anti trim32 antibody  (Proteintech)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: Antiviral and proviral TRIM proteins in SARS-CoV-2 infection. ( A ) Schematic representation of the RNAi screen targeting 71 human TRIM genes to test the effect of TRIM gene silencing on SARS-CoV-2 replication. Caco-2 cells stably expressing human ACE2 (Caco-2-hACE2) were transfected for 40 h with TRIM-specific siRNAs (pool of 4 different siRNAs per TRIM), or nontargeting control siRNA (si.C) (not illustrated), and then infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02). siRNAs targeting TMPRSS2 (proviral) and LY6E (antiviral) were included as controls (not illustrated). Mock-infected cells served as additional controls. Virus replication kinetics in live cells was assessed every hour for a 72-h period by measuring relative fluorescence units (RFU) for EGFP. Parts of this figure were created using Biorender.com. ( B ) Heat map representation of the EGFP-rSARS-CoV-2 replication kinetics from the TRIM RNAi screen in ( A ). ( C and D ) EGFP-rSARS-CoV-2 replication kinetics for selected TRIM proteins identified in the RNAi screen showing antiviral ( C ) or proviral ( D ) activities, presented as mean RFU values. Values were autoscaled to the average values for mock-infected cells at 0 h, set to 200. Mean RFU values for si.C ( C and D ), si.LY6E ( C ), and si.TMPRSS2 ( D ) across all plates are shown. ( E–H ) SARS-CoV-2 genomic RNA (gRNA; Nsp3 ) ( E and G ) and subgenomic RNA (sgRNA; N&Orf9b ) ( F and H ) in Caco-2-hACE2 cells that were transfected for 40 h with either si.C or the indicated TRIM-specific siRNAs and then either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h, determined by RT-qPCR. si.LY6E and si.TMPRSS2 were included as controls. ( I–L ) SARS-CoV-2 genomic RNA (g. Nsp3 ) ( I and K ) and subgenomic RNA (sg. N&Orf9b ) ( J and L ) in Calu-3 cells that were transfected for 40 h with either si.C or siRNAs targeting the indicated TRIM or control genes and then either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h, determined by RT-qPCR. Data are representative of one RNAi screen [conducted in triplicates ( B – D )] or at least two independent experiments [mean ± SD of n = 3 biological replicates ( E – L )]. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s t test). ns, not significant.

Article Snippet: Caco-2 (human colorectal adenocarcinoma, HTB-37, ATCC), Caco-2-hACE2 , Caco-2 TRIM32 -KO, and Caco-2 cells inducibly expressing TRIM32 or TRIM65 were cultured in EMEM supplemented with 20% (v/v) FBS and 1% (v/v) penicillin-streptomycin.

