Deletion of the second immunoglobulin-like domain of nectin-1 alters its intracellular processing and localization and ability to mediate entry of herpes simplex virus

Abstract

Nectin-1 is an immunoglobulin (Ig)-like entry receptor for herpes simplex virus (HSV). Like other nectins, nectin-1 forms dimers and mediates cell adhesion through interactions with other nectins. We constructed a second-domain deletion mutant of nectin-1 (nectin-1-Delta2) to examine the role of the second Ig-like domain in HSV entry. Nectin-1-Delta2 exhibited a severely reduced ability to mediate HSV entry and accumulated in the endoplasmic reticulum but retained the ability to interact with its HSV ligand, gD. The failure of nectin-1-Delta2 to mediate HSV entry probably resulted from its failure to be transported to a membrane targeted by HSV for viral entry.

FIG. 1.

Structure, HSV entry activity, and cell surface expression of nectin-1-Δ2. (A) Schematic representation of the nectin-1-Δ2 mutant, with amino acid numbering (based on GenBank reference sequence NP_002846) shown below. (B) Nectin-1-Δ2 does not mediate entry of HSV-1. CHO-K1 cells transfected with receptor or vector control were grown to confluency in 96-well tissue culture plates and exposed to increasing doses of reporter virus HSV-1(KOS)tk12 that expresses β-galactosidase upon viral entry (23). After 6 h of incubation at 37°C, cells were permeabilized and ONPG substrate was added. Viral entry was quantified by measuring optical densities at 405 nm. Error bars represent 1 standard deviation of triplicate determinations within the same experiment, and the results shown here are representative of three independent experiments. (C) Cell surface expression of nectin-1 and nectin-1-Δ2. CHO-K1 cells were transfected with plasmids expressing nectin-1, nectin-1-Δ2, or vector control, replated in 96-well plates, and then incubated with anti-nectin-1 monoclonal antibodies CK8 or CK41. Following washes to remove unbound antibodies, the cells were fixed and incubated with secondary antibodies and the horseradish peroxidase detection system described elsewhere (6). Results of the cell-based enzyme-linked immunosorbent assays are expressed as optical density (OD) at 380 nm and are means of triplicate measurements, with error bars indicating 1 standard deviation. The results shown are representative of three independent experiments.

FIG. 2.

Western blot analysis of nectin-1 and nectin-1-Δ2 after endoglycosidase treatment. CHO-K1 cells were transfected with tagged versions of nectin-1 and nectin-1-Δ2. The cells were lysed 48 h after transfection, cell lysates were treated with Endo H or PNGase F and run on a denaturing SDS-PAGE gel, and the expressed proteins were visualized by Western blotting using antibodies specific for the tags.

FIG. 3.

Cellular localization and expression of nectin-1 and nectin-1-Δ2. (A) CHO-K1 cells were transfected with plasmids expressing nectin-1 or nectin-1-Δ2 and stained with anti-nectin-1 antibody (top panels) or visualized by phase-contrast microscopy (bottom panels). Nectin-1 localized to cell-cell junctions, whereas nectin-1-Δ2 expression was mainly confined to the cytoplasm. Bars, 10 μm. (B) Similar amounts of nectin-1 (WT) and nectin-1-Δ2 (Δ2) mRNA were detected by reverse transcriptase PCR using primers that amplify a 247-bp region upstream of the deletion in nectin-1-Δ2. Total RNA extracted from transfected CHO cells using the RNeasy kit (QIAGEN) was subjected to DNase I treatment (Invitrogen). RNA samples before (-RT) or after (+RT) reverse transcription (Promega) using an oligo(dT) primer were used as templates in the PCR. (-), negative control (water).

FIG. 4.

Costaining of wild-type and mutant nectin-1 with calreticulin or viral gD. (A) Nectin-1 does not colocalize with calreticulin in CHO cells transfected with nectin-1. (B) Nectin-1-Δ2 colocalizes with calreticulin in CHO cells transfected with nectin-1-Δ2. Primary antibodies used in panels A and B were chicken anti-nectin-1 (green) and rabbit anti-calreticulin (red; Abcam). Bottom right of each panel shows the overlay, and the bottom left shows a phase-contrast image. (C) Colocalization of HSV gD and wild-type or mutant nectin-1. CHO-K1 cells were transfected with plasmid pCJ3, expressing HSV-1 gD (6), or cotransfected with plasmids expressing HSV-1 gD and either nectin-1 or nectin-1-Δ2, stained with chicken anti-nectin-1 antibody (green) and rabbit anti-gD antibody R7 (red), and visualized by confocal microscopy. As in cells transfected with receptor alone, nectin-1 localized to cell-cell junctions, whereas nectin-1-Δ2 localized to the cytoplasm. HSV-1 gD was mostly colocalized with nectin-1 or nectin-1-Δ2 but, when expressed alone, had a different intracellular localization than when expressed with either form of nectin-1. Bars, 10 μm.