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. 2022 Feb;298(2):101584.
doi: 10.1016/j.jbc.2022.101584. Epub 2022 Jan 13.

Host E3 ligase HUWE1 attenuates the proapoptotic activity of the MERS-CoV accessory protein ORF3 by promoting its ubiquitin-dependent degradation

Yuzheng Zhou  1 , Rong Zheng  1 , Sixu Liu  1 , Cyrollah Disoma  1 , Ashuai Du  1 , Shiqin Li  1 , Zongpeng Chen  1 , Zijun Dong  2 , Yongxing Zhang  1 , Sijia Li  1 , Pinjia Liu  1 , Aroona Razzaq  1 , Xuan Chen  1 , Yujie Liao  1 , Siyi Tao  1 , Yuxin Liu  1 , Lunan Xu  1 , Qianjun Zhang  3 , Jian Peng  4 , Xu Deng  5 , Shanni Li  1 , Taijiao Jiang  6 , Zanxian Xia  7
Affiliations

Host E3 ligase HUWE1 attenuates the proapoptotic activity of the MERS-CoV accessory protein ORF3 by promoting its ubiquitin-dependent degradation

Yuzheng Zhou et al. J Biol Chem. 2022 Feb.

Abstract

With the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), coronaviruses have begun to attract great attention across the world. Of the known human coronaviruses, however, Middle East respiratory syndrome coronavirus (MERS-CoV) is the most lethal. Coronavirus proteins can be divided into three groups: nonstructural proteins, structural proteins, and accessory proteins. While the number of each of these proteins varies greatly among different coronaviruses, accessory proteins are most closely related to the pathogenicity of the virus. We found for the first time that the ORF3 accessory protein of MERS-CoV, which closely resembles the ORF3a proteins of severe acute respiratory syndrome coronavirus and SARS-CoV-2, has the ability to induce apoptosis in cells in a dose-dependent manner. Through bioinformatics analysis and validation, we revealed that ORF3 is an unstable protein and has a shorter half-life in cells compared to that of severe acute respiratory syndrome coronavirus and SARS-CoV-2 ORF3a proteins. After screening, we identified a host E3 ligase, HUWE1, that specifically induces MERS-CoV ORF3 protein ubiquitination and degradation through the ubiquitin-proteasome system. This results in the diminished ability of ORF3 to induce apoptosis, which might partially explain the lower spread of MERS-CoV compared to other coronaviruses. In summary, this study reveals a pathological function of MERS-CoV ORF3 protein and identifies a potential host antiviral protein, HUWE1, with an ability to antagonize MERS-CoV pathogenesis by inducing ORF3 degradation, thus enriching our knowledge of the pathogenesis of MERS-CoV and suggesting new targets and strategies for clinical development of drugs for MERS-CoV treatment.

