Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) The nonstructural proteins nsp8, nsp11, and nsp16 could be restored by the proteasome inhibitor MG132. HEK293T cells in 12-well plates were transfected with the plasmids of 16 nonstructural proteins (nsp1–16) encoded by SARS-CoV-2. Thirty-six hours later, the cells were treated with MG132 (10 µM) or DMSO for 12 hr before collection. The protein level was detected by immunoblotting (IB). Quantification of nsp protein levels relative to the control protein is shown. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: *p<0.05; **p<0.01; ***p<0.001. ( B ) Proteasomal inhibitors but no other inhibitors stabilized nsp16 protein. HEK293T cells transfected with the nsp16-Flag expression vector were treated with dimethyl sulfoxide (DMSO), MG132 (10 µM), Bortezomib (10 µM), Carfilzomib (10 µM), Bafilomycin A1 (5 µM), Vinblastine (2.5 µM), or NH 4 Cl (2.5 µM) for 12 hr prior to harvest. The cell lysates were analyzed by anti-Flag antibody. ( C, D ) The half-life of nsp16 was prolonged by the proteasome inhibitor MG132. (C) HEK293T cells were transfected with the nsp16-Flag-expressing plasmids. 12 hr later, the cells were treated with DMSO or MG132 (10 µM) for 12 hr, then 50 µg/ml cycloheximide (CHX) was added. Cells were harvested at the indicated times to detect the level of viral protein by anti-Flag antibody. (D) Quantification of nsp16 protein levels relative to tubulin at different time points is shown. The half-life of the nsp16 protein was determined based on protein quantification using ImageJ, combined with the protein half-life formula for calculation. Results are shown as mean ± SD (n = 3 independent experiments). ***p<0.001 by a two-tailed t-test. ( E ) Samples were prepared for mass spectrometry, and nsp16 interacting proteins were obtained by immunoprecipitation (IP) (created with BioRender.com and the agreement no. is XR281XWMTN). The plasmids were transfected into HEK293T cells for 48 hr. Treat cells with or without MG132 (10 µM) for 12 hr prior to harvest. The whole-cell lysates were incubated with protein G agarose beads conjugated with anti-Flag antibodies and used for IB with anti-Flag antibodies to detect the nsp16 protein. Samples enriched for proteins were analyzed by mass spectrometry. Figure 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 1—source data 2. Original files for western blot analysis displayed in . Figure 1—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Transfection, Western Blot, Control, Two Tailed Test, Expressing, Plasmid Preparation, Mass Spectrometry, Immunoprecipitation, Incubation

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) The DAVID bioinformatics website ( https://david.ncifcrf.gov/tools.jsp ) was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis of the differential proteins bound by nsp16 protein after treating with or without MG132. The bubble plots or circle diagrams depict the results of the analysis separately. In bubble plots, a change from yellow to purple indicates a decreased p-value, while the size of the circles indicates the number of enriched genes. In the circle plot, depicting the BP (Biological Process) analysis in the GO term, the length of the purple rectangle indicates the number of genes included in the term. The length of the green rectangle indicates the number of overlapping genes between the genes included in the term and the genes entered in the gene enrichment analysis, and a change from deep to shallow purple indicates a decreased p-value (created with chiplot.com ). ( B ) Schematic representation of proteins degraded by the proteasome pathway, as well as proteins associated with the proteasome in the MS enrichment analysis, is shown (created with BioRender.com and the agreement no. is PT281XWUL2).

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques:

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Knockdown of MARCHF7 or UBR5 resulted in nsp16 restoration. HEK293T cells were transfected with small interfering RNA (siRNA) of E3 ligase candidates for 24 hr, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr, treated with MG132 (10 µM) for 16 hr before harvesting, lysed, and subjected to immunoblotting (IB) assay using anti-Flag antibody. RT-qPCR was conducted to determine the mRNA expression levels of E3 ligase candidates. The siRNA targeting regions for the candidate E3 ubiquitin ligase proteins and the targeted regions for RT-qPCR are shown in . Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( B ) RNA levels of UBR5 or MARCHF7 from HEK293T cells infected with lentivirus containing control or shRNA targeting UBR5 or MARCHF7 for 48 hr and screened with antibiotics for 48 hr. Knockdown cell lines were transfected with plasmids expressing nsp16-Flag, collected at the indicated times, and the protein levels of nsp16, MARCHF7, and UBR5 were detected by IB. ( C ) MARCHF7 and UBR5 acted separately and did not depend on each other. HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA were transfected with siRNA of MARCHF7 or UBR5 for 24 hr, respectively, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr. The protein levels and the RNA levels of nsp16, UBR5, and MARCHF7 were measured by IB and RT-qPCR, respectively. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( D, E ) In HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA, nsp16 was degraded by overexpressed UBR5 or MARCHF7, respectively, whereas the mutant failed to degrade nsp16. The cell lysates were analyzed by anti-Flag antibody. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Knockdown, Transfection, Small Interfering RNA, Incubation, Expressing, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Two Tailed Test, Infection, Control, shRNA, Stable Transfection, Mutagenesis