Techniques: Infection, Stable Transfection, Expressing, Transfection, Control, Virus, Fluorescence, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: TRIM32 restricts SARS-CoV-2 replication in a RING-dependent but IFN-independent manner. ( A ) Validation of CRISPR TRIM32- KO Caco-2 cells. TRIM32 protein abundance in TRIM32 -KO Caco-2 single cell clones (cl-1 and cl-2) and WT control cells, determined in the whole cell lysates (WCLs) by immunoblot (IB) analysis with anti-TRIM32 and anti-Actin (loading control). The asterisk indicates nonspecific band. ( B ) Validation of CRISPR TRIM32- KO Caco-2 cells (cl-1 and cl-2) by assessing TRIM32 transcripts by RT-qPCR. Values indicate relative mRNA (fold), normalized to cellular HPRT1 . ( C ) Virus replication kinetics over a 72-h period in TRIM32 -KO and WT (control) Caco-2 cells that were infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02) or mock-treated, determined by measuring RFU for EGFP. Values were autoscaled to the average values for mock-infected cells at 0 h, set to 200. ( D ) SARS-CoV-2 titers in the supernatant of TRIM32 -KO and WT (control) Caco-2 cells that were infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h and 60 h, determined by plaque assay. ( E ) SARS-CoV-2 spike (S) and nucleocapsid (N) protein abundances in TRIM32 -KO and WT Caco-2 cells that were mock-treated (−) or infected for 48 h as in ( D ), determined in the WCLs by IB with anti-S and anti-N. ( F ) TRIM32 protein expression in stably reconstituted TRIM32 -KO Caco-2 cells that inducibly [via 2 μg/mL doxycycline (DOX) treatment for 24 h] expressed either empty vector or FLAG-tagged TRIM32 WT or ΔRING, determined in the WCLs by IB analysis. Stable WT Caco-2 cells expressing empty vector (termed “WT control”) served as control. The asterisk indicates nonspecific band. ( G ) Virus replication kinetics over a 72-h period in TRIM32 -KO Caco-2 cells reconstituted as indicated and either mock-treated or infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02), determined by measuring RFU for EGFP. Values were autoscaled as in ( C ). WT control cells were included for comparison. ( H ) SARS-CoV-2 g. Nsp3 transcripts in TRIM32 -KO Caco-2 cells reconstituted as indicated and either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h, determined by RT-qPCR. ( I and J ) SARS-CoV-2 g. Nsp3 and sg. N&Orf9b transcripts in stable Caco-2 cells that inducibly (by 2 μg/mL DOX treatment) expressed either empty vector (Control Caco-2) or TRIM32 WT (Caco-2 TRIM32) and were infected with SARS-CoV-2 (strain WA1; MOI 0.05) for 48 h in the absence or presence of anti-IFNAR2 antibody (2 µg/mL), determined by RT-qPCR. Data are representative of at least two independent experiments [mean ± SD of n = 3 biological replicates ( B – D and G – J )]. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t test). ns, not significant.

Article Snippet: Caco-2 (human colorectal adenocarcinoma, HTB-37, ATCC), Caco-2-hACE2 , Caco-2 TRIM32 -KO, and Caco-2 cells inducibly expressing TRIM32 or TRIM65 were cultured in EMEM supplemented with 20% (v/v) FBS and 1% (v/v) penicillin-streptomycin.

Techniques: Biomarker Discovery, CRISPR, Quantitative Proteomics, Single Cell, Clone Assay, Control, Western Blot, Quantitative RT-PCR, Virus, Infection, Plaque Assay, Expressing, Stable Transfection, Plasmid Preparation, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: TRIM32 binds to and SUMOylates SARS-CoV-2 Nsp14. ( A ) Endogenous TRIM32 binding to Nsp14 in Caco-2 cells that were either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 1) for 48 h, determined by IP with anti-TRIM32 and IB with anti-Nsp14. ( B ) Schematic representation of TRIM32’s domain organization as well as the truncation constructs used in the interaction-mapping experiments. RING, really interesting new gene; BB, B-box; CC, coiled-coil; NHL, NCL-1, HT2A, Lin-41 domain. mutSIM #1 and #2 indicate TRIM32 mutants in which the two identified SIMs are mutated individually. Mutated residues are shown in red. Numbers indicate amino acids. ( C ) Binding of FLAG-tagged Nsp14 (SARS-CoV-2) to V5-tagged TRIM32 WT or truncation proteins in transiently transfected HEK293T cells, assessed by PD with anti-V5 (PD: V5), followed by IB with anti-FLAG. WCLs were probed with anti-FLAG and anti-Actin (loading control). ( D ) SUMOylation of FLAG-tagged Nsp14 (SARS-CoV-2) in HEK293T cells that were cotransfected for 40 h with V5-tagged TRIM32 WT or ΔRING, determined by PD: FLAG and IB with anti-pan-SUMO. ( E ) SUMOylation of FLAG-tagged Nsp14 (SARS-CoV-2) in transiently transfected HEK293T cells that coexpressed either empty vector or V5-tagged TRIM32 WT or ΔRING, along with UBC9-HA and Myc-tagged SUMO1, SUMO2, or SUMO3, determined by PD: FLAG and IB with anti-Myc. ( F ) SUMOylation of Nsp14 in stable Caco-2 cells inducibly expressing FLAG-TRIM32 that were infected for 32 h with SARS-CoV-2 (strain WA1; MOI 2) and then treated for 12 h with DOX (2 µg/mL) to induce TRIM32 expression, assessed by denaturing IP with anti-SUMO2/3 and IB with anti-Nsp14. Hc, antibody heavy chain. The asterisk indicates nonspecific band. ( G ) SUMOylation of FLAG-tagged Nsp14 (SARS-CoV-2) in HEK293T cells that were cotransfected for 40 h with either empty vector or V5-tagged TRIM32 WT or mutants (ΔRING or mutSIM #1 or #2) together with Myc-tagged SUMO2 and HA-tagged UBC9, determined by PD: FLAG and IB with anti-Myc. Data ( A and C – G ) are representative of at least two independent experiments.

