Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global public health threat, with over 641 million coronavirus disease 2019 (COVID-19) cases and 6.6 million deaths as of December 2022 (Carabelli et al., 2023; Markov et al., 2023). The virus encodes four structural proteins, 16 non-structural proteins (nsps) involved in replication and transcription, and several accessory proteins (ORFs) linked to immune evasion (V’kovski et al., 2021; Wang et al., 2020). Many viruses, including Ebola (Valle et al., 2021), dengue (Jung et al., 2018), and reovirus (Furuichi et al., 1975), encode 2’-O methyltransferases (2’-O-MTases) that mimic the host 5’ cap structure to evade innate immune recognition (Daffis et al., 2010; Züst et al., 2011). Similarly, SARS-CoV-2 nsp16, in complex with nsp10, functions as a 2′-O-MTase, modifying capped viral RNA from ‘cap-0’ to ‘cap-1’ (Benoni et al., 2021; Park et al., 2022). This modification protects the virus from host antiviral defences, such as MDA5 recognition and IFIT1 restriction (Bergant et al., 2022; Russ et al., 2022). Beyond immune evasion, nsp16 disrupts global host mRNA splicing, reducing cellular protein and mRNA levels (Banerjee et al., 2020). Nsp16 also enhances SARS-CoV-2 entry by upregulating TMPRSS2 expression (Han et al., 2023), underscoring its role as a virulence factor. Notably, nsp16-deficient strains exhibit low pathogenicity and elicit robust immune responses, suggesting their potential as live-attenuated vaccines (Ye et al., 2022). Given the critical functions of nsp16, inhibitors targeting nsp16 or the nsp16-nsp10 complex have been developed (Klima et al., 2022; Nguyen et al., 2022). Therefore, understanding host factors that regulate nsp16 is essential for identifying new therapeutic strategies.

The ubiquitin-proteasome system (UPS) is a key pathway for targeted protein degradation, often described as ‘the molecular kiss of death’ (Dongdem & Wezena, 2021; Park et al., 2020). Substrate ubiquitination involves a cascade of three enzymes: E1 activating enzyme, E2 conjugating enzyme, and E3 ubiquitin ligase (Zheng & Shabek, 2017). Some E3 ubiquitin ligases function as monomers, whereas others assemble into multi-subunit complexes. Regardless of their structure, all E3 ligases play a crucial role in substrate recognition, dictating UPS specificity. In certain complexes, an additional protein may function as a substrate receptor, mediating substrate-specific recognition (Iconomou & Saunders, 2016; Yang et al., 2021; Li et al., 2021; Wang et al., 2022). Over 600 E3 ligases encoded in the human genome regulate diverse cellular processes in response to various signals (Dongdem & Wezena, 2021; Garcia-Barcena et al., 2020). Dysregulation of their activity is linked to multiple diseases, making them attractive therapeutic targets (Humphreys et al., 2021). Based on conserved domains and ubiquitin (Ub) transfer mechanisms, E3 ligases are categorised into three classes: Really Interesting New Gene (RING), homologous to the E6AP carboxyl terminus (HECT), and RING-between-RING (RBR) ligases (Garcia-Barcena et al., 2020; Morreale & Walden, 2016). RING E3s transfer Ub directly from the E2-Ub complex to substrates (Deshaies & Joazeiro, 2009), whereas HECT-type E3s first attach Ub to their own catalytic cysteine before transferring it (Huibregtse et al., 1995). These distinct mechanisms highlight the complexity and versatility of E3 ligase function in cellular regulation (Walden & Rittinger, 2018; Garcia-Barcena et al., 2020).

To investigate the relationship between SARS-CoV-2 and host factors, we focused on UPS proteins that interact with nsp16. In this study, we demonstrate for the first time that nsp16 undergoes ubiquitination and proteasomal degradation. Notably, two E3 ligases from distinct families—RING-type MARCHF7 and HECT-type UBR5—independently target nsp16 for degradation, disrupting its function and exhibiting strong antiviral activity against SARS-CoV-2. Our findings reveal novel therapeutic targets for combating SARS-CoV-2 and treating COVID-19.

Results

SARS-CoV-2 nsp16 is degraded via the ubiquitin-proteasome pathway

Ubiquitination plays a crucial role in SARS-CoV-2 infection and pathogenesis (Gao et al., 2022; Guo et al., 2021; Li et al., 2023; Zhang et al., 2021; Zhang et al., 2024; Zhang et al., 2023). To determine whether SARS-CoV-2 non-structural proteins (nsps) are UPS-regulated, we examined the effect of the proteasome inhibitor, MG132, on nsps expression. MG132 treatment significantly increased the nsp8, nsp11, and nsp16 abundance in HEK293T cells (Fig. 1A), suggesting UPS-mediated degradation. Nsp8 undergoes TRIM22-mediated ubiquitination and proteasomal degradation (Fan et al., 2024). In this study, we focused on nsp16 for further investigation. To confirm UPS-mediated nsp16 degradation, we tested additional proteasome inhibitors, Bortezomib and Carfilzomib, both of which enhanced nsp16 stability. In contrast, lysosomal inhibitors (Bafilomycin A1 and NH4Cl) and the autophagy-lysosomal inhibitor, Vinblastine, had no such effect (Fig. 1B). To further assess the role of the UPS in regulating nsp16 stability, we treated nsp16-expressing cells with cycloheximide, a protein synthesis inhibitor, and measured the half-life of nsp16. MG132 treatment significantly prolonged nsp16 stability, extending its half-life from 2 h to 15 h (Fig. 1C and 1D).