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A, B ) UBR5 small interfering RNA (siRNA) was transfected into shMARCHF7 cells to knock down UBR5 . After 24 hr, MARCHF7 and nsp16 expression vectors were co-transfected, and the cells were harvested 72 hr later. The levels of nsp16 were characterized by immunoblotting (IB) with anti-Flag antibody. Whether MARCHF7 was dependent on UBR5 to degrade nsp16 was determined by further transfection of MARCHF7 siRNA into shUBR5 cells, followed by co-transfection of UBR5 and nsp16 expression vectors 24 hr later. The other operations are the same as above. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( C ) The siRNA targeting regions and RT-qPCR targeting regions for the E3 ubiquitin ligases—HECTD1, UBR5, MYCBP2, TRIM21, TRIM32, and MARCHF7—are shown. Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Small Interfering RNA, Transfection, Knockdown, Expressing, Western Blot, Cotransfection, Two Tailed Test, Quantitative RT-PCR, Ubiquitin Proteomics

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Nsp16 can be ubiquitinated. HEK293T cells co-transfected with ubiquitin-Myc and nsp16-Flag or transfected with nsp16-Flag alone. The cells were treated with MG132 for 12 hr before collection. The whole-cell lysates were incubated with anti-Flag beads and used for immunoblotting (IB) with anti-Myc or anti-Flag antibodies to detect the polyubiquitination chain of nsp16. ( B ) Assess the endogenous ubiquitination level of nsp16 protein. Cells were transfected with nsp16-Flag or an empty vector and collected 48 hr later. Prior to harvesting, cells were treated with MG132 for 16 hr. Co-immunoprecipitation (Co-IP) experiments were then performed to analyze the endogenous ubiquitination level of nsp16. ( C ) The level of ubiquitination of nsp16 decreased with decreasing the protein levels of MARCHF7 or UBR5. E3 was knocked down by transfection with small interfering RNA (siRNA) targeting UBR5 or MARCHF7 , and 24 hr later, ubiquitin-Myc and nsp16-HA were co-transfected or nsp16-HA alone. Cells were treated with MG132 for 16 hr before collection. Whole-cell lysates were incubated with anti-HA beads, and polyubiquitinated chains of nsp16 were detected by IB with anti-Myc or anti-HA antibodies. ( D ) Nsp16 can be modified by a variety of ubiquitin chains. HEK293T cells were transfected with either nsp16-HA alone or together with plasmids encoding various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). Thirty-six hours later, cells were treated with MG132 for 12 hr. Cell lysates were then subjected to immunoprecipitation, followed by IB to analysis. ( E, F ) MARCHF7 or UBR5 causes nsp16 to be modified by the K27-type or K48-type ubiquitin chain. 293T cell lines with or without MARCHF7 or UBR5 knockdown were co-transfected with plasmids encoding ubiquitin-WT or various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). The other experimental methods were the same as C. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Transfection, Ubiquitin Proteomics, Incubation, Western Blot, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Small Interfering RNA, Modification, Knockdown

