The non-structural protein nsp16 of SARS-CoV-2 was identified that can be degraded through the proteasome pathway.

A. The non-structural 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 h 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 NH4CL (2.5 µM) for 12 h 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 hours later, the cells were treated with DMSO or MG132 (10 µM) for 12 h, 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 Image J, combined with the protein half-life formula for calculation. Results are shown as mean ± SD (n = 3 independent experiments). ***, P < 0.001 by by a two-tailed t-test.

E. Samples were prepared for mass spectrometry, and nsp16 interacting proteins were obtained by immunoprecipitation (IP) (created using BioRender.com). The plasmids were transfected into HEK293T cells for 48 h. Treat cells with or without MG132 (10 µM) for 12 h 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.

MARCHF7 and UBR5 were identified as E3 ubiquitin ligases involves in nsp16 protein degradation.

A. Knockdown of MARCHF7 or UBR5 resulted in nsp16 restoration. HEK293T cells were transfected with siRNA of E3 ligase candidates for 24 h, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 h, treated with MG132 (10 µM) for 16h before harvesting, lysed, and subjected to IB assay using anti-Flag antibody. RT-qPCR was conducted to determine the mRNA expression levels of E3 ligase candidates. The si-RNA targeting regions for the candidate E3 ubiquitin ligase proteins and the targeted regions for RT-qPCR are shown in Appendix-figure S1A. 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 h and screened with antibiotics for 48 h. 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 h, respectively, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 h. The protein levels and the RNA levels of nsp16, UBR5 and MARCHF7 were measured by IB and RT-qPCR, respectively.

(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.

MARCHF7 or UBR5 catalyze the formation of K-27 type or K-48 type ubiquitin chains of nsp16 respectively.

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 h before collection. The whole-cell lysates were incubated with anti-Flag beads and used for 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 hours later. Prior to harvesting, cells were treated with MG132 for 16 hours. 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 siRNA targeting UBR5 or MARCHF7, and 24 h later ubiquitin-Myc and nsp16-HA were co-transfected or nsp16-HA alone. Cells were treated with MG132 for 16 h 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 h. 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.

MARCHF7 and UBR5 directly interact with nsp16 respectively.

(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 siMARCHF7 or siUBR5, respectively. The immunoprecipitates and input were analyzed by IB. The knockdown efficiency was detected by RT-qPCR and IB.

(C-D). MARCHF7 or UBR5 co-localized 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 um. The ratio of colocalization was quantified by measuring the fluorescence intensities using Image J (D).

Knockdown of MARCHF7 or UBR5 promotes viral replication.

A. The virus-encoded nsp16 protein interacts with endogenous MARCHF7 and UBR5 and undergoes ubiquitination modification. In 293T-ACE2 cells, with or without Wuhan strain infection (MOI: 0.01), the medium was changed 2 hours post-infection, and cells were harvested 48 hours later, with MG132 treatment added 16 hours 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 siRNA in Caco2 cells. 24 h after transfection, the cells were infected with Wuhan strain (MOI:0.01) (C-E) or Omicron BA.1 strain (MOI: 0.001) (F-H), respectively. 2 h post infection, the supernatant was discarded, and the cells were cultured in DMEM containing 3% fetal bovine serum for 48 h. The mRNA levels of 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 Wuhan or Omicron viruses were detected by IB (I). Knock-down 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 multiple comparisons posttest: *P < 0.05; **P < 0.01; ***P < 0.001.

Increased levels of nsp16 rescued viral inhibition by UBR5 or MARCHF7’

(A-H) UBR5 or MARCHF7 was transfected in 293T cells stably overexpressed with ACE2, and the increased doses of nsp16-Flag was transfected simultaneously. After 24 h, the cells were infected with Wuhan strains. The mRNA levels of M and E genes of Wuhan 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 was 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 multiple comparisons posttest. P > 0.05; **P < 0.01; ***P < 0.001. Figure 6 — figure supplement 1 shows data related to infection with Omicron BA.1.

In a mouse infection model, overexpression of MARCHF7 or UBR5 exerted inhibitory effects on virus.

(A-G) BLAB/C mice were injected with the corresponding plasmids at 40ug/500ul via the high-pressure tail vein, followed by nasal inoculation with 50µl SARS-CoV-2 virus at a dosage of 105.5 TCID50/mL (created using BioRender.com). IB was used to detect the expression of MARCHF7 or UBR5 in the lung tissues (B). Viral RNA loads in mouse lung tissues were detected by measuring the mRNA levels of the E genes by RT-qPCR (C). Lung tissue was collected, homogenized, and the residue was removed by centrifugation to collect the supernatant. The viral titer was then measured using the TCID50 method (D). Mouse body weight was monitored during the experimental period (E). Representative images of H&E staining of lungs of mice with different treatments. Magnification, ×40. Bars, 20 µm (F). The staining of viral N proteins. Magnification, ×63. Bars, 20 µm. n =3 in each group (G). RT-qPCR was used to measure the expression of cytokines and chemokines in the spleens of mice in each group (H). Statistical significance was analyzed using a one-way analysis of variance with Tukey’s multiple comparisons test. (NS, no significance, *p < 0.05, **p < 0.01, ***p < 0.001).

