key: cord-0991575-9dbtcjhx
authors: Zhou, Yuzheng; Zheng, Rong; Liu, Sixu; Disoma, Cyrollah; Du, Ashuai; Li, Shiqin; Chen, Zongpeng; Dong, Zijun; Zhang, Yongxing; Li, Sijia; Liu, Pinjia; Razzaq, Aroona; Chen, Xuan; Liao, Yujie; Tao, Siyi; Liu, Yuxin; Xu, Lunan; Zhang, Qianjun; Peng, Jian; Deng, Xu; Li, Shanni; Jiang, Taijiao; Xia, Zanxian
title: Host E3 ligase HUWE1 attenuates the pro-apoptotic activity of the MERS-CoV accessory protein ORF3 by promoting its ubiquitin-dependent degradation
date: 2022-01-13
journal: J Biol Chem
DOI: 10.1016/j.jbc.2022.101584
sha: 80dd959b3b51472244969e74583b1037b81435cc
doc_id: 991575
cord_uid: 9dbtcjhx

With the outbreak of SARS-CoV-2, coronaviruses have begun to attract great attention across the world. Of the known human coronaviruses, however, 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 SARS-CoV and SARS-CoV-2, has the ability to induce apoptosis in cells in a dose-dependent manner. Although the functions of these three proteins are similar, the amino acid sequences and structures differ. 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 SARS-CoV 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.

Coronaviruses are a group of related RNA viruses with envelope and a linear single positive strand genome (1) (2) (3) (4) (5) that infect a broad range of hosts, producing symptoms and diseases ranging from the common cold to severe and fatal illnesses. At present, seven coronaviruses can infect humans, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (3, 6, 7) . With the emergence of SARS-CoV-2 at the end of 2019, public attention to coronaviruses has increased in an unprecedented manner (8) (9) (10) . Its global spread has brought great threat to human health and social stability. As of October 8, 2021, the number of confirmed cases has reached nearly 214 million with a case fatality rate of 2% (11). Whilst public health measures and immunization programs have been effective to control the pandemic, there remains a need for a more in-depth and systematic research efforts to understand the pathogenesis of coronaviruses.

Of the seven coronaviruses with ability to infect humans, SARS-CoV, MERS-CoV and SARS-CoV-2 cause severe acute respiratory syndrome that could be fatal (12) (13) (14) . Among these three coronaviruses, MERS-CoV has the highest pathogenicity and mortality of about J o u r n a l P r e -p r o o f 35%. Since the first MERS-CoV outbreak in the Middle East in 2012, there have been confirmed 2468 cases and 851 deaths (15) (16) (17) (18) . Unlike SARS-CoV-2, the distribution of MERS-CoV had been limited with just few countries reporting laboratory-confirmed infections. But because of its high mortality, the pathogenicity of MERS-CoV proteins warrant further investigations since such efforts could provide new strategies and targets for the prevention and treatment of MERS-CoV and other emerging coronaviruses.

The typical genome of a coronavirus is around 30 kb. MERS-CoV genome is 30.12 kb, which can encode 25 viral proteins (19) . Coronavirus proteins can be divided into three categories: non-structural proteins (NSPs) that are translated by frameshift translation of polyproteins under the cleavage of proteases, structural proteins such as spike (S), envelope (E), membrane (M) and nucleocapsid (N), and accessory proteins of varying numbers in different coronavirus (20) . NSPs are generally involved in virus replication, including multiple enzymes such as Papain-like protease (PLpro) (21, 22) , main protease (Mpro) (23, 24) and RNA polymerase (25) . The structural proteins form the protein shell, protecting the genome of the virus and mediating the virus invasion in host cells (26) .

However, the number of accessory proteins in different coronaviruses varies. At present, the prevailing view is that accessory proteins are related to the pathogenicity of coronaviruses (27) (28) (29) (30) .

When the coronavirus invades the host, the viral protein encoded by the viral genome can affect the normal physiological functions or antagonize the immune response of the host .

