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

Abstract

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 and potentially suffer from diverse neurological symptoms. While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from sensory organs, brain, and heart are also susceptible to infection. An understanding of factors critical for viral infection in these tissues may help identify novel therapeutics. 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. We identified both novel as well as previously validated host factors. Notably, we used replication-competent SARS-CoV-2 to validate new viral-entry promoting genes, including potassium channel KCNA6, protease LGMN, and MHC-II component HLA-DPB1. We found that the overexpression of KCNA6 led to a marked increase in infection even in cells with undetectable levels of ACE2 expression. Our analysis of human olfactory neuroepithelium scRNA-seq data revealed that OLIG2+ cells--previously identified as sites of infection in COVID-19 autopsy studies--have high KCNA6 expression and minimal levels of ACE2, suggesting that the presence of KCNA6 may 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 screen hits. We then performed a retrospective analysis of insurance claims of ~8 million patients and found a clinical association between screen-identified drug classes, particularly those targeting potassium channels, and COVID-19 severity. We have thus identified the potassium channel KCNA6 as a SARS-CoV-2 host factor, expanded our understanding of potential viral tropism, and identified promising targets for drug repurposing and development.

Can the SARS-CoV-2 Spike Protein Bind Integrins Independent of the RGD Sequence?

“…The Severe Acute Syndrome Coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, which binds to angiotensin-converting enzyme 2 (ACE2) to initiate viral cell entry, has been shown to contain an exposed RGD motif that has been suggested to be integrin-binding, thus potentially allowing for integrin-mediated cell entry ( Luan et al, 2020 ; Makowski et al, 2021 ). Experimental studies have shown that the SARS-CoV-2 spike protein directly binds α V β 3 and α 5 β 1 integrins and may also interact with α 3 , β 1 , α 4 , and α X integrin subunits ( Aguirre et al, 2020 ; Kotani and Nakano, 2020 ; Beddingfield et al, 2021 ; Nader et al, 2021 ; Wang et al, 2021 ). RGD-based drugs were found to inhibit spike binding to α V β 3 and α 5 β 1 integrins, and mutating the RGD motif to RGE or RGA was also found to decrease binding to α V β 5 integrins – further implicating the spike RGD motif in interactions with integrins ( Sebé-Pedrós et al, 2010 ; Robles et al, 2021 )⁠.…”

DataSheet_1.xlsx

“…A meta-analysis of reported integrin expression levels revealed that several of the putative spike-binding partners are increased during SARS-CoV-2 infection, further adding to their validity. Since a X integrins were both found as putative cell entry receptors for the SARS-CoV-2 spike protein in a CRISPR activation screen, the results presented in this study may help unravel potential binding mechanisms (Wang et al, 2021). The identified motifs may act together with the RGD motif or independently to bind integrins for cell entry or to interfere with cell signaling pathways.…”

“…Binding assays demonstrated direct binding between the spike protein and b 1 integrins (Park et al, 2021). A screening of candidate cell entry receptors for SARS-CoV-2 suggested that a 3 and b 1 integrins may be involved, and a CRISPR activation screening looking at neuronal receptors involved in SARS-CoV-2 infection found that a X integrins may mediate cell entry (Kotani and Nakano, 2020;Wang et al, 2021). The antibody natalizumab, which binds to a 4 integrins, was found to decrease SARS-CoV-2 infection, hinting at the potential use of these integrins in cell entry (Aguirre et al, 2020).…”

“…The Severe Acute Syndrome Coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, which binds to angiotensin-converting enzyme 2 (ACE2) to initiate viral cell entry, has been shown to contain an exposed RGD motif that has been suggested to be integrin-binding, thus potentially allowing for integrin-mediated cell entry (Luan et al, 2020;Makowski et al, 2021). Experimental studies have shown that the SARS-CoV-2 spike protein directly binds a V b 3 and a 5 b 1 integrins and may also interact with a 3 , b 1 , a 4 , and a X integrin subunits (Aguirre et al, 2020;Kotani and Nakano, 2020;Beddingfield et al, 2021;Nader et al, 2021;Wang et al, 2021). RGD-based drugs were found to inhibit spike binding to a V b 3 and a 5 b 1 integrins, and mutating the RGD motif to RGE or RGA was also found to decrease binding to a V b 5 integrinsfurther implicating the spike RGD motif in interactions with integrins (Sebe-Pedroś et al, 2010;Robles et al, 2021).…”

“…The study performed protein-protein docking between the spike protein and a 5 b 1 , a IIb b 3 , and a V b 8 integrins and found that the interactions were not favourable, thus suggesting that other motifs on the spike receptor-binding domain may be involved in interactions with integrins. Also, some of the integrins discovered to be associated with SARS-CoV-2 cell entry, such as a X , are not known to bind RGD peptides (Wang et al, 2021).…”