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with either nsp16-Flag alone or together with MARCHF7-Myc. Thirty-six hours after transfection, the cells were treated with MG132 (10 µM) for 12 hr. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Using immunoblotting (IB) to analyze the precipitates and input. ( B ) HEK293T cells were transfected with nsp16-Flag. Cell lysates were subjected to immunoprecipitation with anti-UBR5 or IgG antibody. ( C, D ) HeLa cells were co-transfected with YFP-nsp16 and CFP-UBR5 or CFP-MARCHF7. A representative image of YFP-nsp16 (yellow) and ECFP-MARCHF7 (cyan) or ECFP-UBR5 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box). Scale bars, 10 µm. The quantization of fluorescence brightness was analyzed by ImageJ. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( E ) HeLa cells transfected with nsp16-Flag were analyzed by confocal microscopy. The Flag-tagged nsp16 labeled with anti-Flag antibody (red). MARCHF7 or UBR5 were labeled with endogenous antibodies (green). Cell nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole) (blue). Representative images were shown. Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ. ( F ) nsp16 was stably transfected into HEK293T cells. The cells were analyzed by confocal microscopy. The other operations are the same as above. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 4—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Fluorescence, Two Tailed Test, Confocal Microscopy, Labeling, Staining, Stable Transfection

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A, B ) The binding of MARCHF7 or UBR5 to nsp16 was not mutually dependent. The binding of nsp16 to UBR5 or MARCHF7 was identified by co-immunoprecipitation in HEK293T cells transfected into si MARCHF7 or si UBR5 , respectively. The immunoprecipitates and input were analyzed by immunoblotting (IB). The knockdown efficiency was detected by RT-qPCR and IB. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( C, D ) MARCHF7 or UBR5 colocalized with nsp16 in the endoplasmic reticulum. Hela cells were co-transfected with YFP-nsp16 (yellow) and CFP-UBR5 (cyan) or CFP-MARCHF7 (cyan). The organelles were labeled with antibodies against marker proteins of endoplasmic reticulum, Golgi apparatus, and mitochondria respectively (red). The cells were analyzed by confocal microscopy ( C ). Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ ( D ). Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Binding Assay, Immunoprecipitation, Transfection, Western Blot, Knockdown, Quantitative RT-PCR, Two Tailed Test, Labeling, Marker, Confocal Microscopy, Fluorescence

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) The schematic represents UBR5 wild-type (WT) or mutants used in the study. ( B ) The homologous to the E6AP carboxyl terminus (HECT) domain of UBR5 is required for nsp16 degradation. After co-transfection with UBR5 WT or mutants and nsp16-HA, cells were harvested 48 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) The HECT domain of UBR5 affects K48-type ubiquitin chain of nsp16. HEK293T cells were transfected with the assigned plasmids. After 36 hr, cells were treated with 10 µM MG132 for 12 hr, harvested, and cell lysates were incubated with protein G agarose beads conjugated with anti-HA antibodies. Cell lysates and precipitated samples were analyzed by IB. ( D ) The schematic represents WT and truncated forms of MARCHF7 used in the study. (E) Only MARCHF7 WT degraded nsp16. ( F ) The N-terminal region of MARCHF7 interacted with nsp16, and only the WT could catalyze the K27-type ubiquitin chain of nsp16. Figure 4—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Cotransfection, Western Blot, Ubiquitin Proteomics, Transfection, Incubation

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) The virus-encoded nsp16 protein interacts with endogenous MARCHF7 and UBR5 and undergoes ubiquitination modification. In 293T-ACE2 cells, with or without IME-BJ01 strain infection (MOI: 0.01), the medium was changed 2 hr post-infection, and cells were harvested 48 hr later, with MG132 treatment added 16 hr before harvesting. nsp16 protein was enriched using protein G beads coupled with the nsp16 antibody, and interactions and ubiquitination were analyzed by immunoblotting (IB) with endogenous antibodies against MARCHF7, UBR5, and ubiquitination. ( B–I ) MARCHF7 and UBR5 were knocked down by small interfering RNA (siRNA) in Caco2 cells. 24 hr after transfection, the cells were infected with IME-BJ01 strain (MOI: 0.01) ( C–E ) or Omicron BA.1 strain (MOI: 0.001) ( F–H ), respectively. 2 hr post-infection, the supernatant was discarded, and the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 3% fetal bovine serum for 48 hr. The mRNA levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M and E genes in the cells ( C, F ) and E genes in supernatant ( D, G ) were detected by RT-qPCR, and the viral titers in supernatant ( E, H ) were measured. The N protein levels of IME-BJ01 or Omicron viruses were detected by IB ( I ). Knockdown efficiencies of MARCHF7 and UBR5 were detected by RT-qPCR or IB ( B, I ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in . Figure 5—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Virus, Ubiquitin Proteomics, Modification, Infection, Western Blot, Small Interfering RNA, Transfection, Cell Culture, Quantitative RT-PCR, Knockdown