This can be achieved through various mechanisms including inhibition of interferon J o u r n a l P r e -p r o o f production, interference with host cell metabolism, or induction of cell apoptosis (31) .

inflammasome by promoting TRAF3-dependent ubiquitination of p105 and apoptosisassociated speck-like protein containing a caspase recruitment domain (ASC) (32) . Another accessory protein of SARS-CoV, ORF9b, induced a strong intracellular autophagic effect (33) . ORF6, ORF8 and N protein of SARS-CoV-2 could inhibit the production of interferon-while ORF3a protein induced apoptosis in cells(37). For MERS-CoV, accessory proteins ORF4a, ORF4b, ORF5 and structural protein M, N demonstrated antiinterferon ability(38), among which ORF4b antagonized type I IFN production in both cytoplasm and nucleoplasm (39, 40) . In addition, MERS-CoV membrane protein triggered apoptosis by activating PERK signaling(41). In contrast to SARS-CoV and SARS-CoV-2, the pathogenesis of MERS-CoV is poorly studied despite its high mortality rate.

An evolutionary tug-of-war exists between a virus and its host. The host seeks to eliminate the pathogen, while the virus aims to achieve immune escape. During virus infection of host cells and disease development, ubiquitination plays a double-edged sword(42). On one hand, viral proteins can induce or promote the ubiquitination and degradation of certain key cytokines, thereby weakening the immune response of cells or tampering their normal functions. For example, N protein of SARS-CoV and SARS-Cov-2 could inhibit the ubiquitination of RIG-I, which in turn weaken its activation(43,44). SASR-CoV ORF8b induced degradation of IRF3 by the ubiquitin proteasome pathway(45), and SARS-CoV-2 M protein induced TBK1 ubiquitination and degradation(46). All of these viral proteins can lead to suppression of the innate immune pathway. Nonetheless, the host cell can also J o u r n a l P r e -p r o o f employ a multitude of mechanisms to achieve viral clearance. Host cells could use its own E3 ligases to specifically degrade certain viral proteins, thereby reducing virulence of the virus. For instance, NSP16 and ORF8b of SARS-CoV and ORF9c of SARS-CoV-2 could be degraded through ubiquitin proteasome system in the cells(47-49).

In our current work, we discovered that MERS-CoV ORF3 protein can induce cell apoptosis. By mass spectrometry analysis and experimental verification, we showed that ORF3 was an unstable protein that could be degraded through the ubiquitin proteasome pathway in the host cell. By examining ORF3 protein interactome, we found that the E3 ligase HUWE1 regulated the ubiquitin-mediated degradation of ORF3. Moreover, HUWE1

weakened the ability of ORF3 to induce apoptosis. Our work reveals a new mechanism for the host cells to antagonize MERS-CoV and provides a new target for drug treatment of MERS-CoV.

MERS-CoV encodes five accessory proteins: ORF3, ORF4a, ORF4b, ORF5 and ORF8b.

To test whether these proteins are related to the pathogenicity of the virus, we tested their anti-interferon ability by luciferase reporter assay and qPCR. Under the condition of Sendai Virus (SeV) stimulation, the four accessory proteins -excluding ORF3-can inhibit the production of IFN- ( Fig. 1A-C) . This suggested that ORF3 may play another role that was distinct from the roles of the other accessory proteins. Because ORF3a protein of SARS-J o u r n a l P r e -p r o o f CoV and SARS-CoV-2 were reported to promote cell apoptosis (37,50), we speculated whether the ORF3 protein of MERS-CoV had a similar function. We overexpressed ORF3a protein of SARS-CoV and SARS-CoV-2 as well as ORF3 protein of MERS-CoV in HEK293T cells. As shown in the results, ORF3 protein of MERS-CoV significantly increased the proportion of apoptotic cells, similar to ORF3a protein of SARS-CoV and SARS-CoV-2 ( Fig. 1D-F ).

