Porcine reproductive and respiratory syndrome virus(PRRSV)is a major economically devastating pathogen that has evolved various strategies to evade innate immunity.Downregulation of antiviral interferon largely promot...Porcine reproductive and respiratory syndrome virus(PRRSV)is a major economically devastating pathogen that has evolved various strategies to evade innate immunity.Downregulation of antiviral interferon largely promotes PRRSV immunoevasion by utilizing cytoplasmic melanoma differentiation-associated gene 5(MDA5),a receptor that senses viral RNA.In this study,the downregulated transcription and expression levels of porcine MDA5 in PRRSV infection were observed,and the detailed mechanisms were explored.We found that the interaction between P62 and MDA5 is enhanced due to two factors:the phosphorylation modification of the autophagic receptor P62 by the upregulated kinase CK2αand the K63 ubiquitination of porcine MDA5 catalyzed by the E3 ubiquitinase TRIM21 in PRRSV-infected cells.As a result of these modifications,the classic P62-mediated autophagy is triggered.Additionally,porcine MDA5 interacts with the chaperonin containing TCP1 subunit 2(CCT2),which is enhanced by PRRSV nsp3.This interaction promotes the aggregate formation and autophagic clearance of MDA5-CCT2-nsp3 independently of ubiquitination.In summary,enhanced MDA5 degradation occurs in PRRSV infection via two autophagic pathways:the binding of MDA5 with the autophagy receptor P62 and the aggrephagy receptor CCT2,leading to intense innate immune suppression.The research reveals a novel mechanism of immune evasion in PRRSV infection and provides fundamental insights for the development of new vaccines or therapeutic strategies.展开更多
The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics.Drugging the multi-functional...The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics.Drugging the multi-functional papain-like protease(PLpro)domain of the viral nsp3 holds promise.However,none of the known coronavirus PLpro inhibitors has been shown to be in vivo active.Herein,we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity,including against the Sarbecoviruses(SARSCoV-1 and SARS-CoV-2),Merbecovirus(MERS-CoV),as well as the Alphacoronavirus(hCoV-229E and hCoVOC43).Importantly,F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice.F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage,as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity.Despite the significant difference of substrate recognition,mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue,whereas an allosteric inhibitor of MERSPLpro interacting with its 271E position.Our proof-ofconcept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anticoronavirus agents.The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.展开更多
The coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection is currently a global pandemic.Extensive investigations have been performed to study the clinical an...The coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection is currently a global pandemic.Extensive investigations have been performed to study the clinical and cellular effects of SARS-CoV-2 infection.Mass spectrometry-based proteomics studies have revealed the cellular changes due to the infection and identified a plethora of interactors for all SARS-CoV-2 components,except for the longest non-structural protein 3(NSP3).Here,we expressed the full-length NSP3 proteins of SARS-CoV and SARS-CoV-2 to investigate their unique and shared functions using multi-omics methods.We conducted interactome,phosphoproteome,ubiquitylome,transcriptome,and proteome analyses of NSP3-expressing cells.We found that NSP3 plays essential roles in cellular functions such as RNA metabolism and immune response(e.g.,NF-κB signal transduction).Interestingly,we showed that SARS-CoV-2 NSP3 has both endoplasmic reticulum and mitochondrial localizations.In addition,SARS-CoV-2 NSP3 is more closely related to mitochondrial ribosomal proteins,whereas SARS-CoV NSP3 is related to the cytosolic ribosomal proteins.In summary,our integrative multi-omics study of NSP3 improves the understanding of the functions of NSP3 and offers potential targets for the development of anti-SARS strategies.展开更多
基金supported by the Tianjin Synthetic Biotechnology Innovation Capability Improvement Project in China(TSBICIP-KJGG-014)the key underprop project of Tianjin Science and Technology Bureau in China(20YFZCSN00340)to Jinhai Huang。
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)is a major economically devastating pathogen that has evolved various strategies to evade innate immunity.Downregulation of antiviral interferon largely promotes PRRSV immunoevasion by utilizing cytoplasmic melanoma differentiation-associated gene 5(MDA5),a receptor that senses viral RNA.In this study,the downregulated transcription and expression levels of porcine MDA5 in PRRSV infection were observed,and the detailed mechanisms were explored.We found that the interaction between P62 and MDA5 is enhanced due to two factors:the phosphorylation modification of the autophagic receptor P62 by the upregulated kinase CK2αand the K63 ubiquitination of porcine MDA5 catalyzed by the E3 ubiquitinase TRIM21 in PRRSV-infected cells.As a result of these modifications,the classic P62-mediated autophagy is triggered.Additionally,porcine MDA5 interacts with the chaperonin containing TCP1 subunit 2(CCT2),which is enhanced by PRRSV nsp3.This interaction promotes the aggregate formation and autophagic clearance of MDA5-CCT2-nsp3 independently of ubiquitination.In summary,enhanced MDA5 degradation occurs in PRRSV infection via two autophagic pathways:the binding of MDA5 with the autophagy receptor P62 and the aggrephagy receptor CCT2,leading to intense innate immune suppression.The research reveals a novel mechanism of immune evasion in PRRSV infection and provides fundamental insights for the development of new vaccines or therapeutic strategies.
