ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a severe global health crisis; its structural protein envelope (E) is critical for viral entry, budding, production, and induction of pathology which makes it a potential target for therapeutics against COVID-19. Here, we find that the E3 ligase RNF5 interacts with and catalyzes ubiquitination of E on the 63rd lysine, leading to its degradation by the ubiquitin-proteasome system (UPS). Importantly, RNF5-induced degradation of E inhibits SARS-CoV-2 replication and the RNF5 pharmacological activator Analog-1 alleviates disease development in a mouse infection model. We also found that RNF5 is distinctively expressed in different age groups and in patients displaying different disease severity, which may be exploited as a prognostic marker for COVID-19. Furthermore, RNF5 recognized the E protein from various SARS-CoV-2 strains and SARS-CoV, suggesting that targeting RNF5 is a broad-spectrum antiviral strategy. Our findings provide novel insights into the role of UPS in antagonizing SARS-CoV-2 replication, which opens new avenues for therapeutic intervention to combat the COVID-19 pandemic.
Subject(s)
COVID-19 , Ubiquitin-Protein Ligases , Animals , Mice , Humans , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , SARS-CoV-2/metabolism , COVID-19/genetics , Pandemics , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Ubiquitin/metabolism , DNA-Binding Proteins/metabolism , Membrane ProteinsABSTRACT
Porcine epidemic diarrhea (PED) indicates the disease of the acute and highly contagious intestinal infection due to porcine epidemic diarrhea virus (PEDV), with the characteristics of watery diarrhea, vomiting, and dehydration. One of the reasons for diarrhea and death of piglets is PEDV, which leads to 100% mortality in neonatal piglets. Therefore, it is necessary to explore the interaction between virus and host to prevent and control PEDV. This study indicated that the host protein, pre-mRNA processing factor 19 (PRPF19), could be controlled by the signal transducer as well as activator of transcription 1 (STAT1). Thus, PEDV replication could be hindered through selective autophagy. Moreover, PRPF19 was found to recruit the E3 ubiquitin ligase MARCH8 to the N protein for ubiquitination. For the purpose of degradation, the ubiquitin N protein is acknowledged by the cargo receptor NDP52 and transported to autolysosomes, thus inhibiting virus proliferation. To conclude, a unique antiviral mechanism of PRPF19-mediated virus restriction was shown. Moreover, a view of the innate immune response and protein degradation against PEDV replication was provided in this study. IMPORTANCE The highly virulent porcine epidemic diarrhea virus (PEDV) emerged in 2010, and causes high mortality rates in newborn pigs. There are no effective and safe vaccines against the highly virulent PEDV. This virus has caused devastating economic losses in the pork industry worldwide. Studying the relationship between virus and host antiviral factors is important to develop the new antiviral strategies. This study identified the pre-mRNA processing factor 19 (PRPF19) as a novel antiviral protein in PEDV replication and revealed its viral restriction mechanisms for the first time. PRPF19 recruited the E3 ubiquitin ligase MARCH8 to the PEDV N protein for ubiquitination, and the ubiquitin N protein was acknowledged by the cargo receptor NDP52 and transported to autolysosomes for degradation. Our findings provide new insights in host antiviral factors PRPF19 that regulate the selective autophagy protein degradation pathway to inhibit PEDV replication.
Subject(s)
Capsid Proteins , Coronavirus Infections , Porcine epidemic diarrhea virus , Swine Diseases , Animals , Capsid Proteins/metabolism , Coronavirus Infections/immunology , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Porcine epidemic diarrhea virus/physiology , Swine , Swine Diseases/immunology , Swine Diseases/virology , Ubiquitin-Protein Ligases/metabolism , Ubiquitins , Virus Replication/genetics , Nuclear Proteins/metabolism , AutophagyABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal pathogen of the ongoing global pandemic of coronavirus disease 2019 (COVID-19). Loss of smell and taste are symptoms of COVID-19, and may be related to cilia dysfunction. Here, we found that the SARS-CoV-2 ORF10 increases the overall E3 ligase activity of the CUL2ZYG11B complex by interacting with ZYG11B. Enhanced CUL2ZYG11B activity by ORF10 causes increased ubiquitination and subsequent proteasome-mediated degradation of an intraflagellar transport (IFT) complex B protein, IFT46, thereby impairing both cilia biogenesis and maintenance. Further, we show that exposure of the respiratory tract of hACE2 mice to SARS-CoV-2 or SARS-CoV-2 ORF10 alone results in cilia-dysfunction-related phenotypes, and the ORF10 expression in primary human nasal epithelial cells (HNECs) also caused a rapid loss of the ciliary layer. Our study demonstrates how SARS-CoV-2 ORF10 hijacks CUL2ZYG11B to eliminate IFT46 and leads to cilia dysfunction, thereby offering a powerful etiopathological explanation for how SARS-CoV-2 causes multiple cilia-dysfunction-related symptoms specific to COVID-19.