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 with BioRender.com and the agreement no. is LC27PCBEOF). 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.

To identify nsp16-interacting proteins, we performed co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS) analysis, comparing nsp16-expressing cells with and without MG132 treatment (Fig. 1E). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses (Figure 1—figure supplement 1A) revealed that inhibiting nsp16 degradation increased its interacting proteins, including 43 associated with viral processes, 22 involved in mRNA stability regulation, and five linked to cellular antiviral defence. Additionally, interactions related to mRNA splicing and RNA methylation were identified (Banerjee et al., 2020), consistent with the function of nsp16 as a 2′-O-MTase. Furthermore, SARS-CoV-2 exploits nsp16 to disrupt host mRNA splicing, which promotes infection (Park et al., 2022; Russ et al., 2022; Züst et al., 2011), further validating our MS data.

As expected, several nsp16-binding proteins were associated with ubiquitination and degradation pathways (Figure 1-figure supplement 1B). We identified four deubiquitinases (DUBs), the E2 ligase UBE2D3, and 14 proteasomal enzymes. Among them, six E3 ligases—UBR5, MARCHF7, HECTD1, TRIM32, MYCBP2, and TRIM21—were selected for further investigation.

E3 ligases, UBR5 and MARCHF7, independently mediate nsp16 degradation

To identify the E3 ligases responsible for nsp16 degradation, we designed small interfering RNAs (siRNAs) targeting six candidate E3 ligases and assessed their effects on nsp16 abundance. UBR5 and MARCHF7 knockdown significantly stabilised nsp16 protein levels (Fig. 2A). To further investigate their roles in nsp16 degradation, we generated stable cell lines with UBR5 or MARCHF7 knockdowns and confirmed silencing efficiency (Fig. 2B). To determine whether UBR5 and MARCHF7 function cooperatively, we performed dual knockdowns. Silencing UBR5 in MARCHF7-knockdown cells, and vice versa, further enhanced nsp16 stability, indicating that these ligases act independently (Fig. 2C). Overexpression studies further supported their independent roles—MARCHF7 overexpression induced nsp16 degradation even in MARCHF7-knockdown and MARCHF7/UBR5 double-knockdown cells. Similarly, UBR5 overexpression promoted nsp16 degradation despite MARCHF7 knockdown. Silencing efficiencies were validated across all experiments (Figure 2 — figure supplement 1A and 1B). Notably, wild-type MARCHF7 and UBR5, but not their mutants lacking functional RING (1-542 aa) or HECT domains, respectively, effectively degraded nsp16 in knockdown cells (Fig. 2D and 2E). These findings confirm that UBR5 and MARCHF7 independently mediate nsp16 ubiquitination via their HECT and RING domains, targeting it for proteasomal degradation.

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.

UBR5 and MARCHF7 mediate distinct Ub linkages on nsp16

We first confirmed nsp16 ubiquitination both with and without exogenous ubiquitin (Ub) expression (Fig. 3A-B). To investigate the role of E3 ligases in this process, we examined the impact of UBR5 and MARCHF7 knockdown on nsp16 ubiquitination. Silencing either ligase reduced nsp16 ubiquitination compared to the negative control, indicating their involvement in this modification (Fig. 3C). Ub contains seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), which form polyubiquitin chains with distinct functions that determine the fate of the attached protein (Grice & Nathan, 2016). To identify the specific polyubiquitin chain types formed on nsp16 by UBR5 and MARCHF7, we used a series of Ub mutants, each retaining only a single lysine residue. Except for K33, all single-lysine Ub mutants supported nsp16 ubiquitination to varying degrees, suggesting a complex ubiquitin architecture potentially regulated by multiple E3 ligases or E2-E3 pairs (Fig. 3D). Further analysis of lysine-specific linkages revealed that MARCHF7 primarily mediates K27-linked ubiquitination of nsp16, whereas UBR5 facilitates K48-linked ubiquitination (Fig. 3E and 3F). These findings establish that UBR5 and MARCHF7 independently regulate nsp16 degradation through distinct ubiquitin linkages.

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.

