The current pandemic of COVID-19 caused by a novel coronavirus,severe acute respiratory syndrome coronavirus-2(SARS-CoV-2),threatens human health around the world.Of particular concern is that bats are recognized as o...The current pandemic of COVID-19 caused by a novel coronavirus,severe acute respiratory syndrome coronavirus-2(SARS-CoV-2),threatens human health around the world.Of particular concern is that bats are recognized as one of the most potential natural hosts of SARS-CoV-2;however,coronavirus ecology in bats is still nascent.Here,we performed a degenerate primer screening and next-generation sequencing analysis of 112 bats,collected from Hainan Province,China.Three coronaviruses,namely bat betacoronavirus(Bat CoV)CD35,Bat CoV CD36 and bat alpha-coronavirus CD30 were identified.Bat CoV CD35 genome had 99.5%identity with Bat CoV CD36,both sharing the highest nucleotide identity with Bat Hp-betacoronavirus Zhejiang2013(71.4%),followed by SARS-CoV-2(54.0%).Phylogenetic analysis indicated that Bat CoV CD35 formed a distinct clade,and together with Bat Hp-betacoronavirus Zhejiang2013,was basal to the lineage of SARS-CoV-1 and SARS-CoV-2.Notably,Bat CoV CD35 harbored a canonical furin-like S1/S2 cleavage site that resembles the corresponding sites of SARS-CoV-2.The furin cleavage sites between CD35 and CD36 are identical.In addition,the receptor-binding domain of Bat CoV CD35 showed a highly similar structure to that of SARS-CoV-1 and SARS-CoV-2,especially in one binding loop.In conclusion,this study deepens our understanding of the diversity of coronaviruses and provides clues about the natural origin of the furin cleavage site of SARS-CoV-2.展开更多
Proteases are enzymes that cleave and hydrolyse the peptide bonds between two specific amino acid residues of target substrate proteins.Protease-controlled proteolysis plays a key role in the degradation and recycling...Proteases are enzymes that cleave and hydrolyse the peptide bonds between two specific amino acid residues of target substrate proteins.Protease-controlled proteolysis plays a key role in the degradation and recycling of proteins,which is essential for various physiological processes.Thus,solving the substrate identification problem will have important implications for the precise understanding of functions and physiological roles of proteases,as well as for therapeutic target identification and pharmaceutical applicability.Consequently,there is a great demand for bioinformatics methods that can predict novel substrate cleavage events with high accuracy by utilizing both sequence and structural information.In this study,we present Procleave,a novel bioinformatics approach for predicting protease-specific substrates and specific cleavage sites by taking into account both their sequence and 3D structural information.Structural features of known cleavage sites were represented by discrete values using a LOWESS data-smoothing optimization method,which turned out to be critical for the performance of Procleave.The optimal approximations of all structural parameter values were encoded in a conditional random field(CRF)computational framework,alongside sequence and chemical group-based features.Here,we demonstrate the outstanding performance of Procleave through extensive benchmarking and independent tests.Procleave is capable of correctly identifying most cleavage sites in the case study.Importantly,when applied to the human structural proteome encompassing 17,628 protein structures,Procleave suggests a number of potential novel target substrates and their corresponding cleavage sites of different proteases.Procleave is implemented as a webserver and is freely accessible at http://procleave.erc.monash.edu/.展开更多
The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL prot...The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL protease.This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein,and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus,Betacoronavirus and Gammacoronavirus.Strong residue preferences were observed at the neighboring positions of the cleavage sites.A random forest(RF)model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes,and the model achieved an AUC of 0.96 in cross-validations.The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts.It achieved an AUC of 0.95 and predicted correctly 80%of the cleavage sites.Then,1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model.These proteins were enriched in several GO terms related to the cytoskeleton,such as the microtubule,actin and tubulin.Finally,a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model.Overall,the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.展开更多
Since mid-2016,the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic(HP)phenotype in China,raising many concerns about public health and poultry industry.The insertion of a“KRTA”motif at hemag...Since mid-2016,the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic(HP)phenotype in China,raising many concerns about public health and poultry industry.The insertion of a“KRTA”motif at hemagglutinin cleavage site(HACS)occurred in the early stage of HP H7N9 variants.During the co-circulation,the HACS of HP-H7N9 variants were more polymorphic in birds and humans.Although HP-H7N9 variants,unlike the H5 subtype virus,exhibited the insertions of basic and non-basic amino acids,the underlying function of those insertions and substitutions remains unclear.The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China.Then,we generated six H7N9 viruses bearing the PEIPKGR/G,PEVPKGR/G,PEVPKRKRTAR/G,PEVPKGKRTAR/G,PEVPKGKRIAR/G,and PEVPKRKRR/G motifs.Interestingly,after the deletion of threonine and alanine(TA)at HACS,the H7N9 viruses manifested decreased thermostability and virulence in mice,and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence.By contrast,the insertion of non-basic amino acid isoleucine and alanine(IA)decreased the transmissibility in chickens and virulence in mice.Remarkably,the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens,suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice.The ongoing evolution of H7N9 increasingly threatens public health and poultry industry,so,its comprehensive surveillance and prevention of H7N9 viruses should be pursued.展开更多