Subject(s)
Cilia , SARS-CoV-2 , Ubiquitin-Protein Ligases , Animals , Cells, Cultured , Cilia/metabolism , Cilia/pathology , Cytoskeletal Proteins , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Mice , SARS-CoV-2/pathogenicity , Smell , Ubiquitin-Protein Ligases/metabolismABSTRACT
Through a screen that combines functional and evolutionary analyses, we identified tripartite motif protein (Trim69), a poorly studied member of the Trim family, as a negative regulator of HIV-1 infection in interferon (IFN)-stimulated myeloid cells. Trim69 inhibits the early phases of infection of HIV-1, but also of HIV-2 and SIVMAC in addition to the negative and positive-strand RNA viruses vesicular stomatitis virus and severe acute respiratory syndrome coronavirus 2, with magnitudes that depend on the combination between cell type and virus. Mechanistically, Trim69 associates directly to microtubules and its antiviral activity is linked to its ability to promote the accumulation of stable microtubules, a program that we uncover to be an integral part of antiviral IFN-I responses in myeloid cells. Overall, our study identifies Trim69 as the antiviral innate defense factor that regulates the properties of microtubules to limit viral spread and highlights the cytoskeleton as an unappreciated battleground in the host-pathogen interactions that underlie viral infections.
Subject(s)
HIV Infections , Tripartite Motif Proteins , Ubiquitin-Protein Ligases , Virus Replication , Humans , Immunity, Innate , Interferons/immunology , Microtubules/metabolism , Tripartite Motif Proteins/genetics , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , HIV Infections/immunologyABSTRACT
Ubiquitination is a highly conserved and fundamental posttranslational modification (PTM) in all eukaryotes regulating thousands of proteins. The RING (really interesting new gene) finger (RNF) protein, containing the RING domain, exerts E3 ubiquitin ligase that mediates the covalent attachment of ubiquitin (Ub) to target proteins. Multiple reviews have summarized the critical roles of the tripartite-motif (TRIM) protein family, a subgroup of RNF proteins, in various diseases, including cancer, inflammatory, infectious, and neuropsychiatric disorders. Except for TRIMs, since numerous studies over the past decades have delineated that other RNF proteins also exert widespread involvement in several diseases, their importance should not be underestimated. This review summarizes the potential contribution of dysregulated RNF proteins, except for TRIMs, to the pathogenesis of some diseases, including cancer, autoimmune diseases, and neurodegenerative disorder. Since viral infection is broadly involved in the induction and development of those diseases, this manuscript also highlights the regulatory roles of RNF proteins, excluding TRIMs, in the antiviral immune responses. In addition, we further discuss the potential intervention strategies targeting other RNF proteins for the prevention and therapeutics of those human diseases.
