Use of Chimeric Nectin-1(HveC)-Related Receptors to Demonstrate That Ability to Bind Alphaherpesvirus gD Is Not Necessarily Sufficient for Viral Entry

Abstract

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.

Function of Herpes Simplex Virus Type 1 gD Mutants with Different Receptor-Binding Affinities in Virus Entry and Fusion

“…By contrast, gD(ٌ243t) had WT affinity for both receptors, yet virus complemented with this form of gD was impaired for entry into cells bearing either receptor. This is consistent with the suggestion, based on studies of nectin-1 mutations, that gD binding is not sufficient for entry (15). We have proposed that gD(ٌ243) might be defective in a postbinding event (61).…”

Use of herpes simplex virus and pseudorabies virus chimeric glycoprotein D molecules to identify regions critical for membrane fusion

“…Binding was quantified as described (21,22). Briefly, CHO-K1 cells in six-well plates were transfected with pCAGGS-based plasmids expressing various forms of gD along with HSV-1 gB, gH, gL, and T7 polymerase for 6 h. The latter plasmids have been described (18).…”

Restoration of E-cadherin-based cell–cell adhesion by overexpression of nectin in HSC-39 cells, a human signet ring cell gastric cancer cell line

“…Nectin is a Ca 2+ -independent immunoglobulinlike cell ± cell adhesion molecule Lopez et al, 1998;Takahashi et al, 1999). This adhesion molecule was originally isolated as the poliovirus receptor-related protein and has recently been shown to serve as an a-herpes virus entry and cell ± cell spread mediator (Cocchi et al, 1998;Geraghty et al, 1998;Warner et al, 1998;Lopez et al, 2000;Sakisaka et al, 2001). Nectin comprises a family consisting of four members, nectin-1, -2, -3, and -4, and each of nectin-1, -2, and -3 has two or three splicing variants: nectin-1a, -1b, -2a, -2d, -3a, -3b, and -3g (Morrison and Racaniello, 1992;Aoki et al, 1994;Lopez et al, 1995;Eberle et al, 1995;Cocchi et al, 1998;Satoh-Horikawa et al, 2000;Reymond et al, 2001).…”