons, but far more often in the brainstem and cervical spinal cord, which are primary sites of virus recovery (5-11). The molecular basis for the differences in neurovirulence between HSV and B virus in humans remains a mystery despite the fact that specific molecular differences between these two viruses have been identified (12)(13)(14)(15)(16)(17)(18)(19).…”

“…The specific interactions of glycoprotein D (gD) with cognate cellular receptors, viz., herpesvirus entry mediator (HVEM), nectin-1, and nectin-2, as well as one of the several isoforms of 3-O-sulfated heparan sulfate, play a key role in initiation of the cascade of events leading to HSV cell entry (24)(25)(26)(27)(28)(29)(30)(31)(32). Similarly, B virus can utilize nectin-1 as a cell entry receptor, and its interaction with B virus gD is required for virus entry into cells bearing nectin-1 as the sole entry receptor (13,14). Unlike HSV, B virus cannot use either the human or monkey HVEM-mediated pathway for cell entry (14).…”

“…Unlike HSV, B virus cannot use either the human or monkey HVEM-mediated pathway for cell entry (14). Another substantial difference between HSV and B virus entry mechanisms is the ability of B virus to enter skin cells by using a gD-independent cell entry pathway(s) (13).…”