Subject(s)
Neoplasms , Ubiquitin-Protein Ligases , Humans , Neoplasms/genetics , Tripartite Motif Proteins/genetics , Tripartite Motif Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/geneticsABSTRACT
In addition to investigating the virology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), discovering the host-virus dependencies are essential to identify and design effective antiviral therapy strategy. Here, we report that the SARS-CoV-2 entry receptor, ACE2, conjugates with small ubiquitin-like modifier 3 (SUMO3) and provide evidence indicating that prevention of ACE2 SUMOylation can block SARS-CoV-2 infection. E3 SUMO ligase PIAS4 prompts the SUMOylation and stabilization of ACE2, whereas deSUMOylation enzyme SENP3 reverses this process. Conjugation of SUMO3 with ACE2 at lysine (K) 187 hampers the K48-linked ubiquitination of ACE2, thus suppressing its subsequent cargo receptor TOLLIP-dependent autophagic degradation. TOLLIP deficiency results in the stabilization of ACE2 and elevated SARS-CoV-2 infection. In conclusion, our findings suggest selective autophagic degradation of ACE2 orchestrated by SUMOylation and ubiquitination as a potential way to combat SARS-CoV-2 infection.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Autophagy , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Sumoylation , Ubiquitin-Protein Ligases/metabolismABSTRACT
Within the past 2 decades, three highly pathogenic human coronaviruses have emerged, namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The health threats and economic burden posed by these tremendously severe coronaviruses have paved the way for research on their etiology, pathogenesis, and treatment. Compared to SARS-CoV and SARS-CoV-2, MERS-CoV genome encoded fewer accessory proteins, among which the ORF4b protein had anti-immunity ability in both the cytoplasm and nucleus. Our work for the first time revealed that ORF4b protein was unstable in the host cells and could be degraded by the ubiquitin proteasome system. After extensive screenings, it was found that UBR5 (ubiquitin protein ligase E3 component N-recognin 5), a member of the HECT E3 ubiquitin ligases, specifically regulated the ubiquitination and degradation of ORF4b. Similar to ORF4b, UBR5 can also translocate into the nucleus through its nuclear localization signal, enabling it to regulate ORF4b stability in both the cytoplasm and nucleus. Through further experiments, lysine 36 was identified as the ubiquitination site on the ORF4b protein, and this residue was highly conserved in various MERS-CoV strains isolated from different regions. When UBR5 was knocked down, the ability of ORF4b to suppress innate immunity was enhanced and MERS-CoV replication was stronger. As an anti-MERS-CoV host protein, UBR5 targets and degrades ORF4b protein through the ubiquitin proteasome system, thereby attenuating the anti-immunity ability of ORF4b and ultimately inhibiting MERS-CoV immune escape, which is a novel antagonistic mechanism of the host against MERS-CoV infection. IMPORTANCE ORF4b was an accessory protein unique to MERS-CoV and was not present in SARS-CoV and SARS-CoV-2 which can also cause severe respiratory disease. Moreover, ORF4b inhibited the production of antiviral cytokines in both the cytoplasm and the nucleus, which was likely to be associated with the high lethality of MERS-CoV. However, whether the host proteins regulate the function of ORF4b is unknown. Our study first determined that UBR5, a host E3 ligase, was a potential host anti-MERS-CoV protein that could reduce the protein level of ORF4b and diminish its anti-immunity ability by inducing ubiquitination and degradation. Based on the discovery of ORF4b-UBR5, a critical molecular target, further increasing the degradation of ORF4b caused by UBR5 could provide a new strategy for the clinical development of drugs for MERS-CoV.
Subject(s)
Coronavirus Infections , Host Microbial Interactions , Middle East Respiratory Syndrome Coronavirus , Proteolysis , Ubiquitin-Protein Ligases , Ubiquitination , Viral Proteins , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cytokines/immunology , Humans , Immunity, Innate , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/metabolism , Molecular Targeted Therapy , Proteasome Endopeptidase Complex/metabolism , Severe acute respiratory syndrome-related coronavirus , SARS-CoV-2 , Ubiquitin-Protein Ligases/metabolism , Ubiquitins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Virus ReplicationABSTRACT
The E3 ligase TRIM7 has emerged as a critical player in viral infection and pathogenesis. However, the mechanism governing the TRIM7-substrate association remains to be defined. Here we report the crystal structures of TRIM7 in complex with 2C peptides of human enterovirus. Structure-guided studies reveal the C-terminal glutamine residue of 2C as the primary determinant for TRIM7 binding. Leveraged by this finding, we identify norovirus and SARS-CoV-2 proteins, and physiological proteins, as new TRIM7 substrates. Crystal structures of TRIM7 in complex with multiple peptides derived from SARS-CoV-2 proteins display the same glutamine-end recognition mode. Furthermore, TRIM7 could trigger the ubiquitination and degradation of these substrates, possibly representing a new Gln/C-degron pathway. Together, these findings unveil a common recognition mode by TRIM7, providing the foundation for further mechanistic characterization of antiviral and cellular functions of TRIM7.
