Expression of the Pseudorabies Virus gB Glycoprotein Triggers NK Cell Cytotoxicity and Increases Binding of the Activating NK Cell Receptor PILRβ

Abstract

Natural killer (NK) cells are components of the innate immunity and are key players in the defense against virus-infected and malignant cells. NK cells are particularly important in the innate defense against herpesviruses, including alphaherpesviruses. Aggravated and life-threatening alphaherpesvirus-induced disease has been reported in patients with NK cell deficiencies. NK cells are regulated by a diversity of activating and inhibitory cell surface receptors that recognize specific ligands on the plasma membrane of virus-infected or malignant target cells. Although alphaherpesviruses have developed several evasion strategies against NK cell-mediated attack, alphaherpesvirus-infected cells are still readily recognized and killed by NK cells. However, the (viral) factors that trigger NK cell activation against alphaherpesvirus-infected cells are largely unknown. In this study, we show that expression of the gB glycoprotein of the alphaherpesvirus pseudorabies virus (PRV) triggers NK cell-mediated cytotoxicity, both in PRV-infected and in gB-transfected cells. In addition, we report that, like their human and murine counterpart, porcine NK cells express the activating receptor paired immunoglobulin-like type 2 receptor beta (PILRβ), and we show that gB expression triggers increased binding of recombinant porcine PILRβ to the surfaces of PRV-infected cells and gB-transfected cells.IMPORTANCE Natural killer (NK) cells display a prominent cytolytic activity against virus-infected cells and are indispensable in the innate antiviral response, particularly against herpesviruses. Despite their importance in the control of alphaherpesvirus infections, relatively little is known about the mechanisms that trigger NK cell cytotoxicity against alphaherpesvirus-infected cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we found that the conserved alphaherpesvirus glycoprotein gB triggers NK cell-mediated cytotoxicity, both in virus-infected and in gB-transfected cells. In addition, we report that gB expression results in increased cell surface binding of porcine paired immunoglobulin-like type 2 receptor beta (PILRβ), an activating NK cell receptor. The interaction between PILRβ and viral gB may have consequences that stretch beyond the interaction with NK cells, including virus entry into host cells. The identification of gB as an NK cell-activating viral protein may be of importance in the construction of future vaccines and therapeutics requiring optimized interactions of alphaherpesviruses with NK cells.

Keywords: NK cells; PILRβ; glycoprotein gB; herpes; natural killer cells; pseudorabies virus.

FIG 1

Expression of PRV gB contributes to NK cell-mediated killing of PRV-infected cells. (A) SK cells were mock infected or infected with WT PRV or isogenic gB-null PRV (MOI, 10), collected at 12 hpi, and subsequently incubated with IL-2-primed porcine primary NK cells at a target/effector cell ratio of 1:25 for 4 h at 37°C. Viability of target cells was assessed by propidium iodide staining and flow cytometric analysis, and the percent NK cell-mediated lysis was calculated. The dot plot shows the results of 8 independent repeats, and the mean value is marked by a horizontal line. Statistically significant differences are indicated with asterisks (**, P < 0.01; ***, P < 0.001). (B) Mock-infected SK cells and SK cells infected with WT PRV or isogenic gB-null PRV were collected at 12 hpi and subsequently analyzed by Western blotting for expression of gB, gD, gE, and tubulin. (C) SK cells were collected at 12 hpi and subsequently analyzed by flow cytometry for the expression of PRV gB (left upper panel), PRV gE (right upper panel), and MHC class I (left lower panel). An overlay of the fluorescence intensities of the different samples (open histograms) and isotype controls (shaded histogram) is shown (black, mock-infected SK cells, blue, WT PRV-infected SK cells, red, gB-null PRV-infected SK cells). The graph (right lower panel) shows the mean fluorescence intensity (MFI) of MHC class I expression on infected SK cells. The data shown in the graph were calculated based on the MFI minus that of the isotype control-labeled cells. The dot plot shows the results of three independent repeats, and the mean value is marked by a horizontal line. Statistically significant differences are indicated with asterisks (**, P < 0.01; ***, P < 0.001).

FIG 2

Expression of PRV gB alone, in the absence of other viral proteins, is sufficient to trigger NK cell-mediated lysis of target cells. (A) RK13 and RN/008 cells were incubated with IL-2-primed porcine primary NK cells at target/effector cell ratios of 1:12, 1:6, and 1:3 for 4 h at 37°C. Viability of target cells was assessed by propidium iodide staining and flow cytometric analysis, and the percent NK cell-mediated lysis was calculated. Data represent mean + standard error of the mean (SEM) of 4 independent repeats. Statistically significant differences are indicated with asterisks (**, P < 0.01). (B) RK13 and RN/008 cells were assessed by Western blotting for expression of PRV gB and tubulin (left panel) and by flow cytometry for surface expression of PRV gB (right panel) (black, RK13 cells; dashed line, RN/008 cells; shaded histogram, isotype control).

FIG 3

Porcine NK cells express PILRβ, and expression of PRV gB increases binding of porcine recombinant PILRβ to the cell surfaces of PRV-infected and gB-transfected cells. (A) RT-PCR shows porcine PILRβ mRNA expression in primary porcine blood NK cells (NTC, control without template RNA; no RT, control reaction wherein reverse transcriptase activity is inhibited; PC, positive control). (B) Recombinant porcine PILRβ was produced, purified, and checked by SDS-PAGE followed by Coomassie blue staining (left panel) and by Western blotting via detection of the human Fc tag (right panel). (C) Swine kidney (SK) cells were mock infected or infected with wild-type PRV (WT) or gB-null PRV and at 14 hpi were incubated with recombinant Fc-tagged porcine PILRβ, followed by staining with fluorescently labeled anti-human-Fc antibodies and flow cytometric analysis. The histogram (left panel) shows PILRβ binding to mock-infected cells (black), WT PRV-infected cells (blue), and gB-null PRV-infected cells (red). The graph (right panel) shows MFI values of cells incubated with recombinant PILRβ minus that of cells that were stained only with secondary antibody. Dot plots show the result of four independent repeats, and the mean value is marked by a horizontal line. Statistically significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01). (D) RK13 cells and gB-expressing RK13 (RN/008) cells were incubated with recombinant Fc-tagged porcine PILRβ, followed by staining with fluorescently labeled anti-human-Fc antibodies and flow cytometric analysis. The histogram (left panel) shows PILRβ binding to RK13 cells (black) and RN/008 cells (blue). The graph (right panel) shows MFI values of cells incubated with recombinant PILRβ minus that of cells that were stained only with secondary antibody. Dot plots show the result of three independent repeats, and the mean value is marked by a horizontal line. Statistically significant differences are indicated with asterisks (*, P < 0.05).