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–H ) UBR5 or MARCHF7 was transfected in 293T cells stably overexpressed with ACE2, and the increased doses of nsp16-Flag were transfected simultaneously. After 24 hr, the cells were infected with IME-BJ01 strains. The mRNA levels of M and E genes of the IME-BJ01 strain in the cells ( A, D ) and E gene in supernatant ( B, E ) were detected by RT-qPCR, as well as the detection of viral titers in supernatant ( C, F ). The N protein of the virus and the overexpression efficiency were detected by IB ( G, H ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. shows data related to infection with Omicron BA.1. Figure 6—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—source data 2. Original files for western blot analysis displayed in . Figure 6—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Transfection, Stable Transfection, Infection, Quantitative RT-PCR, Virus, Over Expression, Western Blot

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–H ) In 293T-ACE2 cells, the Really Interesting New Gene (RING) domain deletion mutant of MARCHF7 (MARCHF7-aa 1–542) or the homologous to the E6AP carboxyl terminus (HECT) domain inactivated mutant of UBR5 (UBR5-ΔHECT) were transfected, along with a gradient of nsp16-Flag overexpression. The cells were infected with the IME-BJ01 strain (MOI: 0.01), medium was changed 2 hr post-infection, and cells and supernatants were collected 48 hr after infection. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 2—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 2—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Mutagenesis, Transfection, Over Expression, Infection, Western Blot

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) This diagram shows the mutation of nsp16 in different virus subtypes. The amino acid sequences of different SARS-CoV-2 strains were obtained from the National Center for Biotechnology Information (NCBI), and the amino acid sequences of nsp16 of different strains were compared by DNAMAN software. ( B ) Nsp16 mutants can still be regulated by MG132. The mutated nsp16 plasmids were transfected into HEK293T cells. After 36 hr of culture, cells were treated with 10 µm MG132 or DMSO, harvested 12 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) MARCHF7 or UBR5 can degrade nsp16 mutants. After transfecting MARCHF7 or UBR5 small interfering RNA (siRNA) and the mutated nsp16 plasmids, the cells were harvested 48 hr later. The cell lysates were detected by IB. Figure 6—figure supplement 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Mutagenesis, Virus, Software, Transfection, Western Blot, Small Interfering RNA

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: Schematic diagram of MARCHF7 and UBR5 ubiquitinate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein nsp16, leading to its degradation via the proteasomal pathway, thereby affecting viral replication (created with BioRender.com and the agreement no. is EV281XWATL).

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques:

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Single-lysine mutation of nsp16 protein can be restored by MG132. Single-lysine mutants of nsp16 protein were obtained through mutagenesis and overexpressed in 293T cells. After 36 hr, cells were treated with either MG132 or DMSO for 16 hr and then harvested. Protein levels were detected by immunoblotting (IB) using the HA antibody. ( B ) nsp16 protein truncates can be restored by MG132. nsp16 protein truncates were obtained through structural analysis and mutations. IB was performed to determine whether the truncates could be restored by MG132. ( C ) The mass spectrometry analysis identified the ubiquitination modification site at lysine 76. nsp16-Flag was overexpressed in 293T cells, followed by MG132 treatment and cell harvest. nsp16 protein was enriched using Flag antibody-conjugated protein G beads. Flag-peptide competition was used to obtain the nsp16-containing solution. The protein and ubiquitination status were visualized by SDS-PAGE and Coomassie staining. Mass spectrometry was used for further analysis. ( D ) Degradation of nsp16-K76R is still regulated by MARCHF7 or UBR5. A plasmid with a mutation at lysine 76 of nsp16 to arginine (nsp16-K76R) was constructed. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in 293T cells. nsp16-WT or nsp16-K76R was transfected the next day, and cells were harvested 48 hr later. Protein levels were detected by IB. ( E ) Ubiquitination levels of nsp16-K76R are reduced but still regulated by MARCHF7 or UBR5. MARCHF7 or UBR5 was knocked down using siRNA in 293T cells. The cells were co-transfected with Ub-K27 or K48, and nsp16-WT or nsp16-K76R mutant. Cells were harvested 48 hr later, with MG132 treatment 16 hr before harvesting. Co-immunoprecipitation (Co-IP) experiments were performed to analyze the ubiquitination status of nsp16-WT or its mutant. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 8—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 8—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 8—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: The antibodies used for IB analysis were as follows: anti-UBR5 monoclonal antibody (mAb) (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig, RRID: AB_2881136 ), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945, RRID: AB_2157974 ), SARS-CoV-2 2’- O -ribose Methyltransferase Antibody (nsp16) (Cell Signaling, Danvers, MA, USA, catalog no. #70811, RRID: AB_2799787 ), anti-Flag mAb (Sigma, catalog no. F1804, RRID: AB_262044 ), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323, RRID: AB_297919 ), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500, RRID: AB_2533987 ), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP, RRID: AB_2877901 ), and anti-GFP mAb (Abcam, catalog no. ab1218, RRID: AB_298079 ).