Article Snippet: Caco-2 (human colorectal adenocarcinoma, HTB-37, ATCC), Caco-2-hACE2 , Caco-2 TRIM32 -KO, and Caco-2 cells inducibly expressing TRIM32 or TRIM65 were cultured in EMEM supplemented with 20% (v/v) FBS and 1% (v/v) penicillin-streptomycin.

Techniques: Binding Assay, Infection, Construct, Transfection, Control, Plasmid Preparation, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: The Nsp14 ExoN domain is SUMOylated at K9 and K200. ( A ) Schematic representation of the affinity purification-mass spectrometry (AP-MS) approach to identify the SUMOylation sites in SARS-CoV-2 Nsp14 as well as Coomassie blue-stained gel of affinity-purified Nsp14. HEK293T cells were transiently transfected with FLAG-tagged Nsp14, V5-tagged TRIM32, HA-tagged UBC9, and Myc-tagged SUMO2 T91R mutant. 40 h later, Nsp14 was affinity-purified by PD: FLAG, followed by SDS-PAGE and Coomassie blue staining ( Center ). Unmodified Nsp14 (arrowhead) and modified Nsp14 versions are indicated. Nsp14 protein was subjected to digestion using trypsin, and peptides modified by SUMO2 T91R Lys-Gly-Gly (KGG) signatures were analyzed using LC–MS/MS. The asterisk indicates antibody heavy chain. Parts of this figure were created using Biorender.com. ( B ) Top : Schematic representation of Nsp14 domain organization with the SUMOylated lysines (Ks) identified by MS analysis indicated. ExoN, exoribonuclease; N7-MTase, N7-methyltransferase. Bottom : Mass spectra of the tryptic peptides of affinity-purified FLAG-tagged Nsp14 (SARS-CoV-2) that identified SUMOylation at K9 and K200, with designated b- and y-ions. Ace denotes acetylated N terminus. ( C ) SUMOylation of FLAG-tagged Nsp14 (SARS-CoV-2) WT or indicated mutants in transiently transfected HEK293T cells that were cotransfected for 40 h with either empty vector or TRIM32-V5 together with UBC9-HA and Myc-SUMO2, determined by PD: FLAG and IB with anti-Myc. ( D ) Binding of FLAG-tagged Nsp14 (SARS-CoV-2) WT or mutants in transiently transfected HEK293T cells that were cotransfected for 40 h with TRIM32-V5, determined by PD: FLAG and IB with anti-V5. (E) Representation of the cryo-EM structure of the SARS-CoV-2 Nsp10–Nsp14–RNA complex (PDB: 7N0B) with Nsp14 (cyan), RNA (orange), and Nsp10 (green). K9 and K200 are depicted in red using spheres. Bound zinc ions are depicted as gray spheres. Data are representative of at least two independent experiments ( C and D ) or are from one unbiased AP-MS analysis ( A and B ).

Article Snippet: Caco-2 (human colorectal adenocarcinoma, HTB-37, ATCC), Caco-2-hACE2 , Caco-2 TRIM32 -KO, and Caco-2 cells inducibly expressing TRIM32 or TRIM65 were cultured in EMEM supplemented with 20% (v/v) FBS and 1% (v/v) penicillin-streptomycin.