Schematic diagram of MARCHF7 and UBR5 ubiquitinate the SARS-CoV-2 non-structural protein nsp16, leading to its degradation via the proteasomal pathway, thereby affecting viral replication (created using BioRender.com).

Analysis of mass spectrometry results.

A. The DAVID bioinformatics website (https://david.ncifcrf.gov/tools.jsp) was used for KEGG pathway analysis and GO analysis of the differential proteins bound by nsp16 protein after treating with or without MG132. The bubble plots or circle diagram 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, a change from deep to shallow of 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 are shown (created using BioRender.com).

MARCHF7 and UBR5 degrade nsp16 independently.

(A-B). UBR5 siRNA was transfected into shMARCHF7 cells to knock down UBR5. After 24 hours, MARCHF7 and nsp16 expression vectors were co-transfected, and the cells were harvested 72 hours later. The levels of nsp16 were characterized by IB with anti-Flag antibody. Whether MARCHF7 was dependent on UBR5 to degrade of nsp16 was determined by further transfection of MARCHF7 siRNA into shUBR5 cells, followed by co-transfection of UBR5 and nsp16 expression vectors 24 h 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.

Interaction of MARCHF7 or UBR5 with nsp16.

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 h. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Using 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), Bars, 10um. The quantization of fluorescence brightness was analyzed by Image J. 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 (blue). Representative images were shown. Scale bars, 20 um. The ratio of colocalization was quantified by measuring the fluorescence intensities using Image J.

F. nsp16 was stably transfected into HEK293T cells. The cells were analyzed by confocal microscopy. The other operations are the same as above.

Domains in which MARCHF7 or UBR5 functions.

A. The schematic represents UBR5 WT or mutants used in the study.

B. The HECT domain of UBR5 is required for nsp16 degradation. After co-transfection with UBR5 WT or mutants and nsp16-HA, cells were harvested 48 hours later, and cell lysates were examined by IB.

C. The HECT domain of UBR5 affects K48-type ubiquitin chain of nsp16.HEK293T cells were transfected with the assigned plasmids. After 36 hours, cells were treated with 10 µM MG132 for 12 hours, 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 wild-type and truncated forms of MARCHF7 used in the study. E. Only MARCHF7 wild-type degraded nsp16.

F. The N-terminal region of MARCHF7 interacted with nsp16, and only the wild type could catalyze the K27-type ubiquitin chain of nsp16.

Effect of MARCHF7 or UBR5 on SARS-CoV-2 trVLP.

(A-B). Knocking down MARCHF7 or UBR5 enhances SARS-CoV-2 trVLP infectivity. MARCHF7 or UBR5 was knocked down by siRNA in Caco2 cells with stable expression of SARS-CoV-2 N protein. Twenty-four hours later, cells were infected with SARS-CoV-2 virus-like-particles (MOI:0.1), the medium was changed two hours after infection, and the eGFP-positive cells were detected by flow cytometry 48 hours later (A). Protein content was determined by RT-qPCR (B).

Effect of MARCHF7 or UBR5 on Omicron BA.1 infectivity.

(A-H). Showing data related to infection with Omicron BA.1. The experimental procedure was the same as Figure 6. 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 multiple comparisons posttest. P > 0.05; **P < 0.01; ***P < 0.001.

The enzyme activity-deficient mutants do not exhibit antiviral activity, and overexpression of nsp16 does not promote viral replication.

(A-H) In 293T-ACE2 cells, the RING domain deletion mutant of MARCHF7 (MARCHF7-1-542) or the HECT domain inactivated mutant of UBR5 (UBR5- Δ HECT) were transfected, along with a gradient of nsp16-Flag overexpression. The cells were infected with the Wuhan strain (MOI: 0.01), medium was changed 2 hours post-infection, and cells and supernatants were collected 48 hours after infection.

MARCHF7 or UBR5 have effects on mutant of nsp16 in different subtypes of SARS-CoV-2.

A. This diagram shows the mutation of nsp16 in different virus subtypes. The amino acids sequences of different SARS-CoV-2 strains were obtained from NCBI, and the amino acids 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 hours of culture, cells were treated with 10 µm MG132 or DMSO, harvested 12 hours late6r, and cell lysates were examined by IB. C. MARCHF7 or UBR5 can degrade nsp16 mutants. After transfecting MARCHF7 or UBR5 siRNA and the mutated nsp16 plasmids, the cells were harvested 48 h later. The cell lysates were detected by IB.

The expression level of MARCHF7 was negatively correlated with the viral titer, while the expression level of UBR5 was increased at low titer and decreased at high titer.

(A-C). The protein and mRNA levels of MARCHF7 or UBR5 upon infection with different titers. Endogenous MARCHF7 and UBR5 RNA levels were detected by RT-qPCR 48 hours after infection with different titers of Wuhan strain (MOI:0, 0.0001, 0.001, 0.01) or omicron BA.1 strain (MOI:0, 0.0001, 0.001). Protein levels were examined by IB.

D. The expression level of UBR5 was negatively correlated with the severity of the disease but MARCHF7 expression levels were not. PBMC cells were extracted from common, severe and critical COVID-19 patients. RT-qPCR was used to detect the mRNA level of UBR5 or MARCHF7 in patients. Significance was determined by one-way ANOVA, followed by a Tukey multiple comparisons posttest. ns, P > 0.05; **P < 0.01; ***P < 0.001.