1. Microbiology and Infectious Disease
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Rare missense variants in the human cytosolic antibody receptor preserve antiviral function

  1. Jingwei Zeng
  2. Greg Slodkowicz
  3. Leo C James  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom
Research Article
Cite this article as: eLife 2019;8:e48339 doi: 10.7554/eLife.48339
10 figures, 1 table and 2 additional files

Figures

TRIM21 is a highly conserved gene.

Conservation of TRIM21 compared to invariant housekeeping genes (HDACs and actins), and immune genes including CGAS, STING and related TRIMs, TRIM5 and TRIM20. Comparison of variants between genes is expressed as number of missense variants (MVs) per 100 residues to normalize for differences in gene length (A-B). Frequency of nonsynonymous variants (A) common variants (≥1%) and (B) rare variants (<1%). Error bars represent standard deviation. Data provided in Figure 1—source data 1.

https://doi.org/10.7554/eLife.48339.002
Domain location of TRIM21 missense variants.

(A) Schematic of TRIM21 showing how variants are distributed amongst component domains. (B–D) Mapping of variants onto structures of each domain. (B) RING (blue) and B-Box (orange) variants are marked on the autoinhibited structure of the TRIM21 RING-B-Box domains (PDB 5OLM). The second copy of the B-Box has been omitted and the location of a bound E2 enzyme (yellow) has been included instead to indicate the location of this functional interface (based on superposition of TRIM25 RING: E2 structure (5FER)). (C) Coiled-coil domain (green) based on TRIM25 (PDB 4CFG). Location of TRIM21 residues are marked based on sequence alignment. (D) PRYSPRY domain (red) with bound IgG Fc (yellow) based on PDB 2IWG. Residues in brackets are not present in the structure.

https://doi.org/10.7554/eLife.48339.004
Figure 3 with 2 supplements
PRYSPRY missense variants maintain antibody binding.

(A) Representative Isothermal titration calorimetry (ITC) trace of IgG Fc against WT TRIM21 PRYSPRY fitted to the one set of sites model. (B) Summary of PRYSPRY variant binding affinities to IgG Fc. Consistent with known binding mode, TRIM21 PRYSPRY binds IgG with a stoichiometry of 2:1 (Keeble et al., 2008). Data provided in Figure 3—source data 1.

https://doi.org/10.7554/eLife.48339.005
Figure 3—figure supplement 1
Representative ITC traces of IgG Fc titration against each PRYSPRY variant.

(A) N297H. (B) A390V. (C) G440R. (D) F446I. (E) K455E. (F) Q470K. (G) G471R. (H) Buffer control. (I) Binding signature plot. In most experiments, 40 µM of IgG Fc was titrated against 8 µM of PRYSPRY except for the F446I (D) where the concentrations were halved. Data provided in Figure 3—source data 1.

https://doi.org/10.7554/eLife.48339.006
Figure 3—figure supplement 2
Purification of TRIM21 PRYSPRY protein.

(A–E) Superdex 75 (HiLoad 16/60) size exclusion chromatograms for a selection of TRIM21 PRYSPRY variants. (A) WT. (B) N297H. (C) A390V. (D) G440R. (E) F446I. (F) Variant TRIM21 PRYSPRY proteins resolved by 4–12% SDS-PAGE developed in MES-SDS buffer and stained using InstantBlue.

https://doi.org/10.7554/eLife.48339.007
PRYSPRY variants have profound differences in intrinsic stability.

(A) Differential scanning fluorimetry of PRYSPRY variants to determine their melting temperature (Tm) using changes in intrinsic tryptophan fluorescence that occur upon unfolding (first derivative of 330/350 nm ratio). (B) Differences between variants are plotted as a ΔTm with respective to wild-type. (C) Correlation between ΔΔG for stability of PRYSPRY variants with predicted degree of deleteriousness from various algorithms. Correlation and p-values were calculated using the Pearson correlation coefficient. Data provided in Figure 4—source data 1.

https://doi.org/10.7554/eLife.48339.009
Figure 5 with 2 supplements
A bespoke system for ectopic TRIM21 expression at endogenous levels.

(A) Map of lentivector containing the endogenous 2 kb upstream promoter sequence of the human TRIM21 gene followed by the 5’UTR (Exons 1–2) and TRIM21 coding sequence (Exons 2–7). (B) Immunoblot of TRIM21 and COX IV (loading control) in WT, TRIM21 KO (KO) or lentivector reconstituted (Reconst.) 293Ts with or without interferon-alpha (IFN-α) pre-treatment. (C) Histograms of mCherry fluorescence intensity in cells transduced with lentivector encoding mCherry-TRIM21 driven by SFFV (Viral; Orange) or native TRIM21 promoter (Blue). Untransduced TRIM21 KO 293Ts were used as negative control (Red). (D) Immunoblot of TRIM21 and COX IV (loading control) in lentivector reconstituted 239Ts expressing the indicated TRIM21 variant, with the variants grouped into their host domains.

https://doi.org/10.7554/eLife.48339.011
Figure 5—figure supplement 1
TRIM21 expression levels in reconstituted cell lines.