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Staining, Transfection, Mutagenesis, SDS Page, Modification, Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Binding Assay, Cryo-EM Sample Prep

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: ExoN SUMOylation by TRIM32 inhibits RNA and Nsp10 binding, suppressing ExoN activity. ( A ) Schematic diagram showing the approach to assess the effect of TRIM32-mediated SUMOylation of Nsp14 on its ExoN activity and RNA–substrate binding. FLAG-tagged Nsp14 (SARS-CoV-2) or vector control was transfected into HEK293T cells together with either empty vector or V5-tagged TRIM32 (WT, ΔRING, or mutSIM) and Myc-SUMO2 and UBC9-HA, followed by affinity purification of Nsp14-FLAG by PD: FLAG and subsequent elution using 3X FLAG peptide. One portion of purified Nsp14-FLAG was incubated in vitro with recombinant His-tagged Nsp10 and FAM-labeled RNA substrate (CoV-RNA1-A) to analyze ExoN activity ( Lower Center ). Furthermore, purified Nsp14-FLAG was used for in vitro RNA binding assay with biotinylated CoV-RNA1-A (Biotin-CoV-RNA1-A) and subjected to PD: Strep ( Lower Right ). Purified Nsp14-FLAG was also analyzed by IB for SUMOylation ( Lower Left ). Parts of this figure were created using Biorender.com. ( B ) In vitro ExoN activity of SARS-CoV-2 Nsp14 purified from cells that coexpressed vector or TRIM32 (WT or mutants) and the indicated SUMOylating machinery components and then incubated in vitro with 5′-FAM-labeled CoV-RNA1-A, determined as described in ( A ). ( C ) Structural modeling of SARS-CoV-2 Nsp14 with K9 and K200 SUMOylated. Nsp10 (light green) and RNA (orange) from the Nsp10–Nsp14–RNA cryo-EM structure (PDB: 7N0B) are depicted using surface representation showing steric clashes with SUMO molecules (K9-SUMO and K200-SUMO in Nsp14 (cyan)). ( D ) RNA binding of SARS-CoV-2 Nsp14 purified from cells coexpressing vector or TRIM32 (WT or mutants) and the indicated SUMOylating machinery components and incubated in vitro with biotinylated CoV-RNA1-A (or control RNA), determined by PD: Strep and IB with anti-FLAG. ( E ) SARS-CoV-2 Nsp14 binding to Nsp10 in HEK293T cells that were transfected for 40 h with Nsp14-FLAG, Nsp10-Strep II, UBC9-HA, Myc-SUMO2, and either empty vector or V5-tagged TRIM32 WT or mutants, determined by PD: FLAG and IB with anti-Strep II. Nsp14 SUMOylation was assessed in parallel by PD: FLAG and IB with anti-Myc. ( F ) SARS-CoV-2 replication in stable Caco-2 cells that inducibly (by 2 µg/mL DOX treatment) expressed empty vector (Control Caco-2) or WT TRIM32 (Caco-2 TRIM32) and were transfected for 40 h with si.C, si.UBC9, or si.SUMO2, followed by infection with SARS-CoV-2 (strain WA1; MOI 0.02). At 48 h postinfection, SARS-CoV-2 g. Nsp3 and sg. N&Orf9b transcripts were determined by RT-qPCR. ( G ) SARS-CoV-2 replication in stable Caco-2 TRIM32 cells that were infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h in the presence of 2.5 µM TAK-981 or DMSO (−), determined by RT-qPCR of the indicated viral g/sgRNAs. Control Caco-2 cells were included for comparison. Data ( B and D – G ) are representative of at least two independent experiments [mean ± SD of n = 3 biological replicates ( F and G )]. ** P < 0.01, *** P < 0.001 (Student’s t test). ns, not significant.

Article Snippet: Caco-2 (human colorectal adenocarcinoma, HTB-37, ATCC), Caco-2-hACE2 , Caco-2 TRIM32 -KO, and Caco-2 cells inducibly expressing TRIM32 or TRIM65 were cultured in EMEM supplemented with 20% (v/v) FBS and 1% (v/v) penicillin-streptomycin.