基金partly supported by funding from Health@InnoHK,Innovation and Technology Commission,the Government of the Hong Kong Special Administrative RegionTheme-Based Research Scheme of the Research Grants Council(T11-709/21-N)+7 种基金the National Program on Key Research Project of China(2020YFA0707500 and 2020YFA0707504)Guangdong Natural Science Foundation(2022A1515010099)the University of Hong Kong Outstanding Young Researcher Awardthe University of Hong Kong Li Ka Shing Faculty of Medicine Research Output Prizethe High Level-Hospital Program,Health Commission of Guangdong Province,Chinathe research project of Hainan Academician Innovation Platform(YSPTZX202004)Emergency Key Program of Guangzhou Laboratory(EKPG22-01)the Swiss National Science Foundation,the National Research Programme Covid-19(No.4078P0_198290/1)。
文摘The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics.Drugging the multi-functional papain-like protease(PLpro)domain of the viral nsp3 holds promise.However,none of the known coronavirus PLpro inhibitors has been shown to be in vivo active.Herein,we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity,including against the Sarbecoviruses(SARSCoV-1 and SARS-CoV-2),Merbecovirus(MERS-CoV),as well as the Alphacoronavirus(hCoV-229E and hCoVOC43).Importantly,F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice.F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage,as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity.Despite the significant difference of substrate recognition,mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue,whereas an allosteric inhibitor of MERSPLpro interacting with its 271E position.Our proof-ofconcept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anticoronavirus agents.The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.
基金supported by the Guangdong Science and Technology Project,China(Grant Nos.2018B030306047 and 2020B1212060052)the National Natural Science Foundation of China(Grant Nos.31770889 and 31801180)+1 种基金the Guangzhou Science and Technology Project,China(Grant No.201904010469)the Guangzhou Regenerative Medicine and Health Guangdong Laboratory project,China(Grant No.2018GZR110104003)。
文摘The coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection is currently a global pandemic.Extensive investigations have been performed to study the clinical and cellular effects of SARS-CoV-2 infection.Mass spectrometry-based proteomics studies have revealed the cellular changes due to the infection and identified a plethora of interactors for all SARS-CoV-2 components,except for the longest non-structural protein 3(NSP3).Here,we expressed the full-length NSP3 proteins of SARS-CoV and SARS-CoV-2 to investigate their unique and shared functions using multi-omics methods.We conducted interactome,phosphoproteome,ubiquitylome,transcriptome,and proteome analyses of NSP3-expressing cells.We found that NSP3 plays essential roles in cellular functions such as RNA metabolism and immune response(e.g.,NF-κB signal transduction).Interestingly,we showed that SARS-CoV-2 NSP3 has both endoplasmic reticulum and mitochondrial localizations.In addition,SARS-CoV-2 NSP3 is more closely related to mitochondrial ribosomal proteins,whereas SARS-CoV NSP3 is related to the cytosolic ribosomal proteins.In summary,our integrative multi-omics study of NSP3 improves the understanding of the functions of NSP3 and offers potential targets for the development of anti-SARS strategies.