Techniques: Mutagenesis, Western Blot, Mass Spectrometry, Ubiquitin Proteomics, Modification, SDS Page, Staining, Plasmid Preparation, Construct, Small Interfering RNA, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Structure (London, England : 1993)

Article Title: Structural insights into the assembly and regulation of 2'-O RNA methylation by SARS-CoV-2 nsp16/nsp10.

doi: 10.1016/j.str.2025.03.009

Figure Lengend Snippet: Figure 3. Putative ligand binding pocket of nsp16 (A) Snapshots of various ligands that bind to the pocket in different nsp16/nsp10 structures: adenosine (PDB: 6WKS),8 m7GDP (PDB: 6WQ3),9

Article Snippet: The following antibodies were used: SARS-CoV-2 nsp10 (ProSci, 9179), SARS-CoV-2 nsp16 (ProSci, 9271), anti-rabbit IgG HRP secondary (Cell Signaling, 7074).

Techniques: Ligand Binding Assay

Journal: Structure (London, England : 1993)

Article Title: Structural insights into the assembly and regulation of 2'-O RNA methylation by SARS-CoV-2 nsp16/nsp10.

doi: 10.1016/j.str.2025.03.009

Figure Lengend Snippet: Figure 5. Sampling oligomeric conforma- tions in solution (A) Coomassie staining of crosslinked nsp16/ nsp10 (±RNA) on SDS-PAGE, with protein bands labeled as 1–6. (B) Immunoblotting with nsp16 antibody shows nsp16 in bands 2, 4–6, and part of wider band 3 (3A). (C) Immunoblotting with nsp10 antibody detects nsp10 in bands 1, 5, 6, and parts of wider band 3 (3B and 3C). Cartoon representations of their olig- omeric states are shown below (blue circle: nsp10, green square: nsp16). (D) Coomassie staining of crosslinked quintuple mutant (±RNA), protein bands labeled as 1–6. (E and F) Immunoblotting with nsp16 and nsp10 antibodies, respectively.

Article Snippet: The following antibodies were used: SARS-CoV-2 nsp10 (ProSci, 9179), SARS-CoV-2 nsp16 (ProSci, 9271), anti-rabbit IgG HRP secondary (Cell Signaling, 7074).

Techniques: Sampling, Staining, SDS Page, Labeling, Western Blot, Mutagenesis

Journal: Structure (London, England : 1993)

Article Title: Structural insights into the assembly and regulation of 2'-O RNA methylation by SARS-CoV-2 nsp16/nsp10.

doi: 10.1016/j.str.2025.03.009

Figure Lengend Snippet: Figure 6. Oligomerization via nsp16-nsp16 interfaces and MP analysis (A) nsp16-nsp16 interface 1 in the current struc- ture. (B) Previously reported interface 2 (PDB: 7JYY). (C) An overlay revealing distinct interfaces on opposite faces of the nsp16/nsp10 heterodimer. (D and E) Mass photometry of the (D) WT apo (gray) and RNA-bound (1:1 M ratio, dark green) and (E) quintuple mutant.

Article Snippet: The following antibodies were used: SARS-CoV-2 nsp10 (ProSci, 9179), SARS-CoV-2 nsp16 (ProSci, 9271), anti-rabbit IgG HRP secondary (Cell Signaling, 7074).

Techniques: Mutagenesis