Techniques: Binding Assay, Activity Assay, Plasmid Preparation, Control, Transfection, Affinity Purification, Purification, Incubation, In Vitro, Recombinant, Labeling, RNA Binding Assay, Cryo-EM Sample Prep, Infection, Quantitative RT-PCR, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: Nsp14 SUMOylation and virus restriction by TRIM32 are broadly conserved for coronaviruses. ( A ) Sequence alignment of the Nsp14 protein regions containing K9 and K200 from the indicated coronaviruses, determined by ClustalW. Asterisks indicate positions which have a single, fully conserved residue; colons illustrate conservation between groups of strongly similar properties; periods indicate conservation between groups of weakly similar properties. ( B ) SUMOylation of Nsp14 from the indicated coronaviruses in HEK293T cells that were transiently transfected with plasmids expressing FLAG-tagged coronaviral Nsp14s together with UBC9-HA, Myc-SUMO2 and either vector or V5-tagged TRIM32 (WT or ΔRING), assessed by PD: FLAG and IB with anti-Myc. ( C ) TRIM32 binding of the indicated coronaviral Nsp14 proteins in HEK293T cells that were transiently transfected with plasmids expressing FLAG-tagged coronaviral Nsp14s (or with empty vector) together with V5-tagged TRIM32, determined by PD: FLAG and IB with anti-V5. FLAG-tagged Nsp10 (SARS-CoV-2) served as control. ( D–F ) MERS-CoV ( D ), HCoV-OC43 ( E ), and MHV ( F ) replication in TRIM32 -KO Caco-2 single cell clones (cl-1 and cl-2) or WT control cells that were either mock-treated (−) or infected with virus as indicated (MOI 0.02 for each) for 48 h, determined by RT-qPCR of sg. M or s g.N transcripts. ( G–I ) MERS-CoV ( G ), HCoV-OC43 ( H ), and MHV ( I ) replication in stable Caco-2 cells that inducibly (by 2 µg/mL DOX) expressed TRIM32 (Caco-2 TRIM32) and were transfected for 40 h with si.C, si.UBC9 or si.SUMO2, followed by virus infection (MOI 0.02 for each) for 48 h, determined by RT-qPCR of sg. M or s g.N transcripts. Control Caco-2 cells were included for comparison. ( J ) Representative knockdown efficiency of endogenous UBC9 and SUMO2 for the experiments in ( G – I ), determined by RT-qPCR. Data ( B – J ) are representative of at least two independent experiments (mean ± SD of n = 3 biological replicates ( D – J ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s t test).

Article Snippet: Caco-2 (human colorectal adenocarcinoma, HTB-37, ATCC), Caco-2-hACE2 , Caco-2 TRIM32 -KO, and Caco-2 cells inducibly expressing TRIM32 or TRIM65 were cultured in EMEM supplemented with 20% (v/v) FBS and 1% (v/v) penicillin-streptomycin.

Techniques: Virus, Sequencing, Residue, Transfection, Expressing, Plasmid Preparation, Binding Assay, Control, Single Cell, Clone Assay, Infection, Quantitative RT-PCR, Comparison, Knockdown