UBR5 and MARCHF7 directly interact and colocalise with nsp16 in the endoplasmic reticulum (ER)

MS analysis suggested interactions between nsp16 and both UBR5 and MARCHF7. Co-IP experiments confirmed that Myc-tagged MARCHF7 and endogenous UBR5 bind to nsp16 (Figure 4—figure supplement 1A and 1B). To assess whether these interactions are direct, we performed fluorescence resonance energy transfer (FRET) assays. When nsp16-YFP was bleached, the fluorescence intensity of CFP-UBR5 and CFP-MARCHF7 increased, confirming direct interactions (Figure 4 — figure supplement 1C). ImageJ software was used to quantify relative fluorescence intensities (Figure 4 — figure supplement 1D). To determine whether UBR5 and MARCHF7 depend on each other for nsp16 binding, we examined the effect of MARCHF7 knockdown on UBR5-nsp16 interactions, and vice versa, using Co-IP. The interactions remained unchanged, indicating that UBR5 and MARCHF7 independently bind to nsp16 (Fig. 4A and 4B). Nsp16 localises to both the nucleus and cytoplasm (Zhang et al., 2020). UBR5 (containing two nuclear localization signals) and MARCHF7 are also present in both compartments (Muñoz-Escobar et al., 2015; Shearer et al., 2018) (Nathan et al., 2008). Immunofluorescence staining revealed that UBR5 and MARCHF7 colocalised with nsp16 predominantly in the cytoplasm, with some nuclear colocalization observed in HeLa cells (Figure 4—figure supplement 1E). Similar results were obtained in nsp16-transfected HEK293T cells using antibodies against endogenous UBR5 and MARCHF7 (Figure 4—figure supplement 1F).

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).

To pinpoint the specific cellular compartment where these interactions occur, we co-transfected UBR5-CFP or MARCHF7-CFP with nsp16-YFP and performed immunostaining using organelle-specific markers: COX5A (mitochondria), PDI (ER), and GM130 (Golgi apparatus). Notably, UBR5 and MARCHF7 both interacted with nsp16 and colocalised with PDI, but not with COX5A or GM130 (Fig. 4C and 4D), indicating that both E3 ligases interact with nsp16 primarily in the ER.

Functional domains of UBR5 and MARCHF7 are required for nsp16 interaction and ubiquitination

UBR5 is a four-domain E3 ligase containing two nuclear localization signals. Its domains include UBA, UBR, PABC, and HECT (Muñoz-Escobar et al., 2015) (Figure 4— figure supplement 2A). Notably, the HECT domain is critical for its E3 ligase activity, as UBR5 must first conjugate to Ub before transferring it to substrates (Kim et al., 2021). To determine which UBR5 domain mediates nsp16 ubiquitination, we used UBR5 mutants with individual domain inactivations. Only the HECT domain mutant failed to degrade nsp16 (Figure 4—figure supplement 2B), consistent with previous studies (Zhou et al., 2022). This mutant also lost the ability to ubiquitinate nsp16, confirming the essential role of the HECT domain in this process (Figure 4—figure supplement 2C).

To identify the MARCHF7 region responsible for nsp16 degradation, we generated a series of truncation mutants as described previously (Figure 4—figure supplement 2D) (Zhao et al., 2018) (Nathan et al., 2008). All mutants lost the ability to degrade nsp16 (Figure 4 — figure supplement 2E). Further analysis revealed that only the mutant containing the intact N-terminal region (aa 1–542) strongly bound to nsp16, while those retaining the active RING domain region (aa 543–616) did not. Consistently, only wild-type MARCHF7 promoted K27-linked ubiquitination of nsp16 (Figure 4—figure supplement 2F).

These results indicate that the N-terminal region of MARCHF7 is essential for nsp16 binding, whereas its RING domain is required for K27-linked ubiquitination activity.

UBR5 and MARCHF7 suppress SARS-CoV-2 replication by targeting nsp16 for degradation

To assess the impact of nsp16 degradation on viral replication, we used a biosafety level 2 system to generate SARS-CoV-2 transmissible virus-like particles capable of infecting Caco2-Nint cells (Ju et al., 2021). Knockdown of either UBR5 or MARCHF7 significantly increased SARS-CoV-2 replication, with simultaneous knockdown further enhancing viral replication (Figure 5—figure supplement 1A and 1B).

To investigate nsp16 modifications following infection, we infected HEK293T-ACE2 cells with the Wuhan strain for 48 h. Co-IP using an anti-nsp16 antibody confirmed its interaction with endogenous MARCHF7 and UBR5, along with ubiquitination (Fig. 5A). In a biosafety level 3 facility, we further validated the antiviral roles of UBR5 and MARCHF7 using the Wuhan (MOI 0.01) and Omicron strains (MOI 0.001). Knockdown of either E3 ligase in Caco2 cells significantly increased intracellular and secreted viral mRNA levels (M and E genes) for both strains. Viral titres in the supernatants also increased, with UBR5 knockdown causing over a 1-log rise for the Wuhan strain and even greater effects for Omicron (Fig. 5C–E and 5F–H for the Wuhan and Omicron strains, respectively). Immunoblotting confirmed increased intracellular and secreted N protein levels upon UBR5 or MARCHF7 knockdown (Fig. 5I), with knockdown efficiencies verified (Fig. 5B). Due to low transfection efficiency in Caco2 cells, we overexpressed UBR5-Myc or MARCH7-Myc in HEK293T-ACE2 cells. Overexpression significantly reduced viral mRNA (M and E genes) and N protein levels in both strains, accompanied by a 0.5-log decrease in viral titres. However, co-transfection with increasing nsp16 levels counteracted these inhibitory effects (Fig. 6A–H and Figure 6—figure supplement 1A–H). To confirm the role of UBR5 and MARCHF7 enzymatic activity, we overexpressed catalytically inactive mutants (UBR5-Δ HECT or MARCHF7-aa 1-542) alongside nsp16. These mutants failed to suppress viral replication. Additionally, gradual increases in nsp16 expression did not further enhance viral replication, with only a slight increase in M mRNA, while E and N protein levels remained unchanged (Figure 6—figure supplement 2A–H). These findings demonstrate that UBR5 and MARCHF7 exert antiviral effects by ubiquitinating and degrading nsp16, thereby significantly inhibiting SARS-CoV-2 replication.

