Requirement of Interaction of Nectin-1α/HveC with Afadin for Efficient Cell-Cell Spread of Herpes Simplex Virus Type 1

Abstract

We recently found a novel cell-cell adhesion system at cadherin-based adherens junctions (AJs), consisting at least of nectin, a Ca 2؉ -independent homophilic immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin is associated with cadherin through afadin and ␣-catenin. The cadherin-catenin system increases the concentration of nectin at AJs in an afadin-dependent manner. Nectin constitutes a family consisting of three members: nectin-1, -2, and -3. Nectin-1 serves as an entry and cell-cell spread mediator of herpes simplex virus type 1 (HSV-1). We studied here a role of the interaction of nectin-1␣ with afadin in entry and/or cell-cell spread of HSV-1. By the use of cadherin-deficient L cells overexpressing the full length of nectin-1␣ capable of interacting with afadin and L cells overexpressing a truncated form of nectin-1␣ incapable of interacting with afadin, we found that the interaction of nectin-1␣ with afadin increased the efficiency of cell-cell spread, but not entry, of HSV-1. This interaction did not affect the binding to nectin-1␣ of glycoprotein D, a viral component mediating entry of HSV-1 into host cells. Furthermore, the cadherin-catenin system increased the efficiency of cell-cell spread of HSV-1, although it also increased the efficiency of entry of HSV-1. It is likely that efficient cell-cell spread of HSV-1 is caused by afadin-dependent concentrated localization of nectin-1␣ at cadherin-based AJs.Herpes simplex viruses (HSVs) are members of the neurotropic subfamily (alphaherpesviruses) of the herpesvirus family. Infection with HSV type 1 (HSV-1) is prevalent. HSV-1 infects cells through initial attachment to the plasma membrane and subsequent fusion of the viral envelope with the plasma membrane or through contiguous cell-cell spread. The entry pathway of HSV-1 is divided into three major processes: binding, fusion, and capsid penetration. These processes require several viral envelope glycoproteins (49-51). The initial attachment is mediated through glycoprotein C (gC) and/or gB to cell surface heparan sulfate proteoglycans (17,18,45), but this attachment is not sufficient for virus penetration (3,11,22,27). The fusion of the viral envelope with the plasma membrane requires gD, gB, gH, and gL (49-51). These four viral glycoproteins also participate in cell-cell spread (3,11,20,28,36,40). Cell-cell spread furthermore requires gE and gI (1, 8-10), but these glycoproteins are not required for entry (8). Thus, entry and cell-cell spread of HSV-1 share similar processes, but these pathways also differ in some significant aspects.Recently, expression cloning has led to the identification and isolation of HSV entry mediators (5,12,33,46,51). The human receptors identified include a lymphotoxin receptor (31), designated HVEM (33, 65) or HSV entry mediator A (HveA), which belongs to the tumor necrosis factor receptor family; two members of the immunoglobulin (Ig) superfamily (5,12,48,62); and 3-O-sulfated heparan ...

Nectin-like Molecule-5/Tage4 Enhances Cell Migration in an Integrin-dependent, Nectin-3-independent Manner

“…To express the extracellular fragment of nectin-3 fused to the human IgG Fc (Nef-3), pFastBac1-Msp-Fc-nectin-3-EC (aa 56 -400) was prepared as described (43). The fusion protein with IgG Fc was produced as a secreted protein by the baculovirus transfer system (Invitrogen, Carlsbad, CA) and purified by use of protein A-Sepharose beads (Amersham Biosciences) as described (42). pEF-BOS-Myc-NWASP-CRIB and pEF-BOS-MycN17Rac1 were prepared as described (44,45).…”

“…Construction-Expression vectors were constructed in pCAGIZeo (41), pFLAG-CMV1 (Sigma), and pFastBac1-Msp-Fc (42). Constructs of Necl-5 contained the following aa: pCAGIZeo-Necl-5-⌬CP, aa 1-374 (deletion of the cytoplasmic region); pFLAG-CMV1-Necl-5-⌬CP, aa 30 -374 (deletion of the signal peptide and the cytoplasmic region); and pFastBac1-Msp-Fc-Necl-5-EC (the extracellular fragment of Necl-5 fused to the human IgG Fc), aa 30 -347 (26).…”

Inhibiting the Arp2/3 Complex Limits Infection of Both Intracellular Mature Vaccinia Virus and Primate Lentiviruses

“…F10, the ecotropic MLV receptor from rat, expression plasmid was created by digesting pcDNA F10-ecoR (Takase-Yoden and Watanabe, 1999) with NaeI followed by the self-ligation. The human nectin 1 alpha (HigR) expression plasmid was generated by inserting EcoRI fragment of pEF-BOS human nectin 1 alpha 3xFLAG (Sakisaka et al, 2001) into EcoRI site of pcDNA3 (Invitrogen). The XhoI-NotI fragment of either pBCMGSneoCD4 or pBCMGSneoCCR5 (a generous gift from Dr. Yamashita) was cloned into XhoI-NotI sites of pcDNA3 (Invitrogen), generating pCD4 and pCCR5, respectively.…”

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 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). Each member of the nectin family forms a homo-cis-dimer, followed by formation of a homotrans-dimer, causing cell ± cell adhesion Miyahara et al, 2000;Satoh-Horikawa et al, 2000;Sakisaka et al, 2001;Reymond et al, 2001). Nectin-3 furthermore forms a hetero-trans-dimer with either nectin-1 or -2 and the formation of each heterotrans-dimer is stronger than that of each homo-transdimer .…”

“…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).…”