(A–C) Immunoblot of TRIM21 and COX IV (loading control) in TRIM21 KO (KO), WT or native promoter lentivector reconstituted (Reconst.) cell lines (A) 293T, (B) Hela, (C) hTERT-RPE-1. (D) Quantification of TRIM21 variant expression in reconstituted 293Ts (from Figure 5D) normalized to wild-type expression level. (E) Correlation between intrinsic stability (Tm) and cellular expression levels of PRYSPRY variants using linear regression analysis in GraphPad Prism7 (R2 = 0.72). Data provided in Figure 5—source data 1.

https://doi.org/10.7554/eLife.48339.012
Figure 5—figure supplement 2
Native promoter driven mCherry-TRIM21 colocalizes with antibody coated AdV5.

(A) Confocal microscopy images staining for human IgG and mCherry in TRIM21 KO 293Ts reconstituted with mCherry-TRIM21 driven by the native TRIM21 promoter. The cells were fixed 30 min after infection by AdV5 coated with either WT 9C12 or 9C12 with the H433A mutation in its Fc. (B) Quantification of colocalization between 9C12-AdV5 and mCherry-TRIM21 using the ComDet plugin in Fiji (Schindelin et al., 2012). (C) Quantification of the mean and median mCherry intensities at 9C12-AdV5 spots using the ComDet plugin in Fiji (Schindelin et al., 2012). Data provided in Figure 5—source data 1.

https://doi.org/10.7554/eLife.48339.013
Figure 6 with 1 supplement
Viral neutralization by TRIM21 natural variants.

(A) Neutralization experiments were carried out in TRIM21 KO 293T cell lines stably reconstituted with TRIM21 variants expressed at endogenous levels. Each stable cell line was challenged with AdV5-GFP in the presence of the anti-hexon monoclonal antibody 9C12. The AdV5 vector contains a copy of the GFP gene and relative infection levels were quantified by flow cytometry and normalized to that of virus only condition. Data compiled from at least two independent experiments (mean ± SEM) and fitted to a one phase exponential decay. (B) Correlation of neutralization efficiency (Kneut, the exponential decay constant calculated from (A)), with cellular protein expression levels (from Figure 5) or thermostability (ΔTm) using linear regression analysis in GraphPad Prism7. Variants are grouped into their host domains. The R234* variant was excluded from correlative analysis. Data provided in Figure 6—source data 1.

https://doi.org/10.7554/eLife.48339.015
Figure 6—figure supplement 1
F446I but not R234* can mediate viral neutralization with IFN-α priming.

(A) Immunoblot of TRIM21 and COX IV (loading control) in lentivector reconstituted 293T cells expressing the indicated TRIM21 variant with or without IFNα pre-treatment. (B) Neutralization of AdV5-GFP in reconstituted 293T cell lines in the presence of anti-hexon 9C12 IgG. Relative infection levels were quantified by flow cytometry and normalized that of virus only condition. Data compiled from three independent experiments (mean ± SEM). (C) Percentage of infected cells in the neutralization experiment (B) in the absence of antibody. Data provided in Figure 6—source data 1.

https://doi.org/10.7554/eLife.48339.016
Unstable TRIM21 PRYSPRY variants function better at 33°C but lost more activity at 39.5°C.

(A) Neutralization of AdV5-GFP in reconstituted 293T cell lines in the presence of anti-hexon 9C12 IgG at the indicated incubation temperatures. Relative infection levels were quantified by flow cytometry and normalized that of virus only condition. Data compiled from two independent experiments (mean ± SEM). (B) Immunoblot of TRIM21 and COX IV (loading control) in lentivector reconstituted 293T cells expressing the indicated TRIM21 variant after 24 hr incubation at the indicated temperatures. Data provided in Figure 7—source data 1.

https://doi.org/10.7554/eLife.48339.018
Viral sensing of by natural TRIM21 variants.