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.

UBR5 and MARCHF7 mediate nsp16 variant degradation

With the continuous emergence of SARS-CoV-2 variants, broad-spectrum antiviral strategies have become essential. To assess whether UBR5 and MARCHF7 mediate various nsp16 variants, we analysed nsp16 amino acid sequences from the National Center for Biotechnology Information (NCBI) (Figure 6 — figure supplement 3A). Most variants had one or two mutations, except for XBB.1.9.1, XBB.1.5, and XBB.1.16, which contained more mutations, suggesting a conserved sequence. We used mutagenesis to synthesise nsp16 sequences for these three variants, along with several single-site mutants from other variants. Treatment with MG132 restored nsp16 protein levels for all variants (Figure 6 — figure supplement 3B), confirming UPS-mediated degradation.

Furthermore, UBR5 or MARCHF7 knockdown stabilised nsp16 proteins from these variants, though to varying degrees (Figure 6 — figure supplement 3C). These results suggest that UBR5 and MARCHF7 contribute to broad-spectrum antiviral activity by targeting nsp16 from diverse SARS-CoV-2 variants for degradation.

SARS-CoV-2 infection reduces UBR5 and MARCHF7 expression

To investigate the impact of SARS-CoV-2 infection on UBR5 and MARCHF7 expression, we analysed their mRNA and protein levels following infection with the Wuhan and Omicron strains at varying MOIs using RT-qPCR and immunoblotting. MARCHF7 expression decreased consistently with increasing viral titres, whereas UBR5 levels initially rose at low titres before declining as infection progressed (Figure 6—figure supplement 4A-4C). This transient UBR5 upregulation may be linked to its role in interferon-γ-mediated pathways, as previously reported (Wu et al., 2022). To confirm these findings in vivo, we examined UBR5 and MARCHF7 mRNA levels in peripheral blood mononuclear cells from SARS-CoV-2-infected patients with varying disease severity. UBR5 expression negatively correlated with disease progression, whereas MARCHF7 levels showed no significant correlation (Figure 6—figure supplement 4D). These results suggest that SARS-CoV-2 may actively suppress host antiviral defences by downregulating UBR5 and MARCHF7 expression.

UBR5 and MARCHF7 protect mice from SARS-CoV-2 challenge

Since no specific activators or inhibitors for E3 ligases are available, we used a transient overexpression method using high-pressure tail vein injection of plasmids in mice (Bonamassa et al., 2011). To evaluate their antiviral effects in vivo, mice were injected with plasmids encoding UBR5 or MARCHF7, followed by SARS-CoV-2 challenge (Fig. 7A). E3 ligase overexpression significantly reduced viral E gene copy numbers in the lungs and markedly decreased viral titres (Fig. 7C-D). Additionally, treated mice experienced less weight loss compared to controls (Fig. 7E). Histopathological analysis at 5 days post-infection showed that lungs from UBR5-or MARCHF7-treated mice exhibited reduced alveolar contraction and pulmonary oedema compared to the severe lesions observed in control mice (Fig. 7F). Immunoblotting further confirmed lower N protein abundance in treated mice (Fig. 7G). These results suggest that UBR5 and MARCHF7 overexpression suppresses SARS-CoV-2 virulence in vivo by promoting nsp16 degradation.

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 with BioRender.com and the agreement no. is FO27O5X39A). 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 with BioRender.com and the agreement no. is FM27O5ZS3U).

SARS-CoV-2 infection can induce a severe cytokine storm, contributing to high mortality rates (Song et al., 2020) (Hojyo et al., 2020). To assess the impact of UBR5 and MARCHF7 on inflammatory responses, we measured key cytokine levels — including interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1RA), and interleukin-1β (IL-1β)—in the spleens of infected mice (Makaremi et al., 2022) (Hu et al., 2021). Consistent with their antiviral activity, UBR5 and MARCHF7 treatment significantly reduced the production of these inflammatory cytokines (Fig. 7H).

Discussion

SARS-CoV-2 nsp16 functions as a 2′-O-methyltransferase (2′-O-MTase), catalysing penultimate nucleotide methylation of the viral RNA cap. This modification allows SARS-CoV-2 to mimic host mRNA, evading immune detection and response (Lin et al., 2020) (Balieiro et al., 2022; Russ et al., 2022). High-resolution structures of nsp16 and nsp16-nsp10 heterodimers have been characterised (Rosas-Lemus et al., 2020) (Klima et al., 2022; Lugari et al., 2010), providing a foundation for antiviral drug development targeting these complexes (Balieiro et al., 2022; Melo-Filho et al., 2022). However, host factors that interact with and regulate nsp16 remain largely unknown.