The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1...The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1 contains the receptor-binding domain(RBD),while the S2 contains the hydrophobic fusion domain for the entry into the host cell.Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various c leavage sites.In this article,we review host proteases including furin,trypsin,transmembrane protease serine 2(TMPRSS2)and cathepsins in the activation of SARS-CoV-2 S.Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin.The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2,and the binding triggers further conformational changes and exposure of the S2'site to proteases such as type Il transmembrane serine proteases(TTPRs)including TMPRSS2.In the presence of TMPRSS2 on the target cells,SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane.In the absence of TMPRSS2,SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry.Additional host proteases involved in the cleavage of the S were discussed.This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2,and discussed the dual roles of such inhibitors in virus replication.展开更多
A novel artificial site specific cleavage reagent, with peptide nucleic acid (PNA) as sequence-recognizing moiety and cerium (IV) ions as “scissors” for cleaving target DNA, was synthesized. Subsequently, it was...A novel artificial site specific cleavage reagent, with peptide nucleic acid (PNA) as sequence-recognizing moiety and cerium (IV) ions as “scissors” for cleaving target DNA, was synthesized. Subsequently, it was employed in the cleavage of target 26-mer single-stranded DNA (ssDNA), which has 10-mer sequence complementary with PNA recognizer in the hybrids, under physiological conditions. Reversed-phase high-performance liquid chromatogram (RPHPLC) experiments indicated that the artificial site specific cleavage reagent could cleave the target DNA specifically.展开更多
基金the National Key R&D Pro-gram of China(2019YFC1200501)Research Units of Discovery of Un-known Bacteria and Function(2018RU010)the Golden Seed Program of Beijing Chaoyang Hospital(CYJZ202220).
文摘The current pandemic of COVID-19 caused by a novel coronavirus,severe acute respiratory syndrome coronavirus-2(SARS-CoV-2),threatens human health around the world.Of particular concern is that bats are recognized as one of the most potential natural hosts of SARS-CoV-2;however,coronavirus ecology in bats is still nascent.Here,we performed a degenerate primer screening and next-generation sequencing analysis of 112 bats,collected from Hainan Province,China.Three coronaviruses,namely bat betacoronavirus(Bat CoV)CD35,Bat CoV CD36 and bat alpha-coronavirus CD30 were identified.Bat CoV CD35 genome had 99.5%identity with Bat CoV CD36,both sharing the highest nucleotide identity with Bat Hp-betacoronavirus Zhejiang2013(71.4%),followed by SARS-CoV-2(54.0%).Phylogenetic analysis indicated that Bat CoV CD35 formed a distinct clade,and together with Bat Hp-betacoronavirus Zhejiang2013,was basal to the lineage of SARS-CoV-1 and SARS-CoV-2.Notably,Bat CoV CD35 harbored a canonical furin-like S1/S2 cleavage site that resembles the corresponding sites of SARS-CoV-2.The furin cleavage sites between CD35 and CD36 are identical.In addition,the receptor-binding domain of Bat CoV CD35 showed a highly similar structure to that of SARS-CoV-1 and SARS-CoV-2,especially in one binding loop.In conclusion,this study deepens our understanding of the diversity of coronaviruses and provides clues about the natural origin of the furin cleavage site of SARS-CoV-2.
基金financially supported by grants from the Australian Research Council(ARC)(Grant Nos.LP110200333 and DP120104460)National Health and Medical Research Council of Australia(NHMRC)(Grant Nos.APP1127948,APP1144652,and APP490989)+2 种基金the National Institute of Allergy and Infectious Diseases of the National Institutes of Health,USA(Grant No.R01 AI111965)a Major Inter-Disciplinary Research(IDR)Grant Awarded by Monash University,Australia(Grant Nos.2019-32 and 2018-28)supported in part by Informatics start-up packages through the School of Medicine,University of Alabama at Birmingham,USA
文摘Proteases are enzymes that cleave and hydrolyse the peptide bonds between two specific amino acid residues of target substrate proteins.Protease-controlled proteolysis plays a key role in the degradation and recycling of proteins,which is essential for various physiological processes.Thus,solving the substrate identification problem will have important implications for the precise understanding of functions and physiological roles of proteases,as well as for therapeutic target identification and pharmaceutical applicability.Consequently,there is a great demand for bioinformatics methods that can predict novel substrate cleavage events with high accuracy by utilizing both sequence and structural information.In this study,we present Procleave,a novel bioinformatics approach for predicting protease-specific substrates and specific cleavage sites by taking into account both their sequence and 3D structural information.Structural features of known cleavage sites were represented by discrete values using a LOWESS data-smoothing optimization method,which turned out to be critical for the performance of Procleave.The optimal approximations of all structural parameter values were encoded in a conditional random field(CRF)computational framework,alongside sequence and chemical group-based features.Here,we demonstrate the outstanding performance of Procleave through extensive benchmarking and independent tests.Procleave is capable of correctly identifying most cleavage sites in the case study.Importantly,when applied to the human structural proteome encompassing 17,628 protein structures,Procleave suggests a number of potential novel target substrates and their corresponding cleavage sites of different proteases.Procleave is implemented as a webserver and is freely accessible at http://procleave.erc.monash.edu/.