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: TRIM32 restricts SARS-CoV-2 replication in a RING-dependent but IFN-independent manner ( A ) Validation of CRISPR TRIM32- KO Caco-2 cells. TRIM32 protein abundance in TRIM32 -KO Caco-2 single cell clones (cl-1 and cl-2) and WT control cells, determined in the whole cell lysates (WCLs) by immunoblot (IB) analysis with anti-TRIM32 and anti-Actin (loading control). Asterisk indicates nonspecific band. ( B ) Validation of CRISPR TRIM32- KO Caco-2 cells (cl-1 and cl-2) by assessing TRIM32 transcripts by RT-qPCR. Values indicate relative mRNA (fold), normalized to cellular HPRT1 . ( C ) Virus replication kinetics over a 72-h period in TRIM32 -KO and WT Caco-2 cells that were either mock-treated or infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02), determined by measuring RFU for EGFP. Values were auto-scaled to the average values for mock-infected cells at 0 h, set to 200. ( D and E ) RT-qPCR analysis of SARS-CoV-2 g. Nsp3 (D) and sg. N&Orf9b (E) transcripts in TRIM32 -KO and WT Caco-2 cells that were either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h. ( F ) SARS-CoV-2 spike (S) and nucleocapsid (N) protein abundances in TRIM32 -KO and WT Caco-2 cells that were mock-treated (−) or infected as in (D), determined in the WCLs by IB with anti-S and anti-N. ( G ) TRIM32 protein expression in stable reconstituted TRIM32 -KO Caco-2 cells that inducibly (via doxycycline (DOX; 2 ug/mL) treatment for 24 h) expressed either empty vector or FLAG-tagged TRIM32 WT or ΔRING, determined in the WCLs by IB with anti-TRIM32, anti-FLAG, and anti-Actin (loading control). WT Caco-2 cells expressing empty vector served as control. Asterisk indicates nonspecific band. ( H ) Virus replication kinetics over a 72-h period in TRIM32 -KO Caco-2 cells that were reconstituted as in (G) and then either mock-treated or infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02), determined by measuring RFU for EGFP. Values were auto-scaled as in (C). WT Caco-2 cells expressing empty vector served as control. ( I ) SARS-CoV-2 g. Nsp3 transcripts in TRIM32 -KO Caco-2 cells reconstituted as indicated and either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h, determined by RT-qPCR. WT Caco-2 cells expressing empty vector served as control. ( J and K ) SARS-CoV-2 g. Nsp3 and sg. N&orf9b transcripts in stable Caco-2 cells that inducibly (via DOX (2 ug/mL) treatment) expressed empty vector (control) or TRIM32 WT and were infected with SARS-CoV-2 (strain WA1; MOI 0.05) for 48 h in the absence or presence of anti-IFNAR2 antibody (2 µg/mL), determined by RT-qPCR. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates (B—E, H—K)). **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). ns, not significant. See also Figure S2.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Biomarker Discovery, CRISPR, Quantitative Proteomics, Clone Assay, Control, Western Blot, Quantitative RT-PCR, Virus, Infection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: TRIM32 binds to and SUMOylates SARS-CoV-2 NSP14 ( A ) Endogenous TRIM32 binding to NSP14 in Caco-2 cells that were either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 1) for 48 h, determined by IP with anti-TRIM32 and IB with anti-NSP14. ( B ) Schematic representation of TRIM32’s domain organization as well as the truncation constructs used in the interaction-mapping experiments. RING, really interesting new gene; BB, B-box; CC, coiled-coil; NHL, NCL-1, HT2A, Lin-41 domain; mutSIM, a mutant of TRIM32 in which the SIM motif located in the NHL domain is mutated. Mutated residues are shown in red. Numbers indicate amino acids. ( C ) Binding of FLAG-NSP14 (SARS-CoV-2) to the indicated V5-tagged TRIM32 truncation proteins in transiently transfected HEK293T cells, assessed by PD with anti-V5 (PD: V5), followed by IB with anti-FLAG. WCLs were probed with anti-FLAG and anti-Actin (loading control). ( D ) SUMOylation of FLAG-tagged NSP14 (SARS-CoV-2) in HEK293T cells that were co-transfected for 40 h with V5-tagged TRIM32 WT or ΔRING, determined by PD: FLAG and IB with anti-pan-SUMO. WCLs were probed by IB with anti-V5 and anti-Actin (loading control). ( E ) SUMOylation of FLAG-tagged NSP14 (SARS-CoV-2) in transiently transfected HEK293T cells that co-expressed either empty vector or V5-tagged TRIM32 WT or ΔRING, along with UBC9-HA and Myc-tagged SUMO1, SUMO2 or SUMO3, determined by PD: FLAG and IB with anti-Myc and anti-FLAG. Expression of co-transfected proteins was determined in the WCLs using the indicated antibodies. ( F ) SUMOylation of NSP14 in stable Caco-2 cells that were infected for 32 h with SARS-CoV-2 (strain WA1; MOI 2) and then treated for 12 h with DOX (2 µg/mL) to inducibly express FLAG-tagged TRIM32, assessed by denaturing IP with anti-SUMO2/3 and IB with anti-NSP14. WCLs were probed by IB with the indicated antibodies. Hc, antibody heavy chain. Asterisk indicates nonspecific band. ( G ) SUMOylation of FLAG-tagged NSP14 (SARS-CoV-2) in HEK293T cells that were transfected for 40 h with either empty vector or V5-tagged TRIM32 WT or mutants (ΔRING or mutSIM) together with Myc-tagged SUMO2 and HA-tagged UBC9, determined by PD: FLAG and IB with anti-Myc. WCLs were probed by IB with the indicated antibodies. Data (A and C—G) are representative of at least two independent experiments. See also Figure S3.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Binding Assay, Infection, Construct, Mutagenesis, Transfection, Control, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: The NSP14 ExoN domain is SUMOylated at K9 and K200 ( A ) Schematic representation of the approach to identify the SUMOylation sites in NSP14 of SARS-CoV-2 by mass spectrometry (MS) analysis. HEK293T cells were transiently transfected with FLAG-tagged NSP14, V5-tagged TRIM32, HA-tagged UBC9, and Myc-tagged SUMO2 T91R mutant. 