Previous studies, including work from our group, have highlighted the role of the UPS in modulating SARS-CoV-2 infection (Xu et al., 2022). Several host E3 ligases—such as RNF5, Cullin4-DDB1-PRPF19, ZNF598, and TRIM7 — ubiquitinate and degrade key SARS-CoV-2 proteins, thereby suppressing viral replication (Li et al., 2023; Liang et al., 2022; Maimaitiyiming et al., 2022; Zhang et al., 2024). In contrast, SARS-CoV-2 uses deubiquitinases (DUBs) to counteract these defences and enhance viral replication (Chen et al., 2024; Gao et al., 2024; Gao et al., 2022; Guo et al., 2021). In this study, we identify nsp16 as a short-lived protein with a half-life of only 2 – 3 h, which is targeted for ubiquitination and degradation via the UPS. Using MS and bioinformatics screening, we identified two E3 ligases, UBR5 and MARCHF7, that directly interact with nsp16 and mediate nsp16 ubiquitination and degradation.

UBR5, a member of the UBR box protein family, contains a HECT domain essential for its E3 ligase activity (Kim et al., 2021). UBR5 regulates tumour growth, metastasis (Xiang et al., 2022) (Qiao et al., 2020), and viral infections. For instance, UBR5 targets the ORF4b protein of Middle East respiratory syndrome coronavirus (MERS-CoV) for degradation, reducing its ability to counteract the host immune response (Zhou et al., 2022). Conversely, UBR5 supports Zika virus replication by assisting TER94/VCP in degrading the capsid protein, allowing viral genome release into the cytoplasm (Gestuveo et al.).

MARCHF7, a RING E3 ligase, plays a role in T cell proliferation, neuronal development, inflammasome regulation, and tumour progression (Zheng, 2021) (Zhang et al., 2016) (Cai et al., 2022) (Zhao et al., 2018). However, its involvement in viral infections remains largely unexplored, with one study identifying it as a host protein interacting with the 2C protein of foot-and-mouth disease virus (Mahajan et al., 2021).

Our findings demonstrate that UBR5 and MARCHF7 independently interact with nsp16 and facilitate its degradation, primarily on the cytoplasmic ER. Our findings highlight the ongoing evolutionary battle between viruses, which evolve strategies to evade host defences, and hosts, which deploy multiple mechanisms to counteract viral infections. One such strategy is the use of multiple E3 ligases targeting the same viral protein for degradation. Functional analyses revealed that knocking down UBR5 or MARCHF7 enhanced SARS-CoV-2 replication, whereas their overexpression impaired it. Moreover, when their enzymatic activity was inactivated, the antiviral effect was abolished, confirming that their E3 ligase activity is essential for viral suppression. Notably, nsp16 overexpression alone did not increase viral levels in the absence of functional UBR5 or MARCHF7. This underscores the critical antiviral role of UBR5 and MARCHF7 in restricting SARS-CoV-2 by promoting nsp16 degradation.

Due to the presence of 18 lysine residues in nsp16, identifying specific ubiquitination sites was challenging. Using structural data (Decroly et al., 2011), we constructed truncated nsp16 mutants to assess their degradation profiles. Our results showed that nsp16 mutants degradation occurred to varying degrees (Appendix-figure S2A-B), suggesting that multiple lysine residues serve as recognition sites for E3 ligases. Furthermore, MG132 recovery experiments provided valuable insights. The Δ2-17 mutant, which lacks this segment, exhibited more than a two-fold increase in nsp16 expression, whereas the Δ204-212 mutant showed significantly reduced recovery after MG132 treatment compared to the wild-type. These observations offer important clues for further investigation into the interaction regions between nsp16 and MARCHF7 or UBR5, as well as potential ubiquitination sites. MS analysis identified K76 as a ubiquitination site (Appendix-figure S2C). However, the nsp16 K76R mutant still underwent degradation, though its ubiquitination level was reduced compared to that of the wild-type, indicating that K76 is one of several ubiquitination sites (Appendix-figure S2D-E). This suggests that nsp16 may undergo ubiquitination at multiple sites or through non-canonical pathways at non-lysine residues (McClellan et al., 2019). Beyond UBR5 and MARCHF7, other E3 ligases may also contribute to nsp16 regulation, warranting further investigation into additional host factors involved in SARS-CoV-2 restriction.

Currently, no specific activators or inhibitors exist for UBR5 or MARCHF7. To assess their antiviral effects in vivo, we used high-pressure tail vein injection, a gene delivery method primarily targeting the liver (Kamimura et al., 2014) but also effective for lung tissues (Bonamassa et al., 2011). Gene expression was successfully detected in the lungs, and while this method induced mild immune activation, its effect was minimal compared to other techniques (Suda et al., 2023). Notably, mice and non-human primates exhibit greater resistance to innate immune activation than humans (Raper et al., 2003), suggesting that immune activation had little influence on our results. However, translating these findings into therapeutic applications remains challenging. For instance, human trials using naked DNA injections (e.g., OTC cDNA) have shown variable immune responses, with one case of fatal immune activation reported (Raper et al., 2003). This underscores the variability of human immune responses and the challenges in translating findings from animal models to clinical applications. Such variability may also explain the inconsistent trends in MARCHF7 mRNA levels observed in peripheral blood samples from patients with different disease severities. Additionally, the small sample size in our study may have contributed to this observation.

