Rare missense variants in the human cytosolic antibody receptor preserve antiviral function

Abstract

The genetic basis of most human disease cannot be explained by common variants. One solution to this ‘missing heritability problem’ may be rare missense variants, which are individually scarce but collectively abundant. However, the phenotypic impact of rare variants is under-appreciated as gene function is normally studied in the context of a single ‘wild-type’ sequence. Here, we explore the impact of naturally occurring missense variants in the human population on the cytosolic antibody receptor TRIM21, using volunteer cells with variant haplotypes, CRISPR gene editing and functional reconstitution. In combination with data from a panel of computational predictors, the results suggest that protein robustness and purifying selection ensure that function is remarkably well-maintained despite coding variation.

Target-induced clustering activates Trim-Away of pathogens and proteins

“…A full list of plasmids used in this study and details of plasmid construction can be found in Supplementary Note 2. For stable expression of TRIM21 at endogenous levels, a native TRIM21 promoter-driven lentiviral vector (pT21MPP) was used 58 . For in vitro mRNA transcription, constructs were cloned into pGEMHE 59 , which contains UTR and polyA sequences for optimal mRNA stability and translation.…”

A functional assay for serum detection of antibodies against SARS‐CoV‐2 nucleoprotein

“…C, D As with A&B, except with a titration of electroporated anti-N antibodies. E Western blot of 293T cells and 293T ACE2 OE/TRIM21 KOs alone or reconstituted with empty vector (EV) or an endogenous promoter-driven TRIM21 vector (T21) (Zeng et al, 2019). F, G 293T ACE2 OE/TRIM21 KO cells reconstituted with EV or TRIM21, electroporated with IgG or anti-N antibodies, and infected with SARS-CoV-2.…”

“…Intracellular neutralization was antibody dose-dependent, consistent with the MHV data and previous ADIN experiments (McEwan et al, 2012) (Fig 2C and D).We next investigated whether intracellular neutralization of SARS-CoV-2 is TRIM21-dependent. We transduced a previously characterized TRIM21 knockout HEK293T clone with ACE2, to make it sensitive to SARS-CoV-2 entry, and then reconstituted TRIM21 expression with lentivirus encoding TRIM21 under its endogenous promoter or transduced with an empty vector control (Fig2E;Zeng et al, 2019). Electroporation/infection experiments were then carried out as before in both cell lines.…”

Substrate-induced clustering activates Trim-Away of pathogens and proteins

“…For stable expression of human TRIM21 in HEK293T cells, a native TRIM21 promoterdriven lentiviral vector (Zeng et al, 2019) was used. Point mutations and deletions within the TRIM21 coding sequence were generated using Quikchange mutagenesis (Agilent).…”

“…Post-translational modifications such as phosphorylation and neddylation add further layers of regulation to control E3 ligase activation and ensure specificity of the ubiquitin transfer reactions (Zheng and Shabek, 2017). Sub-cellular localisation and tissue-specific expression provides the final level of regulation at the cell and organism level (Schapira et al, 2019;Yamada et al, 2013).…”