40 h later, NSP14 was affinity-purified by PD: FLAG, followed by SDS-PAGE and Coomassie blue staining. Unmodified NSP14 (arrowhead) and modified NSP14 versions are indicated (center). NSP14 protein was subjected to digestion using trypsin, and peptides modified by SUMO2 T91R Lys-Gly-Gly (KGG) signatures were analyzed using LC-MS/MS. Asterisk indicates antibody heavy chain. Parts of this figure were created using Biorender.com. ( B ) Top: Schematic representation of SARS-CoV-2 NSP14 domain organization with the SUMOylated lysines (Ks) identified by MS analysis indicated. ExoN, exonuclease domain; N7-MTase, N7-methyltransferase. Bottom: Mass spectra of the tryptic peptides of affinity-purified FLAG-tagged NSP14 (SARS-CoV-2) that identified SUMOylation at K9 and K200, with designated b-and y-ions. Ace denotes acetylated N-terminus. ( C ) SUMOylation of FLAG-tagged NSP14 (SARS-CoV-2) WT or indicated mutants in transiently transfected HEK293T cells that were co-transfected for 40 h with either empty vector or V5-tagged TRIM32 together with HA-tagged UBC9 and Myc-tagged SUMO2, determined by PD: FLAG and IB with anti-Myc. WCLs were probed by IB with the indicated antibodies. ( D ) Binding of FLAG-tagged NSP14 (SARS-CoV-2) WT or mutants in transiently transfected HEK293T cells that were co-transfected for 40 h with V5-tagged TRIM32, determined by PD: FLAG and IB with anti-V5. WCLs were probed by IB with the indicated antibodies. ( E ) Representation of the cryo-EM structure of the SARS-CoV-2 NSP10-NSP14-RNA complex (PDB: 7N0B) with NSP14 (cyan), RNA (orange), and NSP10 (green). K9 and K200 are depicted in red using spheres. Bound zinc ions are depicted as grey spheres. Data are representative of at least two independent experiments (C and D) or are from one unbiased mass spectrometry screen (A and B). See also Figure S3.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Mass Spectrometry, Transfection, Mutagenesis, Affinity Purification, SDS Page, Staining, Modification, Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Binding Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: ExoN SUMOylation by TRIM32 inhibits RNA and NSP10 binding, suppressing ExoN activity ( A ) Schematic diagram of the in vitro exonuclease and RNA-binding assays to assess the effect of TRIM32-mediated SUMOylation on NSP14 ExoN activity and RNA-substrate binding. FLAG-tagged NSP14 or empty vector (control) was expressed in transiently transfected HEK293T cells together with either empty vector or TRIM32-V5 (WT, ΔRING or mutSIM) and Myc-SUMO2 and UBC9-HA, followed by affinity purification of NSP14-FLAG by PD: FLAG and subsequent elution using 3X-FLAG peptide. One portion of purified NSP14-FLAG was incubated with recombinant His-tagged NSP10 (rNSP10) and RNA substrate (CoV-RNA1-A) for the indicated times to analyze ExoN activity (lower center). Furthermore, eluted NSP14-FLAG was used for in vitro RNA binding assay with a biotinylated RNA substrate (CoV-RNA1-A) and subjected to PD: Strep and IB with anti-FLAG (lower right). The third portion of the eluted NSP14-FLAG fraction was analyzed by IB with anti-Myc to assess NSP14 SUMOylation (lower left). Parts of this figure were created using Biorender.com. ( B ) In vitro ExoN activity of purified SARS-CoV-2 NSP14 in the absence or presence of TRIM32 (WT or mutants) and the indicated SUMOylating machinery components, determined as described in (A). ( C ) Structural modeling of SARS-CoV-2 NSP14 with K9 and K200 SUMOylated. NSP10 (light green) and RNA (orange) from the NSP10-NSP14-RNA cryo-EM structure (PDB:7N0B) are depicted using surface representation showing steric clashes with SUMO molecules (K9-SUMO and K200-SUMO in NSP14 (cyan)). ( D ) RNA binding of SARS-CoV-2 NSP14-FLAG purified from mammalian cells co-expressing either empty vector or TRIM32 (WT or mutants) and the indicated SUMOylating machinery components and incubated with biotinylated CoV-RNA1-A (or control RNA), determined by PD: Strep and IB with anti-FLAG. ( E ) SARS-CoV-2 NSP14 binding to NSP10 in HEK293T cells that were co-transfected for 40 h with NSP14-FLAG, NSP10-Strep, UBC9-HA, Myc-SUMO2, and either empty vector or V5-tagged TRIM32 WT or mutants (ΔRING or mutSIM), determined by PD: FLAG and IB with anti-Strep. NSP14 SUMOylation was assessed in parallel by PD: FLAG and IB with anti-Myc. WCLs were probed by IB with anti-FLAG, anti-Strep, anti-V5, anti-Myc, anti-HA, and anti-Actin (loading control). ( F ) SARS-CoV-2 replication in stable Caco-2 cells that inducibly (by DOX treatment (2 µg/mL) for 24 h) expressed FLAG-tagged TRIM32 and were transfected for 40 h with si.C, si.UBC9, or si.SUMO2, followed by infection with SARS-CoV-2 (strain WA1; MOI 0.02). At 48 h post-infection, SARS-CoV-2 g. Nsp3 and sg. N&Orf9b transcripts were determined by RT-qPCR. ( G ) Representative knockdown efficiency of endogenous UBC9 and SUMO2 for the experiment shown in (F), determined by RT-qPCR. Data (B, D, E, F, G) are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates (F and G). **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). ns, not significant. See also Figure S4.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Binding Assay, Activity Assay, In Vitro, RNA Binding Assay, Plasmid Preparation, Control, Transfection, Affinity Purification, Purification, Incubation, Recombinant, Cryo-EM Sample Prep, Expressing, Infection, Quantitative RT-PCR, Knockdown

