PILRalpha is a herpes simplex virus-1 entry coreceptor that associates with glycoprotein B

Abstract

Glycoprotein B (gB) is one of the essential components for infection by herpes simplex virus-1 (HSV-1). Although several cellular receptors that associate with glycoprotein D (gD), such as herpes virus entry mediator (HVEM) and Nectin-1, have been identified, specific molecules that mediate HSV-1 infection by associating with gB have not been elucidated. Here, we found that paired immunoglobulin-like type 2 receptor (PILR) alpha associates with gB, and cells transduced with PILRalpha become susceptible to HSV-1 infection. Furthermore, HSV-1 infection of human primary cells expressing both HVEM and PILRalpha was blocked by either anti-PILRalpha or anti-HVEM antibody. Our results demonstrate that cellular receptors for both gB and gD are required for HSV-1 infection and that PILRalpha plays an important role in HSV-1 infection as a coreceptor that associates with gB. These findings uncover a crucial aspect of the mechanism underlying HSV-1 infection.

Figure 1. PILRα ligand expressed on HSV-1-infected cells is gB

(A) Expression of a ligand for PILRα on HSV-1-infected cells. 293T cells or HSV-1 (strain F, VR3, SC16 or KOS)-infected 293T cells were stained with human PILRα-Ig (solid line) or a control Ig fusion protein (CD200-Ig, dotted line). (B) Immunoprecipitation of PILRα ligand from HSV-1-infected cells. Lysates of HSV-1-infected or non-infected 293T cells were immunoprecipitated with PILRα-Ig and immunoprecipitates were separated by SDS-PAGE, followed by silver staining. (C) Western blot analysis of the HSV-1 PILRα ligand. Lysate from HSV-1-infected cells was immunoprecipitated with PILRα-Ig, Nectin-1-Ig, or CD200-Ig (control). Immunoprecipitates were separated by SDS-PAGE and were blotted with anti-gB or anti-gD Ab. Ig fusion proteins used for immunoprecipitation were detected by anti-human IgG Ab.

Figure 2. Specific interaction between PILRα and gB

(A) Specific binding of human PILRα to gB. Mutant gB that lacks the C-terminus 40 amino acids, gD, or gH and gL was co-transfected with GFP into 293T cells. Transfected cells were stained with PILRα-Ig and Nectin-1-Ig fusion proteins, and anti-gB, anti-gD, and anti-gH mAbs (solid line). Cells were also stained with control Ig fusion protein or control mAb (dotted line). The staining patterns of GFP-positive cells are shown. (B) PILRα-Ig does not recognize cells infected with gB-deficient HSV-1. Non-infected 293T cells and 293T cells infected with wild-type HSV-1, gB-deficient HSV-1, or revertant HSV-1 were stained with PILR-Ig, Nectin-1-Ig, anti-gB mAb, and anti-gD mAb (solid line). Cells were also stained with control Ig fusion protein or control mAb (dotted line).

Figure 3. HSV-1 infection of PILRα-transfected CHO-K1 cells

(A) PILRα-ligand-negative CHO-K1 cells were transiently transfected with the pMx-IRES-DsRed expression vector containing human PILRα. Transfected CHO-K1 cells were infected with HSV-1-GFP, and cells expressing GFP within the DsRed-positive population were analyzed by flow cytometry. (B) Proportions of cells expressing GFP, gated on human PILRα (DsRed)-positive cells, are shown. Mean ± SD of triplicate analyses are shown. (C) CHO-K1 cells were transiently transfected with PILRα-IRES-DsRed or mock-IRES-DsRed expression vectors and cells expressing DsRed were purified by using a cell sorter. The transfected cells were infected with HSV-1-GFP and expression of GFP was analyzed by fluorescence microscopy.

Figure 4. Inhibition of HSV-1 infection by anti-PILRα mAb or by PILRα-Ig

(A) Inhibition of HSV-1 infection by anti-PILRα mAb. CHO-K1 cells were transiently transfected with human PILRα in the pMx-IRES-DsRed expression vector, and cells were infected with HSV-1-GFP in the presence of various concentrations of anti-PILRα or control mAb. Proportions of infected cells were determined by flow cytometry. (B) Inhibition of HSV-1 infection by PILRα-Ig fusion protein. CHO-K1 cells transiently transfected with human PILRα were infected with HSV-1-GFP in the presence of various concentrations of PILRα-Ig or control Ig fusion protein. The proportion of infected cells was determined by flow cytometry. Mean ± SD of triplicate analyses are shown.

Figure 5. Requirement of gD in PILRα-mediated HSV-1 infection

(A) Requirement of gD in HSV-1 infection of PILRα-expressing cells. PILRα- or mock-transfected CHO cells were infected with gD-deficient virus produced by normal Vero cells (gD (−) HSV-1, closed circle) or gD-transfected Vero cells (gD (+) HSV-1, open circle). Proportions of ICP4 (a viral protein produced immediately after infection)-positive cells, detected by flow cytometry, are shown as mean ± SD of triplicate analyses. (B) Inhibition of HSV1 infection by soluble gD. PILRα- or mock-transfected CHO cells were infected with HSV-GFP in the presence of gD-Ig (closed circle) or control Ig (open circle) fusion protein. Proportions of GFP-positive cells are shown as mean ± SD of triplicate analyses.

Figure 6. Cell fusion mediated by interaction between gB and PILRα

Cell fusion assay between CHO cells transfected with HSV-1 glycoproteins and PILRα. gB, gD, gH, and gL (BDHL), gB, gH, and gL (BHL), or gD, gH, and gL (DHL) were co-transfected into CHO-K1 cells with GFP. PILRα or control plasmid (Mock) was co-transfected into CHO-K1 cells with RFP. GFP- and RFP-expressing cells were purified by using flow cytometry and were co-cultured. After 8 h, cells were analyzed by fluorescence microscopy. Photographs taken by green and red filters were overlaid (Overlay). Green and red colors of non-fused cells were converted to gray color, and yellow colors of fused cells were left unchanged (Processed color). Photographs taken using phase contrast are also shown (Phase). Multinuclear cells are circled in red.

Figure 7. PILRα-mediated HSV-1 infection in primary cells

(A) Expression of HVEM, Nectin-1, and PILRα on human PBMC. PBMC were stained with anti-HVEM, anti-Nectin-1, or anti-PILRα mAbs, along with anti-CD14 mAb. (B) PILRα-mediated HSV-1 infection of CD14-positive PBMC. Freshly isolated human CD14-positive or -negative PBMC were infected with various amounts of HSV-1-GFP in the presence or absence of anti-human PILRα mAb or control mAb (10 μg/ml) and the proportion of infected cells was determined by flow cytometry. (C) Role of HVEM in HSV-1 infection of monocytes. Freshly isolated human CD14-positive monocytes were infected with HSV-1 GFP in the presence of anti-HVEM serum or control serum at the indicated concentrations. (D) Role of gD in HSV-1 infection of primary monocytes. Freshly isolated human CD14-positive monocytes were infected with gD-deficient virus produced by normal Vero cells (gD(−) HSV-1) or gD-transfected Vero cells (gD(+) HSV-1). Proportions of ICP4-positive (infected) cells were determined by flow cytometry. All the data are shown as mean ± SD of triplicate analyses.