SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D

Abstract

Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.

Keywords: COVID-19; CP: Immunology; CP: Microbiology; NK cells; NKG2D; Nsp1; SARS-CoV-2; antiviral immunity; immune escape; innate immunity; natural killer cells.

Graphical abstract

Figure 1

SARS-CoV-2-infected target cells evade NK cell killing through a cell-intrinsic mechanism (A) Representative flow plots (left) and boxplot (right) showing the percentage of mNeonGreen+ A549-ACE2 cells following infection with either mNeonGreen SARS-CoV-2 (MOI = 0.5) or media (mock) at an MOI of 0.5 for either 24 or 48 h. Bar plots represent mean of n = 4 technical replicates ± SD values. (B) Schematic illustrating the experimental design of NK cell killing assays. (C) Representative flow plots showing expression of eFluor 780 viability dye in target cells with NK cells (top) and with NK cells (bottom). (D and E) Background-subtracted percentage of A549-ACE2 cell death as measured by eFluor 780 viability dye staining in either infected versus exposed, uninfected cells (D) or mock-infected versus exposed, uninfected cells (E). Background cell death for each experiment and condition was calculated as the average level of death in four wells of the condition of interest. Data are shown from n = 18 unique healthy donors across 4 separate experiments. Lines connect points from individual donors. (F and G) Representative flow plots (F) and quantitations (G) of percentage of NK cells expressing CD107a and IFN-γ upon culture with no targets, mock-infected targets, or SARS-CoV-2-infected targets. Lines connect points from individual donors (n = 6). Significance values for all plots in this figure were determined using a paired Wilcoxon signed-rank test with the Bonferroni correction for multiple hypothesis testing.

Figure 2

SARS-CoV-2 infection downregulates ligands for the activating receptor NKG2D (A) Boxplots showing the mean fluorescence intensity (MFI) of uninfected, bystander, and SARS-CoV-2-infected A549-ACE2 cells expressing CD54, CD112/CD155, HLA-ABC, and NKG2D-L (combination of MICA, MICB, and ULBPs 1, 2, 5, and 6). The cognate receptors recognizing each ligand are noted under each panel. Four technical replicates of each condition were performed. (B) Representative histograms of NKG2D-L, HLA-A/B/C, CD54, and CD112/CD155 expression in SARS-CoV-2-infected cells versus uninfected controls. Isotype controls are shown in gray. Vertical dashed lines represent thresholds for positivity. (C) Representative histograms showing expression of individual ligands for NKG2D in mock-infected (top) and SARS-CoV-2-infected (middle) A549-ACE2s. Isotype controls are shown at the bottom of each histogram for comparison. Dashed vertical lines represent thresholds for positivity. Numbers to the right of vertical lines indicate the percentage of cells positive for each marker. (D) Percentage of NKG2D-L-expressing A549-ACE2s in wells containing only target cells (n = 4 technical replicates) compared with wells containing target cells and NK cells (n = 6 biological replicates). Beginning at 48 h post-infection, target cells were co-cultured with IL-2-activated NK cells for 3 h at an E:T ratio of 5:1. (E) Percentage of mock-infected or SARS-CoV-2-infected (mNeonGreen+) A549-ACE2 expressing NKG2D-L at 24 and 48 h post-infection (n = 4 technical replicates per condition). (F) Background-subtracted target cell death of A549-ACE2 infected for either 24 or 48 h with SARS-CoV-2. Target cells were co-cultured for 3 h with IL-2-activated NK cells at an E:T ratio of 5:1. Lines connect data points from individual donors (n = 6). (G) Correlation between percentage of A549-ACE2s expressing NKG2D-L in target-only wells (mean of four technical replicates per condition) and background-subtracted target cell death in wells containing NK cells (mean of six biological replicates per condition). Significance values for (A, C, and D) were determined using an unpaired Wilcoxon ranked-sum test with the Bonferroni correction for multiple hypothesis testing. Significance value for (E) was determined using a paired Wilcoxon signed-rank test. Best-fit line shown in (F) was calculated using a linear model.

Figure 3

NK cells are able to efficiently kill SARS-CoV-2-infected cells immediately following infection (A) Representative histograms showing expression of mNeonGreen in SARS-CoV-2-exposed A549-ACE2 at 0, 24, or 48 h post-infection at an MOI of 3 (B and C) or 0.5 (D). Vertical dashed lines indicate threshold for positive gating. (B–D) A549-ACE2 were infected with SARS-CoV-2 for 0, 24, or 48 h, then co-cultured with IL-2-activated NK cells for 3 h at an E:T ratio of 5:1. (B) Background-subtracted killing of all single A549-ACE2s by NK cells following infection with SARS-CoV-2 for either 0 or 48 h. Lines connect points from individual donors (n = 12). (C) Fold change in killing of infected target cells compared with mock-infected target cells at 0 and 48 h post-infection. (D) Background-subtracted target cell death of all single A549-ACE2s infected with SARS-CoV-2 at 24 and 48 h post-infection (n = 6 unique donors). Significance values were determined using a paired Wilcoxon signed-rank test.