基金supported by the National Key Plan for Scientific Research and Development of China(2016YFD0500300)National Natural Science Foundation of China(32170651)Hunan Provincial Natural Science Foundation of China(2020JJ3006)。
文摘The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL protease.This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein,and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus,Betacoronavirus and Gammacoronavirus.Strong residue preferences were observed at the neighboring positions of the cleavage sites.A random forest(RF)model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes,and the model achieved an AUC of 0.96 in cross-validations.The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts.It achieved an AUC of 0.95 and predicted correctly 80%of the cleavage sites.Then,1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model.These proteins were enriched in several GO terms related to the cytoskeleton,such as the microtubule,actin and tubulin.Finally,a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model.Overall,the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.
基金supported by National Natural Science Foundation of China(31672586 and 31830097)the Key Research and Development Program of Guangdong Province(2019B020218004)+2 种基金Earmarked Found for China Agriculture Research System(CARS-41-G16)Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme(2018,Wenbao Qi)Young Scholars of Yangtze River Scholar Professor Program(2019,Wenbao Qi)
文摘Since mid-2016,the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic(HP)phenotype in China,raising many concerns about public health and poultry industry.The insertion of a“KRTA”motif at hemagglutinin cleavage site(HACS)occurred in the early stage of HP H7N9 variants.During the co-circulation,the HACS of HP-H7N9 variants were more polymorphic in birds and humans.Although HP-H7N9 variants,unlike the H5 subtype virus,exhibited the insertions of basic and non-basic amino acids,the underlying function of those insertions and substitutions remains unclear.The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China.Then,we generated six H7N9 viruses bearing the PEIPKGR/G,PEVPKGR/G,PEVPKRKRTAR/G,PEVPKGKRTAR/G,PEVPKGKRIAR/G,and PEVPKRKRR/G motifs.Interestingly,after the deletion of threonine and alanine(TA)at HACS,the H7N9 viruses manifested decreased thermostability and virulence in mice,and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence.By contrast,the insertion of non-basic amino acid isoleucine and alanine(IA)decreased the transmissibility in chickens and virulence in mice.Remarkably,the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens,suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice.The ongoing evolution of H7N9 increasingly threatens public health and poultry industry,so,its comprehensive surveillance and prevention of H7N9 viruses should be pursued.
基金National Institutes of Health(NIH)(grants R01 A/130092 and Al161085).
文摘The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1 contains the receptor-binding domain(RBD),while the S2 contains the hydrophobic fusion domain for the entry into the host cell.Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various c leavage sites.In this article,we review host proteases including furin,trypsin,transmembrane protease serine 2(TMPRSS2)and cathepsins in the activation of SARS-CoV-2 S.Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin.The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2,and the binding triggers further conformational changes and exposure of the S2'site to proteases such as type Il transmembrane serine proteases(TTPRs)including TMPRSS2.In the presence of TMPRSS2 on the target cells,SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane.In the absence of TMPRSS2,SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry.Additional host proteases involved in the cleavage of the S were discussed.This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2,and discussed the dual roles of such inhibitors in virus replication.
基金This work was supported by the National Natural Science Foundation of China(No.20443005)the Nanotechnology Special Projects of Shanghai(No.0352nm123)
文摘A novel artificial site specific cleavage reagent, with peptide nucleic acid (PNA) as sequence-recognizing moiety and cerium (IV) ions as “scissors” for cleaving target DNA, was synthesized. Subsequently, it was employed in the cleavage of target 26-mer single-stranded DNA (ssDNA), which has 10-mer sequence complementary with PNA recognizer in the hybrids, under physiological conditions. Reversed-phase high-performance liquid chromatogram (RPHPLC) experiments indicated that the artificial site specific cleavage reagent could cleave the target DNA specifically.