Keywords: HUWE1; MERS-CoV; ORF3; apoptosis; degradation; ubiquitination.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
The ORF3 protein of MERS-CoV induces apoptosis in cells. A, HEK293T cells were transfected with the indicated plasmids expressing HA-tagged MERS-CoV accessory proteins. Forty-eight hours later, cells were collected for immunoblotting. B, HEK293T cells were cotransfected with empty vector or expression plasmids for MERS-CoV accessory proteins as indicated, together with IFNβ-luc and pRL-TK plasmids. Twenty-four hours after transfection, the cells were infected with Sendai virus (100 HAU/ml) for 12 h and lysed for dual luciferase assay. C, HEK293T cells transfected with indicated plasmids were infected with Sendai virus for 12 h. Total RNA was extracted, reverse transcribed, and analyzed by real-time PCR with primers specific for IFNβ. D and E, HEK293T cells were transfected with the indicated plasmids expressing Flag-tagged SARS-CoV ORF3a, MERS-CoV ORF3, and SARS-CoV-2 ORF3a. After 24 h, 90% cells were stained with Annexin V-FITC/PI for flow cytometric analysis (D), and the remaining cells were lysed for immunoblotting (E). F, the percentage of apoptotic cells were measured. Error bars indicate SD of technical triplicates. Statistical significance was calculated using unpaired, two-tailed Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. G, Calu3 cells were transfected with the indicated plasmids expressing MERS-CoV ORF3. After 24 h, cells were stained with Annexin V-FITC/PI for flow cytometric analysis. H, HEK293T cells were transfected with empty vector or Flag-MERS-CoV ORF3 as indicated. After 24 h, cells were subjected for immunoblotting using the indicated antibodies. Cells treated with staurosporine (STS), an apoptosis inducer, for 5 h were used as a positive control. HAU, hemagglutinating unit; IFNβ-luc, interferon beta promoter-driven firefly luciferase reporter; MERS-CoV, Middle East respiratory syndrome coronavirus; pRL-TK, HSV-thymidine kinase promoter-driven Renilla luciferase reporter; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.
Figure 2
Figure 2
MERS-CoV ORF3 is an unstable protein.A, the amino acid sequences of the specified genes were downloaded from NCBI and were compared by clustalW. The phylogenetic tree was constructed by MEGA. B, the protein structures of MERS-CoV ORF3 and SARS-CoV ORF3a were predicated by the iterative threading assembly refinement (I-TASSER) server. MERS-COV ORF3: green, SARS-CoV ORF3a: cyan. C, the structure alignment between ORF3a of SARS-CoV and SARS-CoV-2 was analyzed by PyMOL. SARS-CoV ORF3a: cyan, SARS-CoV-2 ORF3a: yellow. D, top 20 of KEGG terms enrichment. The interacting proteins of ORF3 were identified by mass spectrometry, and KEGG enrichment analysis was performed using the OmicShare tools, a free online platform. E, HEK293T cells transfected with the indicated plasmids were treated with cycloheximide (CHX, 100 μg/ml), and lysates were collected at the indicated times for immunoblotting. F and G, HEK293T cells were transfected with empty vector or increasing amounts of plasmids expressing MERS-CoV ORF3 as indicated. Twenty-four hours later, 90% cells were collected and stained with Annexin V-FITC/PI for flow cytometric analysis (F), and the remaining cells were lysed for immunoblotting (G). H, the percentage of apoptotic cells were measured. Error bars indicate SD of technical triplicates. Statistical significance was calculated using unpaired, two-tailed Student’s t test. ∗p < 0.05; ∗∗p < 0.01. KEGG, Kyoto Encyclopedia of Genes and Genomes; MERS-CoV, Middle East respiratory syndrome coronavirus; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.
Figure 3
Figure 3
ORF3 is degraded by ubiquitin–proteasome system.A, HEK293T cells overexpressing ORF3 protein were treated with DMSO, MG132 (20 μM), bortezomib (BTM, 10 μM), chloroquine (CQ, 20 μM), and NH4Cl (10 mM) for 8 h before collection. Then, ORF3 protein level was detected by immunoblotting. B, ORF3 protein levels normalized to β-actin were quantified by ImageJ. Results were shown as mean ± SD, n = 3 independent experiments. ∗∗∗p < 0.001, Student’s t test. C, HEK293T cells were transfected with the plasmids expressing ORF3 protein. Twelve hours later, cells were split equally into a 12-well plate. After 12 h, cells were then cotreated with MG132 (20 μM) and CHX (100 μg/ml) or DMSO and CHX (100 μg/ml) for 0, 2, 4, or 8 h. The protein level of ORF3 was analyzed by immunoblotting. D, ORF3 protein levels normalized to β-actin were quantified by ImageJ. Data were analyzed by GraphPad Prism 7 as mean ± SD, n = 3 independent experiments. ∗∗p < 0.01, two-way ANOVA test. E, A549, Calu3, and BEAS-2B cells were transfected with the plasmid containing HA-ORF3, split, and cotreated with MG132 (20 μM) and CHX (100 μg/ml) or DMSO and CHX (100 μg/ml) for 0, 4, 8, or 12 h. The protein level of ORF3 were analyzed by immunoblotting. F, ORF3 protein levels normalized to β-actin were quantified by ImageJ. Data were analyzed by GraphPad Prism 7 as mean ± SD, n = 3 independent experiments. ∗∗∗p < 0.001, two-way ANOVA test. G and H, HEK293T cells transfected with the indicated plasmids were treated with MG132 for 8 h before collection. The whole cell lysates were subjected to pulldown with anti-Flag beads and immunoblotting with anti-HA antibody to detect the polyubiquitin chains of ORF3. CHX, cycloheximide; DMSO, dimethyl sulfoxide.
Figure 4
Figure 4