Figure 4

SARS-CoV-2 protein Nsp1 downregulates ligands for NKG2D (A) A schematic illustrating the experimental approach. Plasmids encoding individual SARS-CoV-2 proteins appended with two Strep-Tag domains were transfected into A549-ACE2s. After 48 h, transfected cells could be detected via flow cytometry using a primary antibody against Strep Tag II and a secondary fluorescent antibody. (B) Representative flow plots showing Strep Tag II expression in untransfected and Nsp7-transfected A549-ACE2s. (C) Percentage of transfected (Strep Tag II+) A549-ACE2s that express NKG2D-L by flow cytometry at 48 h post-transfection. 25 SARS-CoV-2 proteins are shown that were successfully transfected into A549-ACE2s, along with GFP as a transfection control. Four technical replicates were performed for each plasmid. Dashed line represents the mean frequency of expression in untransfected (mock) cells. Asterisks represent significance in comparison to mock-transfected controls. Plasmids are ordered by location in the SARS-CoV-2 genome and a schematic of the genome structure is shown below (C). (D) Representative histograms showing expression of individual ligands for NKG2D after transfection with transfection agent alone (top; mock) or Nsp1 (bottom). Dashed vertical lines indicate threshold for positivity. Numbers to the right of dashed lines show percentages of cells positive for each marker. (E) Percentage of A549-ACE2 positive for CD54, DNAM-1-L (CD112/CD155), or HLA-A/B/C at 48 h after transfection with transfection agent alone (mock), Nsp14, GFP, or Nsp1. Significance values for (C–E) were determined using an unpaired Wilcoxon rank-sum test with the Bonferroni correction for multiple hypothesis testing.

Figure 5

SARS-CoV-2 post-transcriptionally downregulates NKG2D-L and does not induce shedding or degradation (A) Boxplots showing the delta CT values of several genes in mock and SARS-CoV-2-infected A549-ACE2s as measured by qRT-PCR. N encodes SARS-CoV-2 nucleoprotein. MICA, MICB, ULBP1, and ULBP2 encode ligands for NKG2D. Each point represents the mean of three qPCR technical replicates. (B) Bar plot showing the fold change in mean fluorescence intensity (MFI) of NKG2D-L in SARS-CoV-2-infected A549-ACE2 compared with mock-infected cells after treatment with PBS (left), proteasome inhibitor MG-132 (middle), or lysosomal inhibitor BAF-A1 (right). Inhibitors were added 24 h after infection and NKG2D-L expression was measured by flow cytometry at 48 h post-infection. Bar plots represent mean values of three technical replicates ± standard deviations. (C) Absorbance values of neat supernatants from mock or SARS-CoV-2-infected cultures at varying dilutions as measured by plate-based ELISAs for soluble MICA (sMICA) and soluble ULBP2 (sUPBP2). Absorbance values were calculated by subtracting absorbance readings taken at 560 nm from those taken at 450 in accordance with the manufacturer’s instructions. Horizontal lines indicate limits of detection (dashed, sMICA; solid, sULBP2). Bar plots represent the means of four technical replicates for each condition ± standard deviations. Significance values in (A and B) were calculated using a Wilcoxon signed-rank test with the Bonferroni correction for multiple hypothesis testing.

Figure 6

Nsp1 is not highly expressed in SARS-CoV-2-infected cells until >24 h post-infection and negatively correlates with NKG2D-L (A) Representative flow plots showing expression of Nsp1 and mNeonGreen in mock-infected A549-ACE2 (left), A549-ACE2 infected with SARS-CoV-2 for 24 h (middle), or A549-ACE2 infected with SARS-CoV-2 for 48 h (right). (B) Representative histograms showing NKG2D-L expression across subpopulations of SARS-CoV-2-infected cells at 48 h post-infection. Dashed vertical line indicates threshold for positivity. Numbers to the right of the dashed vertical line represent the percentage of cells positive. These data are representative of three technical replicates for each condition.

Figure 7

Nsp1 is sufficient to confer significant resistance to NK cell-mediated killing (A) Representative flow plots showing expression of eFluor 780 viability dye in target cells with NK cells (top) and with NK cells (bottom). (B) Background-subtracted target cell death among cells transfected with either GFP or Nsp1 following co-culture with healthy NK cells (E:T = 5:1) for 3 h (n = 22 unique donors). (C) Fold change in killing of target cells transfected with various SARS-CoV-2 proteins compared with untransfected bystander cells. The same healthy NK cell donors were utilized for all killing assays performed in (C). Asterisks represent significance in comparison to Nsp1-transfected cells. Transfected cells were gated by Strep Tag II expression before the percentage of cytotoxicity was determined. n = 6 unique donors. (D) Background-subtracted target cell death in treated (transfected with Nsp1 or infected with SARS-CoV-2) versus bystander A549-ACE2s following co-culture with healthy NK cells (E:T = 5:1) for 3 h. Lines in (B–D) represent individual donors. (E) Boxplot quantifying the fold change in background-subtracted target cell death between bystander (uninfected/untransfected) cells and cells that were positive for either Nsp1 (transfected; n = 22) or SARS-CoV-2 (infected; n = 18). Significance values were determined using a paired Wilcoxon signed-rank test (B–D) or unpaired Wilcoxon ranked-sum test (E) with the Bonferroni correction for multiple hypothesis testing where necessary.