MERS-CoV is a virus that mainly infects the respiratory tract. To explore how ORF3 could affect the respiratory tract, we overexpressed ORF3 protein in normal lung cell BEAS-2B and lung cancer cells Calu3 and A549. Consistent with our results from HEK293T, ORF3 enhanced apoptosis in all of these cell lines (Fig. 1G, S1A , B). To elucidate further this ORF3-induced apoptosis, we determined the levels and activation of proteins involved in apoptosis. We found that ORF3 induced the cleavage/activation of caspase-8 and caspase-3, whereas no apparent changes on BAX and Bcl-2 protein levels. This suggested that ORF3 induced cell death through the extrinsic pathway of apoptosis, not the mitochondrial pathway ( Fig. 1H , S1C). Overall, the results implied that the ORF3 protein of MERS-CoV induced pathological changes in the host by inducing apoptosis in cells.

While ORF3 of MERS-CoV-2 and ORF3a of SARS-CoV and SARS-CoV-2 had similar function, they differed in terms of amino acid sequences. The amino acid sequence homology was higher between ORF3a/b of SARS-CoV and SARS-CoV-2, which is not surprising given the close evolutionary relatedness of the two viruses (5) . But the ORF3 J o u r n a l P r e -p r o o f had low homology with ORF3a/b ( Fig. 2A) . To show structural homology of the three viral proteins, we utilized the resolved 3D structure of SARS-CoV-2 ORF3a deposited in public databases, and used I-TASSER to predict the structures of the SARS-CoV ORF3a and MERS-CoV ORF3 (Fig. 2B ). We found that the structure of both ORF3a were similar (Fig.   2C ). But when the 3D structures of the MERS-CoV ORF3 protein and the two ORF3a proteins were aligned, the results showed they were quite different (Fig. S2A, B) . This implied that compared to the ORF3a protein of SARS-CoV and SARS-CoV-2, ORF3 protein of MERS-CoV had a distinct structure. We then pulled down the ORF3 protein in HEK293T cells (Fig. S2C ). The proteins in the samples were identified by mass spectrometry (Table S1) Based on the earlier finding of pro-apoptotic effect of MERS-CoV ORF3, we overexpressed ORF3 in a dose-dependent manner and found that apoptosis was positively correlated with its expression (Fig. 2F-H) . These results suggested a new mechanism for the host to antagonize MERS-CoV by promoting ORF3 degradation to suppress its proapoptotic activity.

ORF3 is degraded by ubiquitin-proteasome system J o u r n a l P r e -p r o o f There are two common ways to clear damaged and potentially toxic proteins in cells: the ubiquitin-proteasome pathway and the lysosomal pathway. Based on KEGG enrichment analysis, we found that ORF3 might be degraded through the proteasome pathway. To validate this, cells expressing ORF3 were treated with proteasome inhibitors MG132 and bortezomib (BTM) and lysosomal inhibitors NH4Cl and chloroquine (CQ) for 12 hours, and then the changes of ORF3 protein levels were analyzed. The treatment with proteasome inhibitors MG132 and BTM significantly increased the level of ORF3 protein as compared to the control group and the lysosomal inhibitor treatment groups (Fig. 3A, B ). Next, we overexpressed ORF3 in HEK293T and co-treated with CHX and the various inhibitors to analyze ORF3 half-life. After treatment with MG132 and BTM, the protein level of ORF3 remained stable without degradation (Fig. 3C, D, Fig. S3A ), while the treatment of NH4Cl and CQ failed to stabilize ORF3 (Fig. S3B, C) . Similarly, the stability of ORF3 under the influence of MG132 was also seen in A549, Calu3 and BEAS-2B cells (Fig. 3E, F) . If a protein is degraded by the proteasome, it often needs to be ubiquitinated before it can be recognized by the proteasome prior to hydrolysis by proteolytic enzymes. We sought to examine the possibility that ORF3 could be ubiquitinated. When ubiquitin and ORF3 were co-overexpressed, the in vivo ubiquitination assay showed that ORF3 could conjugate with ubiquitin chains (Fig. 3G) . Also, ORF3 could conjugate with endogenous ubiquitin to form polyubiquitin chains (Fig. S3D ). Ubiquitin can form ubiquitin chains through their own lysine and the first methionine. The ubiquitin chains formed by K48 and K63 were most widely studied, in which the K48-linked ubiquitin chain regulated protein degradation. Therefore, we co-expressed ORF3 and wildtype ubiquitin or mutated ubiquitin (K48R and K63R) for in vivo ubiquitination experiments. The results showed that ORF3 mainly J o u r n a l P r e -p r o o f conjugated with K48 ubiquitin chain in cells (Fig. 3H) . These results proved that ORF3 can undergo ubiquitination, resulting to its degradation in host cells.