In conclusion, in this study, we identified the host E3 ligases UBR5 and MARCHF7 as key regulators of SARS-CoV-2 nsp16, facilitating its ubiquitination and degradation. We observed a negative correlation between their expression levels and infection severity, highlighting their antiviral function. Moreover, both ligases effectively suppressed SARS-CoV-2 replication. These findings provide a foundation for developing UPS-targeted therapeutic strategies for COVID-19 treatment.

Materials and Methods

Reagents and antibodies

The drugs used in this study were as follows: MG132 (catalog no. S2619), Bafilomycin A1 (catalog no. S1413), Bortezomib (catalog no. S1013), Carfilzomib (catalog no. S2853) and Vinblastine (catalog no. S4504) were purchased from Selleck (Houston, TX, USA). Cycloheximide (catalog no. 66-81-9) was purchased from Sigma (Saint Louis, MO, USA). The antibodies used for immunoblotting analysis (IB) were as follows: anti-UBR5 mAb (Proteintech, Rosemont, IL, USA, catalog no. 66937-1-Ig), anti-MARCHF7 mAb (Santa Cruz Biotechnology, Dallas, TX, USA, catalog no. sc-166945), SARS-CoV-2 2’-O-ribose Methytransferase Antibody (nsp16) (Cell signaling, Danvers, Massachusetts, USA, catalog no. #70811), anti-Flag mAb (Sigma, catalog no. F1804), anti-tubulin mAb (Abcam, Cambridge, Cambridgeshire, UK, catalog no. ab11323), anti-hemagglutinin (anti-HA) pAb (Invitrogen, catalog no. 71-5500), anti-Myc pAb (Proteintech, Rosemont, IL, USA, catalog no. 16286-1-AP), and anti-GFP mAb (Abcam, catalog no. ab1218). Primary antibody: anti-COX5A pAb (Sangon Biotec, Shanghai, CHN, catalog no. D261450), anti-PDI-mAb (Proteintech, catalog no. 2E6A11) and Human GM130/GOLGA2 Antibody (R&D Systems, Minneapolis, MN, USA, catalog no. AF8199) were used for immunofluorescence, and SARS-CoV-2 nucleocapsid monoclonal antibody (mAb) (GeneTex, Irvine, CA, USA, catalog no. GTX635679) were used for Immunohistochemistry. Fluorescent secondary antibodies: goat anti-Rabbit IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, catalog no. A11001), and goat anti-Rabbit IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 568 (catalog no. A-11011) and immunohistochemical secondary antibodies: Streptavidin-Peroxidase Anti-Rabbit IgG kit (catalog no. KIT-9706) were purchased from Invitrogen and maixin (Fuzhou, CHN) respectively.

Cell lines and viruses

The cell lines used in this study include HEK293T cells (catalog no. CRL-11268), Hela cells (catalog no.CRM-CCL-2), Caco2 cells (catalog no. HTB-37). All cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and tested for mycoplasma. In order to conduct virus related experiments, stable cell lines including Caco2-Nint (stably expressing SARS-CoV-2 N gene) and 293T-ACE2 (stably expressing ACE2) were generated in our laboratory. The cells were cultured in a cell incubator containing 5% CO2 at 37°C using Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) containing 10% heat-inactivated fetal calf serum (FCS, GIBCO BRL, Grand Island, NY, USA), 100 mg/ml penicillin, and 100 ug/ml streptomycin to provide nutrition. We expanded SARS-CoV-2 virus-like-particles (Wuhan-Hu-1, MN908947, Clade:19A) with high replication capacity in Caco2-Nint using a biosafety level-2 (BSL-2) cell culture system. The virus infecting the cells include Omicron BA. 1 strain (human/CHN_CVRI-01/2022) and SARS-CoV-2 Wuhan strain (BetaCoV/Beijing/IME-BJ05-2020, GenBank access no. MT291831.1), and mouse adapted SARS-CoV-2/C57MA14 variant (GenBank: OL913104.1) used for experiments in mice were provided by Key Laboratory of Jilin Province for Zoonosis Prevention and Control, and all experiments for virus infection were performed in Biosafety level 3 (BSL-3) cell culture system.

Plasmids

Eukaryotic expression plasmids encoding SARS-CoV-2 nsp protein were provided by Professor Wang Peihui (Shandong University). The nsp16-HA expression vector was constructed by adding HA tag at the C-terminus. UBR5 (Gnen ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus. The cDNA of 293T cells was used as a template to construct MARCHF7(Gene ID: 64844) and its truncated mutant with a Myc tag at the C-terminus. All the above expression plasmids were inserted into the VR1012 vector. For immunofluorescence (IF) and Fluorescence Resonance Energy Transfer (FRET) experiments, pCDNA3.1-YFP vector (catalog no. 13033) and pECFP-C1 vector (catalog no. 6076-1) were purchased from Addgene and BD (Biosciences Clontech), and MARCHF7 or UBR5 was constructed on pECFP-C1, and nsp16 was constructed on pCDNA3.1-YFP. Single point mutants of nsp16 of different virus subtypes were obtained by point mutagenesis. Multi-site combined mutants were synthesized by Sangon Biotec Company. Human ubiquitin protein and its mutants have been previously described. Primers required for PCR were listed in Appendix Table S1.

