Multiple E3 ligases act as antiviral factors against SARS-CoV-2 via inducing the ubiquitination and degradation of ORF9b

Abstract

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.

Keywords: HUWE1; SARS-CoV-2 ORF9b; UBR4; UBR5; degradation.

Fig 1

Multiple E3 ubiquitin ligases specifically interact and regulate SARS-CoV-2 ORF9b stability. (A) A heatmap of 14 E3 ubiquitin ligases was plotted based on the interacting proteins of SARS-CoV-2 ORF9b as quantified by MS analysis. (B) Identification of the E3 ligases HUWE1, UBR4, and UBR5 mediated ORF9b degradation. Each siRNA of E3 ligases interacting ORF9b or siRNA targeting green fluorescent protein (GFP) as negative control plus the ORF9b-hemagglutinin (ORF9b-HA) expression vector were transfected into HEK293T cells for 48 h. Cells were harvested and analyzed with IB of anti-HA and anti-tubulin antibodies. (C and D) Cells were transfected with siRNA negative control, HUWE1 siRNA, UBR4 siRNA, UBR5 siRNA, or combinations of siRNA plus ORF9b-HA for 48 h. Cells were harvested and the knockdown of the E3 ligases and the protein level of ORF9b were confirmed by IB with the indicated antibodies.

Fig 2

HUWE1 and UBR5 induce the degradation of ORF9b in an enzyme activity-dependent manner. HEK293T cells were transfected with ORF9b-hemagglutinin (ORF9b-HA) and empty vector or HUWE1 wild-type (WT)-Myc (A) or UBR5 WT-Myc plasmid (B) for 48 h, and treated with or without 10 µM MG132 for 12 h prior to harvest and analyzed by IB with the indicated antibodies. (C) Schematic representation of HUWE1 and UBR5 protein structure and their mutants. The domain abbreviations used are as follows: ARM, armadillo repeat-containing domain; BH3, Bcl-2 homology 3 domain; HECT, homologous to the E6AP C-terminus; PABC, polyadenylate-binding protein C-terminal domain; UBA, ubiquitin-associated domain; WWE, Trp-Trp-Glu domain; ZnF, Zinc finger domain. (D) HEK293T cells were transfected with ORF9b-HA and empty vector or HUWE1 WT-Myc or HUWE1 C4341S-Myc mutant for 48 h. IB was performed with anti-HA, anti-Myc, and anti-tubulin. (E) HEK293T cells were transfected with ORF9b-HA and empty vector or UBR5 WT or UBR5 C2768A mutant for 48 h. ORF9b and UBR5 were analyzed with IB of anti-HA or anti-UBR5 or anti-Tubulin antibodies. (F) The interaction of ORF9b and HUWE1 WT-Myc or C4341S-Myc mutant. HUWE1 WT-Myc or C4341S-Myc mutant were co-transfected with ORF9b-HA into HEK293T cells. Cells were treated with 10 µM MG132 for 12 h prior to harvest. HUWE1 was immunoprecipitated with Myc antibody, and the associated ORF9b was detected with HA antibody. (G) The interaction of ORF9b and UBR5 WT or C2768A mutant. UBR5 WT or C2768A mutant were co-transfected with ORF9b-HA into HEK293T cells. Cells were treated with 10 µM MG132 for 12 h prior to harvest. UBR5 was immunoprecipitated with endogenous UBR5 antibody, and the associated ORF9b was detected with HA antibody.

Fig 3

siHUWE1, siUBR4, or siUBR5 alone or combination all reduced the polyubiquitination of ORF9b. (A and B) Negative control (NC) siRNA, siHUWE1, siUBR4, siUBR5, or combination of three siRNAs were co-transfected with ORF9b-hemagglutinin (HA) plus K48-only ubiquitin-Flag (Ub-K48-Flag) into HEK293T cells. Lysates of cells were subjected to Flag IP, and then analyzed by IB. (C) Quantification of ORF9b ubiquitination assay results was shown in (B). The intensity of the Ub band in IP lanes was quantified using ImageJ software and the siNC group was set to 100%. All data are representative of three independent experiments. The data are presented as means ± SD (***, P < 0.001).

Fig 4

Lysine 4 in ORF9b is ubiquitination site for HUWE1 and UBR4, and lysine 40 is for UBR5. (A–D) HEK293T cells were transfected with negative control (NC) siRNA or HUWE1 siRNA or UBR4 siRNA or UBR5 siRNA plus ORF9b-K4R, K40R, or KK4/40RR mutant as indicated. Knockdown of E3 ligases and the protein level of ORF9b were confirmed by IB with the indicated antibodies. (E–G) HEK293T cells were transfected with NC siRNA, siHUWE1, siUBR4, or siUBR5 plus ORF9b-hemagglutinin (HA) mutant and Ub-K48-Flag as indicated. Lysates of cells were subjected to Flag IP and analyzed by IB.

