TRIM7 Restricts Coxsackievirus and Norovirus Infection by Detecting the C-Terminal Glutamine Generated by 3C Protease Processing

Figure 2. TRIM7 binds diverse substrates using a C-terminal helix-ΦQ motif. (A) MNV1 polyprotein (pale blue) and the relevant proteins in a darker shade (NS3 and NS6), CVB3 polyprotein in pale green and the relevant 2C protein in a darker shade. (B) Main peptide sequences used in the binding and structural experiments. Outlined are the sequences synthesized, whilst the shaded residues are those resolved in the crystal structures. Color coding is maintained throughout the figure. (C) hisTRIM7-PRYSPRY:Peptide complex structures superposed. Shows PRYSPRY as a transparent surface representation with a few key residues in stick. Peptides are color coded as described. PDB accession codes are 7OW2, 7OVX, 8A5L and 8A5M). (D) Detail of the PRYSPRY pocket with the recognition motif bound. Several key residues are highlighted. Dashes indicate charged or H-bonding between the peptide and the PRYSPRY residues. Underlined residues are essential for binding (see Figure S3). (E) Peptide substitutions based on the TIEALFQ peptide and possible polyprotein processing ends. Binding experiments with the minimal LQ motif, sequence is shown and the derived KD from ITC titrations. No binding* denotes where the…

Figure 1. A linear epitope of GYG1 is sufficient to explain TRIM7 binding. (A) Sequence and domain organization of TRIM7 (grey) and Glycogenin1 (pale orange). TRIM7 is also depicted as a cartoon (circle—RING, rectangle—B-Box, line—Coiled-coil, cut-out circle—PRYSPRY) used throughout this paper. Highlighted are sequences determining TRIM7 binding. (B) ITC binding experiments with sequential truncations of rbGYG1 (shown above each titration) titrated into human hisTRIM7-PRYSPRY. Representative traces and their accompanying fitted KD’s are shown. (C) Cartoon overview of the crystal structure of hisTRIM7-PRYSPRY (grey) with the GYG1 peptide (orange) on left (PDB accession 7OVX). Comparison with TRIM21:Fc structure (2IWG, PRYSPRY wheat, Fc magenta) on the right.

Figure 3. TRIM7 co-localizes with helix-ΦQ containing substrates inside cells and degrades them. (A–E) Schematic representation of in vitro ubiquitination experiments. (A) TRIM7-RING (grey circle) was mixed with combinations of Ubiquitin (Ub, blue circle), Ube2N/Ube2V2 (purple semi-circle) and Ube2W (green teardrop) as shown and the reactions followed by immunoblotting (C) with an anti-TRIM7 antibody. A representative blot from at least three independent experiments is shown. (B) E2 discharge experiment: Ube2N~Ub complex was incubated with TRIM7-RING, and the reaction followed over time as indicated in (D). Blots were probed with anti-ubiquitin antibody. A single asterisk denotes the charged Ube2N~Ub, whereas a double asterisk denotes the uncharged Ube2N. A representative blot from at least three independent experiments is shown. (E) Densitometry quantification of band intensities from 3D was plotted to show the kinetics of E2-Ub discharge. Error bars in all graphs depict the mean +/− SEM. Data represent three independent replicates. (F) Western blots from cells overexpressing indicated constructs of epitope-tagged TRIM7 (probed with anti-HA antibody). Ubiquitin-laddering is lost when the…

Figure 4. TRIM7 restriction of MNV1 and CVB3 infection is driven by helix-ΦQ binding. (A–C) Co-transfection of TRIM constructs and reporter plasmids. Overexpression of TRIM7 induces strong signaling by AP-1 and NF-κB that is dependent on the RING domain. (D) Overexpression of TRIM7 induces signaling, as measured by qPCR; values are normalized to actin. (E) Point mutation that prevents binding to targets does not impact signaling. For all signaling experiments (A–E), representative examples of at least two independent experiments are shown, with values normalized to cells transfected with empty vector. ANOVA was used for statistical analysis and significant differences indicated (p < 0.0005 (***), p < 0.0001 (****)). (F,G) Schematic overview of the experiments shown in H and I. Lentivirus generation and stable transfection of HeLa-CD300lf cells (F), with the expression confirmed by Western blotting (Figure S6). Cells were infected with recombinant CVB3 virus-producing EGFP in host cells or MNV1 (G). (H) Quantification of CVB3 infection by measuring the fraction of EGFP-expressing cells. Data shown are from three independent experiments. (I) Quantification of MNV1 replication by TCID50…