Subject(s)
COVID-19 , Ubiquitin-Protein Ligases , Humans , Ubiquitin-Protein Ligases/metabolism , Glutamine/metabolism , SARS-CoV-2 , Ubiquitination , Antiviral Agents , Tripartite Motif Proteins/metabolismABSTRACT
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), triggers enhanced accumulation of lipids to generate foamy macrophages (FMs). This process has been often attributed to the surge in the expression of lipid influx genes with a concomitant decrease in those involved in lipid efflux. Here, we define an Mtb-orchestrated modulation of the ubiquitination of lipid accumulation markers to enhance lipid accretion during infection. We find that Mtb infection represses the expression of the E3 ubiquitin ligase, ITCH, resulting in the sustenance of key lipid accrual molecules viz. ADRP and CD36, that are otherwise targeted by ITCH for proteasomal degradation. In line, overexpressing ITCH in Mtb-infected cells was found to suppress Mtb-induced lipid accumulation. Molecular analyses including loss-of-function and ChIP assays demonstrated a role for the concerted action of the transcription factor YY1 and the arginine methyl transferase PRMT5 in restricting the expression of Itch gene by conferring repressive symmetrical H4R3me2 marks on its promoter. Consequently, siRNA-mediated depletion of YY1 or PRMT5 rescued ITCH expression, thereby compromising the levels of Mtb-induced ADRP and CD36 and limiting FM formation during infection. Accumulation of lipids within the host has been implicated as a pro-mycobacterial process that aids in pathogen persistence and dormancy. In line, we found that perturbation of PRMT5 enzyme activity resulted in compromised lipid levels and reduced mycobacterial survival in mouse peritoneal macrophages (ex vivo) and in a therapeutic mouse model of TB infection (in vivo). These findings provide new insights into the role of PRMT5 and YY1 in augmenting mycobacterial pathogenesis. Thus, we posit that our observations could help design novel adjunct therapies and combinatorial drug regimen for effective anti-TB strategies.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Animals , Lipids , Mice , Mycobacterium tuberculosis/genetics , Protein-Arginine N-Methyltransferases , Tuberculosis/genetics , Tuberculosis/therapy , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Ubiquitin is a small protein that is conjugated to target proteins to signal a great number of critical biological processes. Impaired ubiquitin signaling and defects in the ubiquitin proteasome system (UPS) surveillance are implicated in many human diseases, including cancer. Characterization of the physiological roles of UPS components and their regulatory mechanisms is therefore vital for the identification of therapeutic targets and the development of tools and paradigms to better understand and treat human diseases. In this Special Issue, we assembled seven original research and review articles to provide insights on the multifaceted role of the UPS in pathogenesis and disease, covering the areas of molecular and cellular mechanisms of UPS enzymes, biochemical and biophysical characterization strategies, drug development, and targeted protein degradation.
Subject(s)
Neoplasms , Ubiquitin , Humans , Neoplasms/genetics , Proteasome Endopeptidase Complex/metabolism , Signal Transduction , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Wang et al. report in this issue (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202108015) that the SARS-CoV-2 protein ORF10 increases the activity of the E3 ligase CUL2ZYG11B, leading to the degradation of multiple ciliary proteins. The resulting loss of cilia may facilitate the spread of SARS-CoV-2 in the respiratory tree.
Subject(s)
COVID-19 , Cilia , Ubiquitin-Protein Ligases , COVID-19/pathology , Cell Cycle Proteins , Cilia/pathology , Cullin Proteins , Genes, Viral , Humans , Proteins/metabolism , SARS-CoV-2 , Ubiquitin-Protein Ligases/metabolismABSTRACT
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a major threat to human health. As a unique putative protein of SARS-CoV-2, the N-terminus of ORF10 can be recognized by ZYG11B, a substrate receptor of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we elucidated recognition mechanism of ORF10 N-terminus by ZYG11B through presenting the crystal structure of ZYG11B bound to ORF10 N-terminal peptide. Our work expands the current understanding of ORF10 interaction with ZYG11B, and may also inspire the development of novel therapies for COVID-19.
Subject(s)
COVID-19 , Cell Cycle Proteins , Open Reading Frames , Ubiquitin-Protein Ligases , COVID-19/metabolism , COVID-19/virology , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cullin Proteins , Humans , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
The p53-dependent ubiquitin ligase Pirh2 regulates a number of proteins involved in different cancer-associated processes. Targeting the p53 family proteins, Chk2, p27Kip1, Twist1 and others, Pirh2 participates in such cellular processes as proliferation, cell cycle regulation, apoptosis and cellular migration. Thus, it is not surprising that Pirh2 takes part in the initiation and progression of different diseases and pathologies including but not limited to cancer. In this review, we aimed to summarize the available data on Pirh2 regulation, its protein targets and its role in various diseases and pathological processes, thus making the Pirh2 protein a promising therapeutic target.