In the KEGG pathway enrichment analysis of ORF3 interacting proteins, we created a network map of the first 20 pathways to acquire an intuitive understanding of their relationship (Fig.4A ). Because we sought to demonstrate the ubiquitination-mediated degradation of ORF3, we took out the proteins related to the ubiquitin-proteasome pathway, such as proteasome subunits, ubiquitin mediated proteolysis and E3 ligases to create a protein interaction network diagram (Fig. 4B ). Meanwhile, based on the number of peptides of the interacting proteins identified by mass spectrometry, we made a heat map of the E3 ligases and ubiquitin mediated proteolysis identified in the control group and the experimental group (Fig. 4C) . As a key link in the ubiquitin proteasome system, E3 ligase can specifically recognize the target protein, and conjugate ubiquitin to the protein to form polyubiquitin chains, and finally make it recognized by the proteasome and degraded (51) .

In our results, five E3 ligases were identified to interact with ORF3 protein. Among them, HUWE1 had the most identified peptides and was most likely to be the key E3 ligase for ORF3 ubiquitination. To verify whether ORF3 can interact with HUWE1 at the cellular level, we overexpressed ORF3 and HUWE1 in HEK293T cells. ORF3 could bind to both exogenous and endogenous HUWE1 ( Fig. 4D and 4E ). These results confirmed the interaction of ORF3 and HUWE1 in cells, suggesting that HUWE1 was the key E3 ligase that regulated the ubiquitination and degradation of ORF3.

Since our previous result confirmed the interaction of ORF3 with HUWE1, we next investigated whether HUWE1 was indeed the E3 ligase that regulated the ubiquitination and degradation of ORF3. As the expression of HUWE1 increased, the expression level of ORF3 protein gradually decreased (Fig. 5A, B) , while the other identified E3 ligases (UBR5, TRIM33, Cullin5, Cullin3, and UBR4) had no effect on the stability of ORF3 ( shRNAs. The knockdown of HUWE1 increased ORF3 protein level (Fig. S4F ). Next, we tested the stability of ORF3 in control cells and stable HUWE1-knockdown cell lines, respectively. We found that compared with the control group, the reduction of HUWE1 stabilized ORF3 with no apparent degradation (Fig. 5F , G). And HUWE1 knockdown significantly reduced the ubiquitin chains (Fig. 5H ). In addition, we overexpressed HUWE1-C4341A, the ubiquitin ligase activity-deficient mutant, to test its effect on the stability of ORF4b. With the increase of HUWE1-C4341A expression, the protein level of ORF4b was not reduced (Fig. 5I) . Also, overexpression of C4341A didn't shorten the halflife of ORF4b (Fig. 5J , K). Furthermore, we performed an in vitro ubiquitination assay using GST-ORF3, HUWE1-HECT and the enzyme-deficient HUWE1-HECT-C4341A purified from bacteria (Fig. S4G) . These results confirmed that HUWE1 mediated ORF3 ubiquitination to direct its degradation by the proteasome. Unlike MERS-CoV ORF3, neither the ORF3a protein of SARS-CoV nor SARS-CoV-2 could interact with HUWE1 J o u r n a l P r e -p r o o f ( Fig. S4H) . Moreover, the overexpression of HUWE1 attenuated ORF3-induced apoptosis, while overexpression of HUWEI alone had little effect on apoptosis (Fig. 5L, M) . All in all, these results strongly indicated that HUWE1 was an E3 ligase that specifically regulated the degradation of ORF3 via ubiquitination.

