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

SARS-CoV-2 evades cytotoxic responses from natural killer cells

In a recent study published on bioRxiv*preprint server, Stanford University School of Medicine researchers have revealed the mechanism via severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evading natural killer (NK) cell cytotoxic reactions.


The immune response to viral infection is significantly influenced by NK cells, which are innate lymphocytes. These cytotoxic effector cells respond rapidly to viral infection by targeting and lysing infected cells.

Studies evaluating the immune response in CoV disease 2019 (COVID-19) during the SARS-CoV-2 pandemic found that NK cells are less prevalent in the peripheral blood of patients with severe SARS-CoV-2 compared to to healthy donors. Additionally, immune profiling revealed substantial, severity-related transcriptional and phenotypic alterations in peripheral NK cells that persist in the blood of SARS-CoV-2 patients.

Although several studies have shown that NK cells can inhibit the replication of SARS-CoV-2 in vitro, the mechanism by which NK cells respond immediately to SARS-CoV-2 infected cells remains unclear. This is all the more crucial as several viruses adopt techniques to avoid being detected and eliminated by NK cells.

Study: SARS-CoV-2 evades direct NK cell killing through Nsp1-mediated ligand downregulation for NKG2D

About the study

The present research aimed to assess the ability of SARS-CoV-2 to adjust NK cell identification and lysis of virus-infected cells. The team developed a in vitro model approach that analyzes NK cell response to SARS-CoV-2 infected cells using primary NK cells derived from healthy donors and replicating SARS-CoV-2. To further elucidate how the balance between SARS-CoV-2 detection and evasion leads to COVID-19, they focused on examining the direct killing of SARS-CoV-2-infected target cells by NK cells.

Adenocarcinoma cells of human alveolar basal epithelium (A549)-angiotensin-converting enzyme 2 (ACE2), identified and lysed by NK cells and infectable by SARS-CoV-2, have been used as a model by the authors to study the response of NK cells to COVID -19. They introduced NK cells obtained from healthy donors preactivated overnight with interleukin 2 (IL-2) to treat SARS-CoV-2 infected cells for 48 hours to understand how exposure to Target cells infected with SARS-CoV-2 affects NK cell activity and phenotype.

The researcher assessed whether SARS-CoV-2 alters the ability of NK cells to annihilate infected target cells. Additionally, they explored how SARS-CoV-2-infected cells managed to evade lysis and recognition by NK cells.

The authors examined the expression of NK group ligand 2D (NKG2D-L) on cells that persisted after being co-cultured with NK cells to investigate the relationship between NKG2D-L expression and destruction of cells infected with SARS-CoV-2. The team then attempted to understand how SARS-CoV-2 affects the expression of the NKG2D-L protein in cells infected with SARS-CoV-2.

NK cells respond similarly to SARS-CoV-2-infected and fake-infected target cells.  A) Representative flow plots (left) and box plot (right) showing the percentage of mNeonGreen-positive A549-ACE2 cells following infection with mNeon Green SARS-CoV-2 (MOI 0.5) or media (“sham”) at an MOI of 0.5 for 24 or 48 hours.  Bar charts representNK cells respond similarly to SARS-CoV-2-infected and fake-infected target cells. A) Representative flow plots (left) and box plot (right) showing the percentage of mNeonGreen-positive A549-ACE2 cells following infection with mNeon Green SARS-CoV-2 (MOI 0.5) or media (“sham”) at an MOI of 0.5 for 24 or 48 hours. Bar plots represent n = 4 technical replicates ∓ SD values. B) Schematic illustrating the experimental design of NK cell killing assays. CD) Plots showing median fluorescence intensity (C) and % positive NK cells (D) for eight different NK cell markers by flow cytometry when cultured without targets, mock-infected targets, or targets infected with SARS-CoV-2. EF) Representative flow plots (E) and quantifications (F) of the percentage of NK cells expressing CD107a and IFNγ when cultured without targets, mock-infected targets, or SARS-CoV-2 infected targets. Significance values ​​were determined using a paired Wilcoxon ranked sum test with Bonferroni’s correction for multiple hypothesis testing.


Overall, the study results showed that NK cells have weak cytotoxic responses towards SARS-CoV-2 infected targets, selectively killing uninfected bystander cells. Additionally, the team showed that the significant downregulation of SARS-CoV-2-infected cells of ligands for the stimulating NKG2D receptor mediated this escape from NK cell killing.

Indeed, NK cells can efficiently identify and kill infected cells in the early stages of SARS-CoV-2 infection before NKG2D-L downregulation. However, NK cells lose this ability following the expression of viral proteins inside infected cells. Current data show that when introduced into culture at later timestamps after SARS-CoV-2 infection, after expression of viral proteins that attenuate the innate immune response, NK cells cannot effectively kill infected cells. Since there is a small window for removal of infected cells before bystander cell death occurs, the researchers noted that the timing of NK cell migration to the site of infection could be crucial in deciding whether NK cells were pathogenic or protective in COVID-19.

Finally, the authors found that SARS-CoV-2 non-structural protein 1 (Nsp1) causes the downregulation of NKG2D-L. They also illustrated that transfection with just Nsp1 was sufficient to confer tolerance to NK cell killing. This observation has crucial implications for NK cell-mediated regulation of SARS-CoV-2, as the preferred escape of infected cells with death of bystander cells could lead to the pathogenesis of SARS-CoV-2.


Collectively, the current investigation takes an in-depth look at the NK cell response to SARS-CoV-2 and offers new insights into NK cell function in COVID-19. The researchers found that cells infected with SARS-CoV-2 evade killing by healthy NK cells in a cell-intrinsic manner, leading to the preferential killing of uninfected bystander cells. The ability of infected cells to evade NK cell identification requires that the infection last long enough to allow an infected cell to present proteins encoded by SARS-CoV-2. Furthermore, the present results highlight the importance of studying the temporal dynamics of NK cell response to SARS-CoV-2 infected cells.

Additionally, the team showed that downregulation of NKG2D-L drives the SARS-CoV-2 NK cell recognition evasion process. The main hallmark of the NK cell response to SARS-CoV-2, according to the results, was the loss of NKG2D-L. The study data further demonstrated that the SARS-CoV-2 protein Nsp1 was responsible for this ligand downregulation and that Nsp1 itself was adequate to induce direct NK cell escape. Current work has shown that NK cell responses to SARS-CoV-2 infected cells could be partially or fully rescued by reducing the activity of Nsp1, proving this protein to be an even more desirable target than one would expect. previously believed.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

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