ORF3 interacts with E3 ligase HUWE1. A, KEGG network was constructed based on KEGG enrichment analysis. B, the interacting proteins related to proteasome pathway, apoptosis, E3 ligase, and ubiquitin-mediated proteolysis were clustered as a protein–protein network. C, a heatmap of the E3 ligases and ubiquitin-mediated proteolysis were plotted based on the interacting proteins of MERS-CoV ORF3 protein. D, HEK293T cells were cotransfected with plasmids containing Flag-ORF3 and GFP-HUWE1. WCLs were precipitated with the anti-Flag beads. WCLs and precipitated proteins were detected by immunoblotting with indicated antibodies to analyze the interaction between ORF3 and HUWE1. E, HEK293T cells were transfected with plasmids containing Flag-ORF3. WCLs were precipitated with the anti-Flag beads to analyze the interaction between ORF3 and endogenous HUWE1. MERS-CoV, Middle East respiratory syndrome coronavirus; WCLs, whole cell lysates.
Figure 5
Figure 5
Proapoptotic activity of ORF3 is weakened by HUWE1-mediated degradation. A, HEK293T cells overexpressing the ORF3 protein were transfected with increasing amounts of plasmids containing GFP-HUWE1. Cells were collected 48 h after transfection, and the protein level of ORF3 was analyzed by immunoblotting. B, ORF3 protein levels normalized to β-actin were quantified by ImageJ. Data were analyzed by GraphPad Prism 7 as mean ± SD, n = 3 independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Student’s t test. C, HEK293T cells expressing ORF3 protein were transfected with vector or GFP-HUWE1 plasmids, treated with CHX, and collected at indicated time for immunoblotting to test the protein level of ORF3. D, quantification of ORF3 protein level was normalized to β-actin. Results were shown as mean ± SD, n = 3 independent experiments. ∗∗p < 0.01, two-way ANOVA test. E, HEK293T cells transfected with the indicated plasmids were treated with MG132 for 8 h before collection. The whole cell lysates were subjected to pulldown with anti-Flag beads and to detect the polyubiquitin chains of ORF3 by immunoblotting. F, HEK293T cells stably expressing shNC or shHUWE1 were transfected with ORF3-containing plasmid, treated with CHX, and collected at indicated time to test the protein level of ORF3. G, quantification of ORF3 protein level was normalized to β-actin. Results were shown as mean ± SD, n = 3 independent experiments. ∗∗∗p < 0.001, two-way ANOVA test. H, HEK293T cells stably expressing shNC and shHUWE1 were transfected with the ORF3 expressing plasmid and treated with MG132 for 8 h before collection. The whole cell lysates were subjected to pulldown and detect the polyubiquitin chains of ORF3. I, HEK293T cells overexpressing the ORF3 protein were transfected with increasing amounts of plasmids containing HA-HUWE1-C4341A. Cells were collected 48 h after transfection, and the protein level of ORF3 was analyzed by immunoblotting. J, HEK293T cells expressing ORF3 protein were transfected with vector or HA-HUWE1-C4341A plasmids, treated with CHX, and collected at indicated time for immunoblotting to test the protein level of ORF3. K, quantification of ORF3 protein level was normalized to β-actin. Results were shown as mean ± SD, n = 3 independent experiments. NS, not significant, two-way ANOVA test. L, HEK293T cells were transfected with indicated plasmids. Twenty-four hours later, cells were stained with Annexin V-FITC/PI for flow cytometric analysis. M, the percentage of the apoptotic cells was measured. Error bars indicate SD of technical triplicates. Statistical significance was calculated using unpaired, two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, NS, not significant.
Figure 6
Figure 6
Ubiquitination-resistant mutant could increase the stability of ORF3. A, the amino acid sequence of MERS-CoV ORF3 was downloaded from NCBI, and all the lysine residues were marked in red. B, the structure of ORF3 protein was visualized by PyMOL with two lysine residues highlighted in red. C, HEK293T cells were transfected with ORF3-expressing plasmid for 48 h. ORF3 protein was purified and analyzed by tandem mass spectrometry. One peptide containing glycine residue was identified, K45 (in red). D, HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 μM) for 8 h before collection. The whole cell lysates were subjected to pulldown with anti-Flag beads and later immunoblotting with indicated antibodies to detect the polyubiquitin chains of ORF3-WT and ORF3 mutants. E, single ubiquitination-resistant mutants, including K24R and K45R were constructed. HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 μM) for 8 h before collection. Cell lysates were used to detect the indicated protein level by immunoblotting. F, Quantification of ORF3 protein level was normalized to β-actin. Results were shown as mean ± SD, n = 3 independent experiments. ∗∗∗p < 0.001, NS, not significant, Student’s t test. G, HEK293T cells were transfected with plasmids expressing ORF3-WT, ORF3-K24R, and ORF3-K45R. Twelve hours later, cells were evenly divided into 12-well plates. After 24 h, cells were treated with CHX (100 μg/ml) and collected at indicated time to detect the protein level of ORF3. H, quantification of ORF3 protein level was normalized to β-actin. Results were shown as mean ± SD, n = 3 independent experiments. ∗∗∗, p < 0.001, NS, not significant, two-way ANOVA test. I, HEK293T cells overexpressing the ORF3-K45R protein were transfected with increasing amounts of plasmids containing GFP-HUWE1. Cells were collected 48 h after transfection of the GFP-HUWE1 plasmid, and the protein level of ORF3-K45R was analyzed by immunoblotting. J, HEK293T cells were transfected with indicated plasmids. Twenty-four hours later, cells were stained with Annexin V-FITC/PI for flow cytometric analysis. K, the percentage of the apoptotic cells was measured. Error bars indicate SD of technical triplicates. Statistical significance was calculated using unpaired, two-tailed Student’s t test. ∗∗p < 0.01, NS, not significant. CHX, cycloheximide; MERS-CoV, Middle East respiratory syndrome coronavirus.
Figure 7
Figure 7
A pattern diagram of HUWE1 mediating the ubiquitination and degradation of ORF3 to weaken its proapoptotic activity. DPP4, dipeptidyl peptidase 4; FADD, Fas-associated death domain; FAS, tumor necrosis actor receptor superfamily member 6.
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