A proteome-wide genetic investigation identifies several SARS-CoV-2-exploited host targets of clinical relevance

Background: The virus SARS-CoV-2 can exploit biological vulnerabilities (e.g. host proteins) in susceptible hosts that predispose to the development of severe COVID-19.Methods: To identify host proteins that may contribute to the risk of severe COVID-19, we undertook proteome-wide genetic colocalisation tests, and polygenic (pan) and cis-Mendelian randomisation analyses leveraging publicly available protein and COVID-19 datasets.Results: Our analytic approach identified several known targets (e.g. ABO, OAS1), but also nominated new proteins such as soluble Fas (colocalisation probability > 0.9, p = 1 x 10-4), implicating Fas-mediated apoptosis as a potential target for COVID-19 risk. The polygenic (pan) and cis-Mendelian randomisation analyses showed consistent associations of genetically predicted ABO protein with several COVID-19 phenotypes. The ABO signal is highly pleiotropic and a look-up of proteins associated with the ABO signal revealed that the strongest association was with soluble CD209. We demonstrated experimentally that CD209 directly interacts with the spike protein of SARS-CoV-2, suggesting a mechanism that could explain the ABO association with COVID-19.Conclusions: Our work provides a prioritised list of host targets potentially exploited by SARS-CoV-2 and is a precursor for further research on CD209 and FAS as therapeutically tractable targets for COVID-19.Funding: MAK, JSc, JH, AB, DO, MC, EMM, MG, ID were funded by Open Targets. J.Z. and T.R.G were funded by the UK Medical Research Council Integrative Epidemiology Unit (MC_UU_00011/4). JSh and GJW were funded by the Wellcome Trust Grant 206194. This research was funded in part by the Wellcome Trust [Grant 206194]. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas–/–)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL–Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL–DR5 interaction-mediated CX3CL1–CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL–DR5 interaction promotes inflammation and is a potential therapeutic target.

The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region

We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using site-directed mutagenesis we cloned a mutant form of sFasL in which all the charged amino acids of the stalk region were changed to neutral alanines (mut-sFasL). We used the Fas-sensitive Jurkat T-cell line and mouse and human alveolar epithelial cells to test the bioactivity of sFasL complexes, using caspase-3 activity and Annexin-V externalization as readouts. Finally, we tested the effects of mut-sFasL on lipopolysaccharide-induced lung injury in mice. We found that mutation of all the 8 charged amino acids of the stalk region into the non-charged amino acid alanine (mut-sFasL) resulted in reduced apoptotic activity compared to wild type sFasL (WT-sFasL). The mut-sFasL attenuated WT-sFasL function on the Fas-sensitive human T-cell line Jurkat and on primary human small airway epithelial cells. The inhibitory mechanism was associated with the formation of complexes of mut-sFasL with the WT protein. Intratracheal administration of the mut-sFasL to mice 24 hours after intratracheal Escherichia coli lipopolysaccharide resulted in attenuation of the inflammatory response 24 hours later. Therefore, the stalk region of sFasL has a critical role on bioactivity, and changes in the structure of the stalk region can result in mutant variants that interfere with the wild type protein function in vitro and in vivo.

Mendelian randomisation identifies alternative splicing of the FAS death receptor as a mediator of severe COVID-19

Severe COVID-19 is characterised by immunopathology and epithelial injury. Proteomic studies have identified circulating proteins that are biomarkers of severe COVID-19, but cannot distinguish correlation from causation. To address this, we performed Mendelian randomisation (MR) to identify proteins that mediate severe COVID-19. Using protein quantitative trait loci (pQTL) data from the SCALLOP consortium, involving meta-analysis of up to 26,494 individuals, and COVID-19 genome-wide association data from the Host Genetics Initiative, we performed MR for 157 COVID-19 severity protein biomarkers. We identified significant MR results for five proteins: FAS, TNFRSF10A, CCL2, EPHB4 and LGALS9. Further evaluation of these candidates using sensitivity analyses and colocalization testing provided strong evidence to implicate the apoptosis-associated cytokine receptor FAS as a causal mediator of severe COVID-19. This effect was specific to severe disease. Using RNA-seq data from 4,778 individuals, we demonstrate that the pQTL at the FAS locus results from genetically influenced alternate splicing causing skipping of exon 6. We show that the risk allele for very severe COVID-19 increases the proportion of transcripts lacking exon 6, and thereby increases soluble FAS. Soluble FAS acts as a decoy receptor for FAS-ligand, inhibiting apoptosis induced through membrane-bound FAS. In summary, we demonstrate a novel genetic mechanism that contributes to risk of severe of COVID-19, highlighting a pathway that may be a promising therapeutic target.

A proteome-wide genetic investigation identifies several SARS-CoV-2-exploited host targets of clinical relevance

The virus SARS-CoV-2 can exploit biological vulnerabilities in susceptible hosts that predispose to development of severe COVID-19. Previous reports have identified several host proteins related to the interferon response (e.g. OAS1), interleukin-6 signalling (IL-6R), and the coagulation cascade (linked via ABO) that were associated with risk of COVID-19. In the present study, we performed proteome-wide genetic colocalisation tests leveraging publicly available protein and COVID-19 datasets, to identify additional proteins that may contribute to COVID-19 risk. Our analytic approach identified several known targets (e.g. ABO, OAS1), but also nominated new proteins such as soluble FAS (colocalisation probability > 0.9, p = 1 × 10−4), implicating FAS-mediated apoptosis as a potential target for COVID-19 risk. We also undertook polygenic (pan) and cis-Mendelian randomisation analyses that showed consistent associations of genetically predicted ABO protein with several COVID-19 phenotypes. The ABO signal was associated with plasma concentrations of several proteins, with the strongest association observed with CD209 in several proteomic datasets. We demonstrated experimentally that CD209 directly interacts with the spike protein of SARS-CoV-2, suggesting a mechanism that could explain the ABO association with COVID-19. Our work provides a prioritised list of host targets potentially exploited by SARS-CoV-2 and is a precursor for further research on CD209 and FAS as therapeutically tractable targets for COVID-19.