Ubiquitination generally occurs on the lysine residues of the substrate protein (52) . ORF3 is a small accessory protein (only103 amino acids long) with only two lysine residues: K24 and K45 (Fig. 6A ). Using PyMOL to analyze the predicted structure of the ORF3 protein, we found that K24 was located on the random coil while K45 was located on the -fold;

both of which were located on the surface of the protein (Fig. 6B ). The tandem mass spectrometry results further showed that K45 could be a ubiquitination site (Fig. 6C ). To validate the MS data, we constructed ubiquitination-resistant mutants of ORF3-K24R and K45R. The polyubiquitin chains conjugated on the ORF3-K45R was significantly weaker than ORF3-WT and ORF3-K24R (Fig. 6D) . Further, ORF3-WT and ORF3-K24R protein levels with MG132 treatment were significantly higher than that of the control group without MG132, while the protein level of ORF3-K45R was not affected by MG132 (Fig.   6E, F) . Moreover, we went on to analyze the half-live of ORF3-WT, ORF3-K24R and ORF3-K45R. With CHX treatment, the protein levels of ORF3-WT and ORF3-K24R gradually decreased with time, while K45R remained relatively stable with a longer halflife (Fig. 6G, H) . To further determine that lysine 45 was the site in which HUWE1 conjugated ubiquitin, we overexpressed HUWE1 in a gradient and tested its effect on the ubiquitination-resistant mutant ORF3-K45R. The protein level of ORF3-K45R was not J o u r n a l P r e -p r o o f affected by the overexpression of HUWE1 (Fig. 6I) . Also, ORF3-K45R could still induced apoptosis and was not affected by HUWE1 (Fig. 6J, K) . These results demonstrated that K45 was the site regulated by HUWE1 to facilitate ubiquitination that affected ORF3 stability.

The SARS-CoV-2 outbreak is so far the most serious global health crisis in the recent history. Because of its cosmopolitan distribution as well as its detrimental impact to the healthcare system and economies, SARS-CoV-2 has received an unparalleled attention. In a way, the COVID-19 pandemic somehow led to increasing concerns on coronaviruses. Mpro(38, 55, 56) . In particular, our study has several highlights. First, we have identified for the first time that the viral protein ORF3 of MERS-CoV could induce apoptosis in host cells, revealing a new pathogenic mechanism that can be capitalized as new viral target protein for clinical treatment of MERS-CoV. Second, we found that ORF3 protein was unstable in host cells and could be degraded through the ubiquitin proteasome pathway. Third, among the interacting proteins of ORF3, HUWE1 was an E3 ligase with the most peptides identified by mass spectrometry, implying that HUWE1 was likely to be the specific E3 ligase. The interaction of HUWE1 and ORF3 was further validated experimentally and HUWE1 ubiquitinated ORF3 at lysine 45 to tag it for proteasomal degradation. Fourth, overexpression of HUWE1 can effectively attenuate the induction of apoptosis by ORF3-WT, but not the ubiquitination-resistant mutant ORF3-K45R. This suggested that HUWE1 was an anti-MERS-CoV factor in host cells that could effectively antagonize the pathogenicity of ORF3 (Fig. 7) . Whereas HUWE1 had been reported to play roles in various pathologies (57) (58) (59) (60) (61) (62) and was rarely studied as antiviral factor in host immunity (63) (64) (65) , this was the first time that HUWE1 was found to play an antagonistic role in the pathogenesis of coronavirus infection. Our discovery of ORF3 degradation by HUWE1 might offer therapeutic implications. One is, regulating ORF3 by ubiquitination and degradation may confer better resistance to viral release during its life cycle. This can be achieved by designing small compounds to enhance the binding between HUWE1 and ORF3, thereby increasing the degradation of ORF3. In recent years, an emerging protein hydrolysis targeting chimera (PROTAC) strategy has provided a new approach to drug development (80) (81) (82) . PROTAC has been widely used in drug development in the field of cancer and is expanding into areas such as immune abnormalities and neurodegenerative disease (83) . However, PROTAC strategies J o u r n a l P r e -p r o o f for viral infections are rarely addressed (84) . Since viral proteins are exogenous, degradation of viral proteins by PROTACs has higher targeting efficiency and specificity.

