Figures and data
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. 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). 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, lysed, and subjected to IB assay using anti-Flag antibody. RT-qPCR was conducted to determine the mRNA expression levels of E3 ligase candidates. 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 nsp16 protein levels were measured.
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 of nsp16 and the RNA levels of 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 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.
(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). The ratio of colocalization was quantified by measuring the fluorescence intensities using Image J(D).
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. 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-Flag were co-transfected or nsp16-Flag alone. Cells were treated with MG132 for 12 h before collection. Whole cell lysates were incubated with anti-Flag beads, and polyubiquitinated chains of nsp16 were detected by IB with anti-Myc or anti-Flag antibodies.
C. 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.
(D-E) 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 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 B.
Knockdown of MARCHF7 or UBR5 promotes viral replication.
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) (A-C) or Omicron BA.1 strain (MOI: 0.001) (D-F), 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 (A, D) and E genes in supernatant (B, E) were detected by RT-qPCR and the viral titers in supernatant (C, F) were measured. The N protein levels of Wuhan or Omicron viruses were detected by IB (G). Knock-down efficiencies of MARCHF7 and UBR5 were detected by RT-qPCR (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.
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. Viral RNA loads in mouse lung tissues were detected by measuring the mRNA levels of the E genes by RT-qPCR (B). IB was used to detect the expression of MARCHF7 or UBR5 in the lung tissues(C). Mouse body weight was monitored during the experimental period (D). Representative images of H&E staining of lungs of mice with different treatments. Magnification, ×40. Bars, 20 µm(E). The staining of viral N proteins. Magnification, ×63. Bars, 20 µm. n =3 in each group(F). RT-qPCR was used to measure the expression of cytokines and chemokines in the spleens of mice in each group(G). 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).