(A) Stable 293T cell lines expressing TRIM21 variants were infected with AdV5 in the presence of anti-hexon 9C12 antibody and immune activation was measured 6 hr post infection using an NF-κB luciferase reporter. Data compiled from at least two independent experiments and expressed as fold change over that of virus only condition. EV (empty vector); mean ± SEM. (B) Correlation between NF-κB induction and cellular expression levels or thermostability by domain using linear regression analysis in GraphPad Prism7. The R234* variant was excluded from correlative analysis. Data provided in Figure 8—source data 1.

https://doi.org/10.7554/eLife.48339.020
Figure 9 with 1 supplement
Natural missense variants do not exert dominant negative effect.

(A–D) Immunoblot (IB) for TRIM21 and COX IV (loading control) in (A) selected LCLs (B) Transduced WT 293T cell lines stably expressing TRIM21 variants under the native TRIM21 promoter. (C–D) CRISPR gene-edited 293T clones expressing the R234* and R118Q variant respectively. (E–H) AdV neutralization in the presence of anti-adenovirus hexon monoclonal IgG 9C12 in (E) selected LCLs. (F) WT 293T cells expressing the R234* and F446I variant. (G–H) CRISPR gene-edited 293T clones. Relative infection levels quantified by flow cytometry and normalized to virus only condition. Data compiled from at least two independent experiments (mean ± SEM). (I–L) Activation of NF-κB signaling by 9C12 coated AdV5 (I–J) or human TNF-α (K–L) in the respective cell lines. Data compiled from two independent experiments (mean ± SEM). Data provided in Figure 9—source data 1.

https://doi.org/10.7554/eLife.48339.022
Figure 9—figure supplement 1
Sanger sequencing chromatograms of the TRIM21 gene in LCLs.

Codons with heterozygous mutation are highlighted in blue.

https://doi.org/10.7554/eLife.48339.023
Natural rare variants are less deleterious than expected by chance.

The fraction of deleterious variants calculated using the indicated algorithms for all possible variants and naturally occurring variants that are present at a frequency of ≥10−5 or <10−5.