Subject(s)
Tumor Suppressor Protein p53 , Ubiquitin-Protein Ligases , Cell Cycle Checkpoints , Tumor Suppressor Protein p53/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Adenoviruses (AdVs) are widespread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver, and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID-19 vaccination. AdV vectors are physically and genetically stable and generally safe in humans. The particles have an icosahedral coat and a nucleoprotein core with a DNA genome. We describe the concept of AdV cell entry and highlight recent advances in cytoplasmic transport, uncoating, and nuclear import of the viral DNA. We highlight a recently discovered "linchpin" function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3 ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover mechanisms controlling capsid stability and premature uncoating and provide insight into nuclear transport of nucleic acids.
Subject(s)
Adenoviridae , COVID-19 , Animals , Humans , Active Transport, Cell Nucleus , Adenoviridae/genetics , Adenoviridae/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Proteasome Endopeptidase Complex/metabolism , Kinesins , COVID-19 Vaccines , Nuclear Pore/genetics , Nuclear Pore/metabolism , Capsid Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , Nucleoproteins/metabolism , Mammals/genetics , Mammals/metabolismABSTRACT
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.
Subject(s)
Apoptosis , Coronavirus Infections/metabolism , Middle East Respiratory Syndrome Coronavirus/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Viral Nonstructural Proteins/metabolism , A549 Cells , Cell Line , Computational Biology , Coronavirus Infections/physiopathology , Coronavirus Infections/virology , Epithelial Cells/physiology , Epithelial Cells/virology , HEK293 Cells , Host-Pathogen Interactions , HumansABSTRACT
Ubc13-catalyzed K63 ubiquitination is a major control point for immune signaling. Recent evidence has shown that the control of multiple immune functions, including chronic inflammation, pathogen responses, lymphocyte activation, and regulatory signaling, is altered by K63 ubiquitination. In this review, we detail the novel cellular sensors that are dependent on K63 ubiquitination for their function in the immune signaling network. Many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can target K63 ubiquitination to inhibit pathogen immune responses; we describe novel details of the pathways involved and summarize recent clinically relevant SARS-CoV-2-specific responses. We also discuss recent evidence that regulatory T cell (Treg) versus T helper (TH) 1 and TH17 cell subset regulation might involve K63 ubiquitination. Knowledge gaps that merit future investigation and clinically relevant pathways are also addressed.
Subject(s)
COVID-19 , Lysine , Humans , Lysine/metabolism , SARS-CoV-2 , Signal Transduction , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
The three classes of interferons (IFNs) share the ability to inhibit viral replication, activating cell transcriptional programs that regulate both innate and adaptive responses to viral and intracellular bacterial challenge. Due to their unique potency in regulating viral replication, and their association with numerous autoimmune diseases, the tightly orchestrated transcriptional regulation of IFNs has long been a subject of intense investigation. The protective role of early robust IFN responses in the context of infection with SARS-CoV-2 has further underscored the relevance of these pathways. In this viewpoint, rather than focusing on the downstream effects of IFN signaling (which have been extensively reviewed elsewhere), we will summarize the historical and current understanding of the stepwise assembly and function of factors that regulate IFNß enhancer activity (the "enhanceosome") and highlight opportunities for deeper understanding of the transcriptional control of the ifnb gene.
Subject(s)
Epigenesis, Genetic , Gene Expression Regulation , Host-Pathogen Interactions/physiology , Interferon-beta/genetics , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , DNA Methylation , Enhancer Elements, Genetic , Host-Pathogen Interactions/genetics , Humans , Influenza A Virus, H5N1 Subtype/pathogenicity , Interferon-beta/metabolism , Promoter Regions, Genetic , SARS-CoV-2/pathogenicity , Transcription, Genetic , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.
Subject(s)
Autophagy/physiology , Blood Proteins/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus/growth & development , Poly(A)-Binding Proteins/metabolism , Virus Replication/physiology , Animals , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Infectious bronchitis virus/growth & development , Murine hepatitis virus/growth & development , Nuclear Proteins/metabolism , Porcine epidemic diarrhea virus/growth & development , Proteolysis , Sp1 Transcription Factor/metabolism , Swine , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Vero CellsABSTRACT
SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.
Subject(s)
ADP-Ribosylation , COVID-19/virology , Interferons/metabolism , Neoplasm Proteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , SARS-CoV-2/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/metabolism , HumansABSTRACT
COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.