Pseudorabies Virus Infection Causes Downregulation of Ligands for the Activating NK Cell Receptor NKG2D

Abstract

Herpesviruses display a complex and carefully balanced interaction with important players in the antiviral immune response of immunocompetent natural hosts, including natural killer (NK) cells. With regard to NK cells, this delicate balance is illustrated on the one hand by severe herpesvirus disease reported in individuals with NK cell deficiencies and on the other hand by several NK cell evasion strategies described for herpesviruses. In the current study, we report that porcine cells infected with the porcine alphaherpesvirus pseudorabies virus (PRV) display a rapid and progressive downregulation of ligands for the major activating NK cell receptor NKG2D. This downregulation consists both of a downregulation of NKG2D ligands that are already expressed on the cell surface of an infected cell and an inhibition of cell surface expression of newly expressed NKG2D ligands. Flow cytometry and RT-qPCR assays showed that PRV infection results in downregulation of the porcine NKG2D ligand pULBP1 from the cell surface and a very substantial suppression of mRNA expression of pULBP1 and of another potential NKG2D ligand, pMIC2. Furthermore, PRV-induced NKG2D ligand downregulation was found to be independent of late viral gene expression. In conclusion, we report that PRV infection of host cells results in a very pronounced downregulation of ligands for the activating NK cell receptor NKG2D, representing an additional NK evasion strategy of PRV.

Keywords: NKG2D; ligands; pMIC2; pULBP1; pig; pseudorabies virus.

Figure 1

Pseudorabies virus (PRV) infection suppresses NKG2D ligand expression on the surface of infected cells. (A) Flow cytometric histograms of mock-infected (left) and PRV-infected (right) swine kidney (SK) cells at 14 hpi, stained with antibodies against viral proteins gB (orange) and gD (purple), incubated with recombinant Fc-tagged human NKG2D protein (black) or a secondary antibody control (gray). (B) SK cells in suspension were mock-infected or infected with PRV strain Kaplan and at 14 h post-inoculation (hpi) incubated with recombinant Fc-tagged human NKG2D protein and analyzed by flow cytometry. (C) SK cells were cultivated in suspension for 8 h before mock or PRV inoculation. If indicated, cycloheximide (CHX) was added at 8 h of cultivation at a concentration of 5 µg/mL. At the different time points, indicated in the graph, cells were incubated with recombinant Fc-tagged human NKG2D protein and analyzed using flow cytometry. Graphs show the median fluorescence intensity (MFI) for each condition. Bars represent the mean value ± SD; different symbols correspond to individual data points from different independent experiments, n = 3. Statistically significant differences are indicated with asterisks (* p < 0.05 ** p < 0.01, *** p < 0.001).

Figure 2

Time course of the effect of PRV or mock inoculation on NKG2D binding to SK cells. SK cells were cultivated in suspension for 8 h before mock or PRV inoculation. At different time points, as shown in the graph, SK cells were incubated with recombinant Fc-tagged human NKG2D protein and analyzed using flow cytometry. The graph shows the median fluorescence intensity (MFI) for each condition. Representative graph of 2 independent experiments is shown, n = 2.

Figure 3

Viral late gene expression is not required for PRV-induced downregulation of NKG2D ligand expression. SK cells were cultivated in suspension for 8 h before mock or PRV infection. SK cells were treated or untreated with phosphonoacetic acid (PAA, 400 µg/mL) starting 30 min before inoculation. All samples were incubated with recombinant Fc-tagged human NKG2D protein at 14 hpi and analyzed using flow cytometry. Graph shows the median fluorescence intensity (MFI) for each condition. Bars represent the mean value ± SD; different symbols correspond to individual data points from different independent experiments, n = 3.

Figure 4

pULBP1 expression on the cell surface is reduced after PRV infection. SK cells were cultivated in suspension for 8 h before mock or PRV inoculation. At the indicated time points, pULBP1 expression on the cell surface was measured using flow cytometry. Graphs show the median fluorescence intensity (MFI) for each condition. Bars represent the mean value ± SD; different symbols correspond to individual data points from different independent repeats, n = 4. Statistically significant differences are indicated with asterisks (* p < 0.05).

Figure 5

mRNA expression of pULBP1 and pMIC2 in SK cells upon mock or PRV infection. Gene expression of (A) pULBP1 and (B) pMIC2 were analyzed in mock-infected SK cells or PRV-Kaplan-infected SK cells at different time points as shown in the graph, and were normalized to gene expression in SK cells cultivated in suspension for 8 h. Graphs represent the mean and SD values of relative mRNA expression obtained from five (A,B) or three (C,D) independent repeats of the experiment, n = 5 or n = 3. Statistically significant differences are indicated with asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).