Fig 1: Effects of RBPs with Antiviral Potential in SINV Infection(A) UV crosslinking and immunoprecipitation of TRIM25-EGFP, GEMIN5-EGFP, XRCC6-EGFP, or unfused EGFP in cells infected or not with SINV for 18 h. The presence of SINV RNA in eluates and inputs was detected by RT-PCR using specific primers against SINV RNAs.(B) Relative mCherry fluorescence produced in cells overexpressing TRIM25-EGFP (top left), TRIM56-eGFP (top middle), GEMIN5-eGFP (top right), and infected with SINV-mCherry (measured as in Figure 5C). mCherry expression is represented as the mean ± SD of three independent infections in each of the three biological replicates (n = 9). Overexpression was assessed by western blotting. Bottom: western blots of SINV C at 18 hpi, indicating below the average inhibition of C relative to control cells. ***p < 0.001; **p < 0.01.(C) Volcano plots comparing the intensity of proteins in GEMIN5-EGFP versus unfused EGFP IPs in uninfected (left) and infected cells (middle); every dot represents a protein. Dark green dots are proteins enriched with p < 0.01, blue dots are those enriched with p < 0.1, and gray dots represent nonenriched proteins. Pink dots represent ribosomal proteins. Right: a volcano plot comparing the intensity of proteins in GEMIN5 IPs in infected versus uninfected cells.(D) iCLIP analysis of GEMIN5-binding sites on SINV RNA. Top: coverage pileup of 5' first base of unique molecules mapping to the SINV genome, shown as 20-nt sliding mean of five replicates after GFP background subtraction. Each position is given relative to total SINV count (RPM). Middle: key features of SINV annotation. Bottom: the top track shows iCLIP coverage but as a heatmap representation. The middle heatmap shows GEMIN5 binding sites along SINV divided into five groups according to strength of binding. The bottom heatmap shows the number of replicates supporting each binding site when binding sites are called independently for each replicate.See also Figure S7 and Table S5.
Fig 2: Salmonella infection induces proteasomal degradation of TRIM56 and TRIM65.(a) TRIM56 and TRIM65 protein levels decrease in SopA activity-dependent manner. Lysates from HCT116 cells infected with Salmonella SL1344 WT, ?SPI1 or indicated sopA mutant strains were subjected to SDS–PAGE and immunoblotting. (b) SopA-mediated degradation of TRIM56 and TRIM65 on infection. Lysates from HCT116 cells treated with 100 µg ml-1 cycloheximide and infected with the indicated sopA mutant Salmonella strains for indicated time points were subjected to SDS–PAGE and immunoblotting. (c) SopA-mediated degradation of TRIM56 and TRIM65 on heterologous expression. Lysates from inducible HeLa Flp-In T-REx GFP-SopA cells left untreated or treated with indicated combinations of 1 µg ml-1 doxycycline and 20 µM MG132 were subjected to SDS–PAGE and immunoblotting.
Fig 3: TRIM56 and TRIM65 are substrates of degradative SopA ubiquitination.(a) Workflow for SopA-dependent ubiquitinome analysis using SILAC diGly proteomics of Salmonella SL1344 WT and ?sopA-infected HCT116 cells 30 min post infection. (b) SopA ubiquitinates TRIM56 and TRIM65. Scatter plot of replicate SopA ubiquitinome experiments. (c,d) SopA-mediated ubiquitination of TRIM56 on infection is MG132 sensitive and depends on SopA catalytic activity (c) and on SopA–TRIM56 binding (d). Lysates from HeLa cells infected with Salmonella SL1344 WT or indicated mutant strains in the absence or presence of 20 µM proteasome inhibitor MG132 were subjected to TUBE pulldown followed by SDS–PAGE and immunoblotting. (e) SopA ubiquitinates TRIM56 RING domain in vitro. Purified SopA WT and catalytic mutant (C753A) were incubated with E1, UbcH7, ubiquitin, ATP and TRIM56 RING domain. TRIM56 ubiquitination is seen with WT SopA but not in the presence of catalytic-dead SopA. (f) Testing SopA ubiquitin chain specificity. WT SopA was incubated with TRIM56 (1–207) and WT ubiquitin or various ubiquitin mutants that contain only a single surface lysine. E1, UbcH7, ubiquitin and ATP were added to all the reactions. TRIM56 alone is not active under these conditions (lane 1).
Fig 4: Structural features of TRIMs define specificity of SopA towards TRIM56.(a) Multiple sequence alignment of the first Zn2+-binding loop region from various TRIMs and the closely related RING domain-containing proteins reveal Glu25 and Leu26 (residues in TRIM56 that are involved in binding to SopA) are fairly conserved (coloured in red). Zn2+-coordinating cysteines are indicated using an asterisk. (b,c,d,e) TRIM56 RING domain was aligned with various closely related RINGs showing the clashes (circled) of SopA with the central a-helix of various RING domains (b, TRIM32; c, TRAF6; d, RNF4; e, TRIM39). PDB codes of the structures used in the alignment are indicated in brackets: (2JMD)47, (4AP4)30. (f) SopA does not bind TRIM32, TRIM39 and RNF4. Lysates from cells co-expressing GFP-SopA C753A and indicated FLAG-RING E3 ligase constructs were subjected to anti-FLAG IP, followed by SDS–PAGE and immunoblotting.
Fig 5: Identification of TRIM56 and TRIM65 as SopA-interacting proteins.(a) Workflow for SILAC-coupled SopA interactome analysis from inducible HeLa Flp-In T-REx GFP-SopA-expressing cells. (b) SopA interacts with TRIM56 and TRIM65. Scatter plot of forward and reverse SILAC SopA interactome. Proteins situated in the upper left quadrant include contaminants. (c) Endogenous TRIM56 and TRIM65 specifically interact with SopA. Lysates from HEK293T cells expressing GFP, GFP-SopA or GFP-NleL constructs were subjected to anti-GFP IP, followed by SDS–PAGE and immunoblotting. (d) Bacterially translocated SopA interacts with TRIM56/65. Scatter plot of forward and reverse SILAC interactome experiments from Salmonella-infected HeLa cells. Proteins situated in the upper left quadrant include contaminants. (e) Endogenous TRIM56 interacts with bacterially secreted SopA during infection. Lysates from HeLa cells infected with SL1344 WT, SopA–HA or catalytic-dead SopA C753A-HA-expressing strains were subjected to anti-HA IP, followed by SDS–PAGE and immunoblotting.
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