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: NSP14 SUMOylation by TRIM32 is broadly conserved for coronaviruses ( A ) SUMOylation of NSP14 from the indicated coronaviruses in HEK293T cells that were transiently transfected with plasmids expressing FLAG-tagged coronaviral NSP14s together with UBC9-HA and Myc-SUMO2 and either empty vector or V5-tagged TRIM32, assessed by PD: FLAG and IB with anti-Myc. WCLs were probed by IB with the indicated antibodies. ( B ) TRIM32 binding of the indicated coronaviral NSP14 proteins in HEK293T cells that were transiently transfected with plasmids expressing FLAG-tagged coronaviral NSP14s together with either empty vector or V5-tagged TRIM32, determined by PD: FLAG and IB with anti-V5. FLAG-tagged NSP10 (SARS-CoV-2) served as negative control. ( C ) MHV replication in stable Caco-2 cells that inducibly (by DOX treatment (2 µg/mL) for 24 h) expressed FLAG-tagged TRIM32 and were transfected for 40 h with si.C, si.UBC9 or si.SUMO2, followed by infection with MHV (strain A59; MOI 0.02) for 48 h, determined by RT-qPCR of g.N transcripts. ( D ) Representative knockdown efficiency of endogenous UBC9 and SUMO2 for the experiment in (C), determined by RT-qPCR. Data (A—D) are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates (C and D). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). See also Figure S4.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Transfection, Expressing, Plasmid Preparation, Binding Assay, Negative Control, Infection, Quantitative RT-PCR, Knockdown