Fig 5

HUWE1 and UBR5 attenuate ORF9b-mediated IFN inhibition. (A–F) HEK293T cells were transfected with ORF9b WT-hemagglutinin (WT-HA) and empty vector VR1012 (VR), HUWE1 WT-Myc, HUWE1 C4341S-Myc mutant, UBR5 WT, or UBR5 C2768A mutant expressing plasmids for 24 h. Then, cells were transfected with RIG-I(N)-Myc for another 8 h. RT-qPCR was conducted to determine the mRNA levels of IFN-β (A), IL-29 (B), OAS2 (C), ISG15 (D), ISG56 (E), and CXCL10 (F). (G) Protein samples in Fig. 5A through F were analyzed using IB. (H–J) HEK293T cells were transfected with ORF9b KK4/40RR-HA mutant and empty vector VR1012 (VR), HUWE1 WT-Myc, or UBR5 WT-Myc expressing plasmids for 24 h. Then, cells were transfected with RIG-I(N)-Myc for another 8 h. RT-qPCR was conducted to determine the mRNA levels of ISG15 (H), ISG56 (I), and CXCL10 (J). (K) Protein samples in Fig. 5H through J were analyzed using IB. All data are representative of three independent experiments. The data are presented as means ± SD (ns, not significant, **, P < 0.01; ***, P < 0.001).

Fig 6

Knockdown of HUWE1, UBR4, or UBR5 further attenuated the mRNA levels of IFNs and ISGs stimulated by RIG-I(N) in cells infected with SARS-CoV-2. (A–F) Caco2 cells transfected with individual E3 ligase siRNA as indicated were transfected with RIG-I(N)-Myc for 8 h with and without Omicron strain infection at 0.001 multiplicity of infection (MOI）for 48 h. RT-qPCR was conducted to determine the mRNA levels of IFN-β (A), IL-29 (B), OAS2 (C), ISG15 (D), ISG56 (E), and CXCL10 (F). All data are representative of three independent experiments. The data are presented as means ± SD [ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with negative control (NC)].

Fig 7

The E3 ligases HUWE1, UBR4, and UBR5 suppressed SARS-CoV-2 viral replication. (A–E) Knockdown of any one of three E3 ligases promoted the replication of SARS-CoV-2 Omicron strain. Caco2 cells transfected with individual E3 ligase siRNA as indicated were infected with Omicron strain at 0.001 MOI for 48 h and harvested. RNAs of SARS-CoV-2 M and E were detected by RT-qPCR (A, B, and D), and the virus titer in supernatant (E). The expression of SARS-CoV-2 N of intracellular and cell-free supernatant, HUWE1, UBR4, UBR5, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected by IB (C). (F–J) HEK293T-ACE2 cells that stably expressed HUWE1, UBR5, or empty vector VR1012 were infected with Omicron strain at 0.001 MOI for 48 h, and cells and supernatants were harvested for the further detections. The expression of SARS-CoV-2 N of intracellular and cell-free supernatant, HUWE1, UBR5, and GAPDH were detected by IB (F). RNA of SARS-CoV-2 M and E were detected by RT-qPCR (G–I), and the virus titer in supernatant (J). All data are representative of three independent experiments. The data are presented as means ± SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 8

Induction of HUWE1, UBR4, and UBR5 by IFNs. (A to C) Caco2 cells were treated with the indicated IFNs (500 U/mL each) or the vehicle (phosphate-buffered saline) for 8 h, and the mRNA levels of HUWE1, UBR4, and UBR5 were analyzed by RT-qPCR. (D) Caco2 cells were treated with Omicron strain viral infection at 0.001 MOI for 48 h. The mRNA levels of HUWE1, UBR4, and UBR5 were analyzed by RT-qPCR. All data are representative of three independent experiments. The data are presented as means ± SD (ns, not significant; **, P < 0.01; ***, P < 0.001).

Fig 9

Pattern diagram of HUWE1, UBR4, and UBR5 as antiviral factors against SARS-CoV-2 by promoting degradation of ORF9b (created with BioRender.com.) E3, ubiquitin protein ligase, refers to HUWE1, UBR4, and UBR5; E2, ubiquitin-conjugating enzyme; Ub, ubiquitin; IFN, interferon; NF-κB, nuclear factor kappa B; IRF, interferon regulatory factor; ISG, interferon-stimulated gene.