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

SummaryNatural 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 interrogated the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We found 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 found 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 reveals that SARS-CoV-2 evades NK cell cytotoxicity and describes a mechanism by which this occurs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/496341v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@bcffeeorg.highwire.dtl.DTLVardef@469b0eorg.highwire.dtl.DTLVardef@16dd205org.highwire.dtl.DTLVardef@f78070_HPS_FORMAT_FIGEXP M_FIG C_FIG

Activated interstitial macrophages are a predominant target of viral takeover and focus of inflammation in COVID-19 initiation in human lung

Early stages of deadly respiratory diseases such as COVID-19 have been challenging to elucidate due to lack of an experimental system that recapitulates the cellular and structural complexity of the human lung, while allowing precise control over disease initiation and systematic interrogation of molecular events at cellular resolution. Here we show healthy human lung slices cultured ex vivo can be productively infected with SARS-CoV-2, and the cellular tropism of the virus and its distinct and dynamic effects on host cell gene expression can be determined by single cell RNA sequencing and reconstruction of "infection pseudotime" for individual lung cell types. This revealed the prominent SARS-CoV-2 target is a population of activated interstitial macrophages, which as infection proceeds accumulate thousands of viral RNA molecules per cell, comprising up to 60% of the cellular transcriptome and including canonical and novel subgenomic RNAs. During viral takeover, there is cell-autonomous induction of a specific host interferon program and seven chemokines (CCL2, 7, 8, 13, CXCL10) and cytokines (IL6, IL10), distinct from the response of alveolar macrophages in which neither viral takeover nor induction of a substantial inflammatory response occurs. Using a recombinant SARS-CoV-2 Spike-pseudotyped lentivirus, we show that entry into purified human lung macrophages depends on Spike but is not blocked by cytochalasin D or by an ACE2-competing monoclonal antibody, indicating a phagocytosis- and ACE2-independent route of entry. These results provide a molecular characterization of the initiation of COVID-19 in human lung tissue, identify activated interstitial macrophages as a prominent site of viral takeover and focus of inflammation, and suggest targeting of these macrophages and their signals as a new therapeutic modality for COVID-19 pneumonia and progression to ARDS. Our approach can be generalized to define the initiation program and evaluate therapeutics for any human lung infection at cellular resolution.

CRISPRa screening with real world evidence identifies potassium channels as neuronal entry factors and druggable targets for SARS-CoV-2

Although vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been successful, there are no good treatments for those who are actively infected. While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from sensory organs and the brain are also susceptible to infection. While many patients suffer from diverse neurological symptoms, the viruss neuronal entry remains mysterious. To discover host factors involved in SARS-CoV-2 viral entry, we performed CRISPR activation (CRISPRa) screens targeting all 6000+ human membrane proteins in cells with and without overexpression of ACE2 using Spike-pseudotyped lentiviruses. This unbiased gain-of-function screening identified both novel and previously validated host factors. Notably, newly found host factors have high expression in neuronal and immune cells, including potassium channel KCNA6, protease LGMN, and MHC-II component HLA-DPB1. We validated these factors using replication-competent SARS-CoV-2 infection assays. Notably, the overexpression of KCNA6 led to a marked increase in infection even in cells with undetectable levels of ACE2 expression. Analysis of human olfactory epithelium scRNA-seq data revealed that OLIG2+/TUJ1+ cells--previously identified as sites of infection in COVID-19 autopsy studies-- have high KCNA6 expression and minimal levels of ACE2. The presence of KCNA6 may thus explain sensory/neuronal aspects of COVID-19 symptoms. Further, we demonstrate that FDA-approved compound dalfampridine, an inhibitor of KCNA-family potassium channels, suppresses viral entry in a dosage-dependent manner. Finally, we identified common prescription drugs likely to modulate the top identified host factors, and performed a retrospective analysis of insurance claims of ~8 million patients. This large cohort study revealed a statistically significant association between top drug classes, particularly those targeting potassium channels, and COVID-19 severity. Taken together, the potassium channel KCNA6 facilitates neuronal entry of SARS-CoV-2 and is a promising target for drug repurposing and development.