Cell-to-cell spread of wild-type herpes simplex virus type 1, but not of syncytial strains, is mediated by the immunoglobulin-like receptors that mediate virion entry, nectin1 (PRR1/HveC/HIgR) and nectin2 (PRR2/HveB)

Abstract

The immunoglobulin-like receptors that mediate entry of herpes simplex virus type 1 (HSV-1) into human cells were found to mediate the direct cell-to-cell spread of wild-type virus. The receptors here designated Nectin1alpha and -delta and Nectin2alpha were originally designated HIgR, PRR1/HveC, and PRR2alpha/HveB, respectively. We report the following. (i) Wild-type HSV-1 spreads from cell to cell in J cells expressing nectin1alpha or nectin1delta but not in parental J cells that are devoid of entry receptors. A monoclonal antibody to nectin1, which blocks entry, also blocked cell-to-cell spread in nectin1-expressing J cells. Moreover, wild-type virus did not spread from a receptor-positive to a receptor-negative cell. (ii) The antibody to nectin1 blocked transmission of wild-type virus in a number of human cell lines, with varying efficiencies, suggesting that nectin1 is the principal mediator of wild-type virus spread in a variety of human cell lines. (iii) Nectin1 did not mediate cell fusion induced by the syncytial strains HSV-1(MP) and HFEM-syn. (iv) Nectin2alpha could serve as a receptor for spread of a mutant virus carrying the L25P substitution in glycoprotein D, but not of wild-type virus, in agreement with its ability to mediate entry of the mutant but not of wild-type virus.

FIG. 1

Cell-to-cell spread of HSV-1 occurs in nectin1α- or nectin1δ-expressing cells (B) but not in the receptor-negative J or CHO cells (A). (a) Micrograph of J cells transfected with HSV-1(F) DNA shows only singly infected cells or small aggregates. (b) Micrograph of stable transformants of J cells expressing nectin1α transfected with HSV-1(F) DNA shows presence of plaques. (c and d) Infectious center assay. Monolayers of nectin1α cells infected with R8102 were trypsinized and seeded onto recipient monolayers of J (c) or CHO (d) cells. Note the absence of plaques. (B) Plaque formation in nectin1α or nectin1δ cells infected with R8102 and inhibition by MAb R1.302. (e and f) Micrographs of nectin1α-expressing J cells infected with R8102, unexposed (e) or exposed to MAb R.1302 (f). (g and h) Micrographs of nectin1δ-expressing J cells infected with R8102, unexposed (g) or exposed to MAb R1.302 (h). Note that plaque size is greatly reduced in panels f and h relative to size in panel e or g. Infected cells were detected by immunostaining with polyclonal antibody to gM (a and b) or by X-Gal staining (c to h). Pictures were taken in an Axioplan Zeiss microscope. All pictures are at the same magnification.

FIG. 2

MAb R1.302 (A) and soluble nectin1 (B) inhibit plaque formation in nectin1α-expressing J cells infected with R8102. (A) Nectin1α-expressing J cells in 96 wells, infected with R8102, were exposed to the indicated concentrations of purified IgGs from MAb R1.302 (●), control mouse IgG (▴), or R1.302 ascites fluid (■), from 4 h after infection. Infection was detected 48 h later by permeabilization with 0.5% NP-40 and quantitative detection of β-galactosidase activity with ONPG, followed by reading of the optical density (OD) at 405 nm in a Bio-Rad enzyme-linked immunosorbent assay reader. The reading in untreated cultures was in the linear range (around 1 optical density unit) and was made equal to 100%. Each point represents the average of triplicate samples. The values in the abscissa represent the concentrations of purified IgGs (● and ▴) or dilution of ascites fluid (■). (B) Nectin1α-expressing J cells infected with R8102 in 96 wells were exposed to the indicated concentrations of the soluble form of nectin1 containing the entire ectodomain fused to the Fc portion of human IgG (VCC1-Fc) or containing the single V domain (V1-Fc) or to bovine serum albumin (BSA), from 4 h after infection. The soluble forms of nectin1 were described previously (6). Infection was detected as described for panel A. The values in the ordinate represent the optical densities (OD) at 405 nm (×100). Each point represents the average of triplicate samples. (C) Micrograph of R8102-infected J cells expressing a chimeric form of nectin1 [V(HIgR)-PVRα] that contains the V domain of nectin1 fused to C domains and the transmembrane and cytoplasmic regions of poliovirus receptor.

FIG. 3

(A) MAb R1.302 inhibits R8102 plaque formation in HEp-2 and 143tk⁻ cells. Shown are micrographs of HEp-2 (a to c) and 143tk⁻ (d to f) cells infected with R8102, unexposed (a and d) or exposed to MAb R1.302 (b and e) or to an irrelevant MAb (c and f), from 4 to 48 h after infection. Plaques were detected by X-Gal straining. (B) MAb R1.302 reduces infectious center formation in human cell lines. Nectin1α cells infected with R8102 were trypsinized at 4 h after infection and plated onto monolayers of recipient cells in the absence (control) (left) or presence (right) of MAb R1.302 in six-well plates. Recipient cells were nectin1α cells, 143tk⁻, HEp-2, 5637, and Lan5 cells. Monolayers were stained with X-Gal. The stained six-well dishes were scanned in an Agfa StudioStar scanner, equipped with incident light, and the image was imported by means of Photoshop software. The number of stained cells was determined by the histogram program. This measure accounts for all the stained cells in the monolayer. The overall number of stained cells in the monolayer treated with MAb R1.302 was expressed as a percentage relative to the corresponding untreated monolayer. All pictures are at the same magnification.

FIG. 4

MAb R1.302 does not reduce plaque formation by HSV-1(MP) or HFEM-syn. Shown are micrographs of nectin1α (A and B) and 143tk⁻ (C and D) cells infected with HSV-1(MP) or HFEM-syn, respectively, and unexposed (A and C) or exposed to MAb R1.302 (B and D) from 4 h after infection till fixation. Plaques were detected by immunostaining with polyclonal antibody to gM. All pictures are at the same magnification.

FIG. 5

(a and b) Plaque formation in nectin2α-expressing cells infected with HSV-1(U10) and inhibition by MAb R2.525. Shown are micrographs of nectin2α cells, which harbor a lacZ gene driven by the α27 promoter, infected with HSV-1(U10) and maintained in the absence (a) or presence (b) of MAb R2.525 from 4 h after infection. (c) Micrograph of nectin2α cells transfected with HSV-1(F) DNA. Note singly infected cells or small aggregates and the absence of plaques. (a and b) Infection was monitored as β-Gal activity. (c) Cells were stained with polyclonal antibody to gM.