RNA extraction and real-time quantitative PCR

We used the way of Trizol (Invitrogen, catalog no. 15596018CN) to extract RNA. The RNA was subsequently reverse transcribed using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA, catalog no. 4368814). Finally, PrimeScript RT Master Mix (Takara, Shiga, JPN, catalog no. RR036A) and relative real-time primer were used for qPCR on an Mx3005P instrument (Agilent Technologies, Stratagene, La Jolla, CA, USA). Amplification procedure of the target fragment is as follows: predenaturation at 95°C for 2min, denaturation at 95 °C for 30s, annealing at 55 °C for 30s, and extension at 72 °C for 30s, total of 40 cycles. Real-time primer sequences used in this study were shown in appendix table S1.

Immunoblotting analysis (IB)

Cells or supernatant cultured for a certain time were collected, cell precipitates were resuspended with lysis buffer (50 mM Tris–HCl [pH 7.8], 150 mM NaCl, 1.0% NP-40, 5% glycerol and 4 mM EDTA), 4xloading buffer was added (0.08M Tris [pH 6.8], 2.0%SDS, 10% glycerol, 0.1 M dithiothreitol, and 0.2% bromophenol blue), and samples were lysed by heating at 100℃ for 30min. After removal of cell debris by centrifugation, the supernatant was taken to separate proteins of different sizes by SDS-PAGE. The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, incubated overnight with the indicated primary antibody, and after 1 hour at room temperature with HRP-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, NJ, USA, catalog no. 115-035-062 for anti-mouse and 111-035-045 for anti-rabbit), the proteins were visualized through Ultrasensitive ECL Chemiluminescence Detection Kit (Proteintech, catalog no. B500024).

Stable cell line generation

We purchased the lentiviral vector pLKO.1-puro (catalog no. 8453) from Adggen. ShRNA targeting target genes were designed and synthesized by Sangon Biotech. After annealing, it was inserted into the vector. Lentivirus was generated by transfection with shRNA (cloned in pLKO.1) or control vector, RRE, VSVG and REV, and the cells were screened with 1/1000 puromycin (3 μg/ml, Sigma, catalog no. P8833) after infection 48 hours. The knockdown efficiency was detected by RT-qPCR or IB. The shRNA target sequences used in this study are shown in appendix table S1.

RNAi

Short interfering RNA (siRNA) used in this study were purchased from RiboBio Co. Ltd. (Guangzhou, CHN). The siRNA sequences for knockdown of MARCHF7 or UBR5 are provided in appendix table S1. The siRNA was transfected into the cells by transfection reagent Lipofectamine 2000 (Invitrogen, catalog no. 11668-019), and the corresponding plasmids were transfected 24 hours later using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA, catalog no. L3000-008). The expression of target genes was detected by RT-qPCR or immunoblotting (IB) analysis 48 hours later.

Immunoprecipitation

After 10 hours of MG132 (10 µM) treatment, the cells were harvested, resuspended in 1ml lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40) containing protease inhibitor (Roche, catalog no. 11697498001), placed on the shaker oscillator for 4 hours to lyse, then centrifuged to remove cell debris, and the supernatant was incubated with the antibody and protein G agarose beads (Roche, Basel, Basel-City, CH, catalog no. 11243233001) at 4°C overnight. Protein G agarose beads was washed 6-8 times with wash buffer (20 mM Tris-HCL [pH 7.5], 100 mM NaCl, 0.1 mM EDTA, and 0.05% Tween 20) at 4°C, centrifuged at 800 g for 1 min. The proteins were eluted by adding loading buffer (0.08 M Tris [pH 6.8], 2.0% SDS, 10% glycerol, 0.1 M dithiothreitol, and 0.2% bromophenol blue), the samples were boiled at 100°C for 10min to elate the proteins. The lysates and immunoprecipitations were detected by IB.

Mass spectrometry (MS)

HEK293T were transfected with SARS-CoV-2-nsp16-Flag for 36 hours, and then harvested after 10 hours of treatment with MG132 or DMSO. Proteins were enriched by co-immunoprecipitation assay. And the elutions were analyzed by MS. MS analysis was performed by the national center for protein science (Beijing, CHN).

Immunofluorescence

At 48 hours after transfection, the solution was discarded and cells were washed twice with preheated PBS for 5min each, followed by fixation with 4% paraformaldehyde at 37°C for 10min. After three washes with PBS, permeabilized with 0.2% Triton X-100 for 5 min at 37°C, and then immediately followed by blocking with 10% fetal bovine serum at room temperature for 1 hour. The blocked cells were incubated overnight with the indicated primary antibodies. The next day, after three washes with PBS, the cells were incubated with corresponding secondary antibodies for 1 hour at room temperature in the dark. The nuclei were stained with DAPI (49,6-diamidino-2-phenylindole, Sigma, catalog no. 9542) and then stored in 90% glycerol. The fluorescence was detected by a laser scanning confocal microscope (FV 3000, Olympus, Tokyo, Japan).