https://doi.org/10.7554/eLife.48339.025

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene
(Homo sapiens)
TRIM21HGNCHGNC: 11312
Strain, strain background (Escherichia coli)C41(DE3)Sigma-AldrichCat#: CMC0017Chemically competent cells
Strain, strain background (Human adenovirus type 5)AdV5-GFPViraquestN/A
Strain, strain background (Human adenovirus type 5/35 chimera)AdV5/35-GFPViraquestN/A
Cell line
(Homo sapiens)
293TATCCCat#: CRL-3216;
RRID:CVCL_0063
Cell line
(Homo sapiens)
TRIM21
Knockout
293T
Dickson et al., 2018DOI: 10.7554/eLife.32660CRISPR/Cas9 gene knockout
Cell line
(Homo sapiens)
TRIM21
Knockout
HeLa
Bottermann et al., 2019DOI: 10.1016/j.chom.2019.02.016CRISPR/Cas9 gene knockout
Cell line
(Homo sapiens)
Lymphoblastoid cell linesNHGRI Repository at Coriell Institute for Medical ResearchRRID:SCR_004528
Recombinant DNA reagentpGL4.32PromegaCat#: E8491NF-kB-RE-firefly-luciferase reporter construct (Photinus pyralis)
Recombinant DNA reagentpCR/V1
(HIV-1 Gag-Pol)
Zennou et al., 2004DOI: 10.1128/JVI.78.21.12058–12061.2004Lentivirus packaging vector
Recombinant DNA reagentpMD2.GAddgeneRRID:Addgene_12259VSV-G env for making pseudotyped lentiviral vector
Recombinant DNA reagentpHR’Demaison et al., 2002DOI: 10.1089/10430340252898984HIV-1 based lentiviral vector
AntibodyHuman plasma IgG FcAthens Research and TechnologyCat#: 16-16-090707-FC;
RRID:AB_575814
AntibodyAnti-Human TRIM1 (Mouse monoclonal)Santa Cruz BiotechnologyCat#: sc-25351;
RRID:AB_628286
Immunoblot (1:1000)
AntibodyAnti-Human COX IV (Rabbit monoclonal)LI-CORCat#: 926–42214;
RRID:AB_2783000
Immunoblot (1:5000)
AntibodyAnti-Mouse-HRP (Goat polyclonal)SigmaCat#: A0168;
RRID:AB_257867
Immunoblot (1:5000)
AntibodyAnti-Rabbit-HRP (Goat monoclonal)SigmaCat#: A0545;
RRID:AB_257896
Immunoblot (1:5000)
AntibodyAnti-mCherry (Rabbit polyclonal)AbcamCat#: ab167453;
RRID:AB_2571870
IF (1:500)
AntibodyAnti-Human IgG Alexa Fluor 488 (Goat polyclonal)ThermoFisherCat#: A-11013;
RRID:AB_2534080
IF (1:500)
AntibodyAnti-Rabbit IgG Alexa Fluor 568 (Goat polyclonal)ThermoFisherCat#: A-11036;
RRID:AB_10563566
IF (1:500)
AntibodyIRDye 680RD Goat anti-Rabbit IgGLI-CORCat#: 926–68071;
RRID:AB_10956166
Immunoblot (1:5000)
AntibodyIRDye 800CW Goat anti-Mouse IgGLI-CORCat#: 926–32210;
RRID:AB_621842
Immunoblot (1:5000)
AntibodyHumanized anti-AdV5 hexon monoclonal IgGFoss et al., 2016DOI: 10.4049/jimmunol.1502601
Peptide, recombinant proteinHuman TNF-αPeproTechCat#: 300-01A10 ng/ml
Peptide, recombinant proteinHuman IFN-α1SigmaCat#: SRP45961000U/ml
Sequence-based reagentAlt-R CRISPR-Cas9 tracrRNAIDTCat#: 1072532
Sequence-based reagentAlt-R CRISPR-Cas9 crRNA
(TRIM21 KO)
IDTN/AATGCTCACAGGCTCCACGAA
Sequence-based reagentAlt-R CRISPR-Cas9 crRNA
(TRIM21 R118)
IDTN/AGTCACGGTGTTTCCGAGACT
Sequence-based reagentAlt-R CRISPR-Cas9 crRNA
(TRIM21 R234)
IDTN/ATCATCTCAGAGCTAGATCGA
Sequence-based reagentssODN template for HDR (TRIM21 R118Q)IDTN/ACACAGGGGGAACGGTGTGCAGTGCATGGAGAGAGACTTCACCTGTTCTGTGAGAAAGATGGGAAGGCCCTTTGCTGGGTATGTGCTCAGTCTCAGAAACACCGTGACCACGCCATGGTCCCTCTTGA
Sequence-based reagentssODN template for HDR (TRIM21 R234*)IDTN/AAGCCAGGCCCTACAGGAGCTCATCTCAGAGCTAGATTGAAGATGCCACAGCTCAGCACTGGAACTGCTGCAGGTGAGACAGGGAGGGGTTTCCTTCTACAATTCAGGGAATAACTGAAAAAGACCAG
Sequence-based reagentForward primer for TRIM21 promoter cloningSigmaN/AggatcgataagcttgatatcgaattcGCATGTTGTGCACA
Sequence-based reagentReverse primer for TRIM21 promoter cloningSigmaN/AtgcttaacgcgTGTCAAGTGTGCCGTTAAACAG
Commercial assay or kitGibson Assembly Master MixNEBCat#: E2611L
Commercial assay or kitQIAprep Spin Miniprep KitQIAGENCat#: 27106
Commercial assay or kitQIAquick PCR Purification KitQIAGENCat#: 28104
Commercial assay or kitQIAquick Gel Extraction KitQIAGENCat#: 28704
Commercial assay or kitNeon Transfection System 10 µL KitInvitrogenCat#: MPK1096
Software, algorithmsGraphPad Prism 7GraphPadRRID:SCR_002798
Software, algorithmsRR Project for Statistical ComputingRRID:SCR_001905
OtherHoechst 33342 SolutionThermo ScientificCat. #: 62249(1 µg/mL)
OtherSteadylite plus Reporter Gene Assay SystemPerkinElmerCat. #: 6066751
OtherFugene 6 Transfection ReagentPromegaCat. #: E2691
OtherGenome Aggregation DatabaseBroad InstituteRRID:SCR_014964
Other1000 Genomes Project1000 Genomes ProjectRRID:SCR_006828

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Previously published data from the 100 Genomes Project (2015; http://www.internationalgenome.org/data#download) and the Genome Aggregation Datatbase (2016; https://gnomad.broadinstitute.org/downloads) was used as part of this work.

Additional files

Supplementary file 1

Rare missense variants in TRIM21 present in the 1000 Genomes study.

All variants listed have an allele frequency of <1%. The number of heterozygous and homozygous haplotypes are indicated for each variant, together with its domain location, predicted deleteriousness (using various algorithms, see main text) and measured neutralization (Kneut) and signaling activity (Ksig). Kneut is a measure of the efficiency of TRIM21-mediated adenovirus neutralization as defined by the exponential decay constant calculated from Figure 6A. Ksig is a measure of signaling ability defined by the fold-change in NF-κB reporter activity upon adenovirus infection in the presence of antibody (Figure 8).

https://doi.org/10.7554/eLife.48339.027
Transparent reporting form
https://doi.org/10.7554/eLife.48339.028

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