Moreover, the antigenic fragments generated by the degradation of proteins induced by PROTACs also effectively promotes the immune response of the organism (85) . Therefore, targeting the MERS-CoV ORF3 protein by PROTAC small molecule drugs will have more advantages and better prospects for clinical applications.

There were several limitations in this study that requires follow-up research. Firstly, ORF3

induced apoptosis through the extrinsic pathway and the KEGG enrichment analysis identified several host proteins in the apoptosis pathway to interact with ORF3.Whether ORF3 protein affects the functions of these proteins to induce apoptosis remains to be investigated. In addition, it has not been addressed whether the replicative capacity of MERS-CoV could be enhanced when expression of HUWE1 in host cell is repressed or when mutation is introduced at lysine K45 of ORF3. This would require experiments in BSL3. Live virus assays can also further clarify the antagonistic effect of HUWE1 as an antiviral host factor against MERS-CoV.

Our study reveals a novel mechanism for host antagonism to the pathogenicity of MERS-CoV. We found that MERS-CoV ORF3 protein can induce apoptosis in host cells. Further, ORF3 protein was unstable and can be degraded by the ubiquitin proteasome system, resulting in the inhibition of its ability to induce apoptosis. We uncovered the E3 ligase HUWE1 mediated the ubiquitination of ORF3 protein to facilitate its degradation. This was the first time that HUWE1 had been shown to play an antiviral role in host immunity. Apoptosis detection. The Flow cytometry assay was used to detect cell apoptosis. The cells seeded on the 6-well plates were transfected with indicated plasmids. After 36h, cells were washed with PBS for three times and collected by centrifugation at 1000 g for 5min.

The apoptosis assay was performed using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) and the steps were described as follows: Washed cells twice with cold PBS and then resuspend cells in 1X Annexin V binding buffer at 1x106 cells/ml. Transferred 150l of the solution to a 5ml culture tube. Then added 5μL of FITC Annexin V and 5μL

Propidium iodide staining solution. Gently vortexed the cells and incubated for 15min at room temperature (20-25°C) in the dark. Added 300l of binding buffer to each tube and performed Flow cytometry analysis within one hour.

Caspase-3/8/9 activity detection assays. The cells seeded on the plates were transfected with indicated plasmids. 5h before the collection, cells were treated with staurosporine (1M, Sigma) or DMSO, then were lysed for Western blot. The indicated primary antibodies were used to detect the full length and cleaved caspase3/8/9. Antibodies used in immunoblotting: anti-caspase3 (abcam, ab32351), anti-caspase8 (proteintech, 13423-1-AP), anti-caspase9 (proteintech, 10380-1-AP), anti-Bcl-2 (abcam, ab32124) and anti-BAX (abcam, ab32503).

Each experiment was repeated at least three times. All results were shown as the mean±s.d., Student's t-test (unpaired, two-tailed) was used to compare two independent groups, and two-way ANOVA test was performed for comparisons of multiple groups. All statistical analyses were performed with GraphPad Prism 7. P < 0.05 was considered statistically significant.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE repository with the dataset identifier PXD030024. All other data are included in this article and its supporting information.

This article contains supporting information. 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. WCL were precipitated with the anti-Flag beads to analyze the interaction between ORF3 and endogenous HUWE1. 

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Abbreviations The abbreviations used are: SARS-CoV, severe acute respiratory syndrome coronavirus

Middle East respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2

ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain

We like to thank Professor Wenjie Tan for the plasmids containing coronavirus proteins.We also thank Professor Genze Shao for providing the HUWE1-expressing plasmid.Shanghai Applied Protein Technology is acknowledged for Mass spectrometry, and the Nursing Development, Central South University is acknowledged for performing the Flow cytometry assay.