Viral infectivity assay

The Caco2 cells were transfected with siRNA by Lipofectamine RNAiMAX Reagent (Invitrogen, catalog no. 13778150) to knockdown UBR5 or MARCHF7 and infected with Wuhan strain (MOI=0.01) or Omicron-BA.1 strain (MOI=0.001) at 24 hours after transfection. After two hours of infection, the Caco2 cells were cultured in fresh medium containing 2% fetal bovine serum for 48 hours. The cells and supernatants were collected. Viral levels were characterized by RT-qPCR and IB analysis of viral structural proteins. The overexpression plasmid UBR5-Myc, MARCHF7-Myc and nsp16-Flag was transfected in 293T-ACE2 by Lipofectamine 3000 Reagent and infected with virus 24 hours later.

Mouse lines and infections

BALB/C mice, 6 weeks, were purchased from Charles River Laboratories, Beijing. Mice were cultured in groups according to experimental groups. Our study examined male and female animals, and similar findings are reported for both sexes. To reduce animal suffering, all welfare and experimental procedures were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals and relevant ethical regulations during the experiment. The mice were randomly divided into 6 groups and injected with 40 µg/500 µl MARCHF7 or UBR5 plasmids via the tail vein at high pressure. Three groups of mice were infected 50ul with SARS-CoV-2 isolates by nasal challenge at a dose of 105.5 TCID50/mL, three groups were not infected with virus as control, and the empty vector injection group was also used as control.

Immunohistochemical analysis

Three mice in each group, a total of 18 mice, were sacrificed after anesthesia. The lungs of mice were collected and fixed in 4% paraformaldehyde fixative. After 2 days, the lungs were progressively dehydrated through different concentrations of ethanol solution, transparently treated, soaked in xylene, and finally embedded in paraffin. Some thin sections obtained through a microtome for hematoxylin and eosin (H&E) staining. The other fraction was incubated with 3% hydrogen peroxide for 5-10 min at room temperature to eliminate endogenous peroxidase activity. SARS-CoV-2 nucleocapsid monoclonal antibody (mAb) (GeneTex, catalog no. GTX635679) and a Streptavidin-Peroxidase Anti-Rabbit IgG kit (Maixin, Fuzhou, CHN, catalog no. KIT-9706) was used to quantify the viral level in lung tissue.

Statistical analysis

The statistical analyses used in the figures have been described in detail in the figure legends. All data were expressed as mean ± standard deviations (SDs). Statistical comparisons were performed using student’s t-test, one-way ANOVA, or repeated measurement ANOVA. The differences were statistically significant as follows: *P < 0.05, **P < 0.01, ***P < 0.001; ns is for no meaning

Ethics statement

Collection of inpatient blood was approved by the Ethics Committee of the First Hospital of Jilin University (21K105-001) in accordance with the guidelines and principles of WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. All study participants signed an informed consent form. PBMCS were obtained from 9 COVID-19 patients with mild disease, 6 patients with severe disease, and 5 patients with critical disease (Appendix table S2). All animal experiments were approved by the ethics committee of Research Unit of Key Technologies for Prevention and Control of Virus Zoonoses, Chinese Academy of Medical Sciences, Changchun Veterinary Research Institute, Chineses Academy of Agricultural Sciences (IACUC of AMMS-11-2020-006).

Figure legends

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 with BioRender.com and the agreement no. is VO27O5XHHC).

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-aa 1-542) or the HECT domain inactivated mutant of UBR5 (UBR5-ΔHECT) 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 later, 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.

Data availability

The authors declare that [the/all other] data supporting the findings of this study are available within the paper [and its supplementary information files]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identified PXD053961.

Acknowledgements

We thank C.Y. Dai for providing critical reagents.

Additional information

Funding information

This work was supported by funding from the National Natural Science Foundation of China (82341072 and 82272316 to ZWY, 82341062 to LZL), the National Key R&D Program of China (2021YFC2301900 and 2301904, 2023YFC2306603), the Science and Technology Department of Jilin Province (YDZJ202301ZYTS521 and YDZJ202201ZYTS587), the Key Laboratory of Molecular Virology, Jilin Province (20102209), and Bethune Project, Jilin University (2023B03). The funding sources were involved in study design, data collection and interpretation, and the decision to submit the work for publication.

Authors’ contributions

L.T.: Conceptualization; Data curation; Formal Analysis; Investigation; Methodology; Writing – original draft. Z.Z.: Methodology; Data curation; Writing – review & editing. Z.L.: Conceptualization; Data curation; Formal Analysis; Investigation; Methodology; Writing – review & editing; Funding acquisition. X.L., Z.L. and W.G.: Methodology; Resources. W.Z.: Conceptualization; Formal Analysis; Funding acquisition; Methodology; Project administration; Resources; Writing – review & editing.

Additional files

Appendix figures and tables