Avian influenza virus(AIV) nonstructural 1(NS1) gene was amplified by real-time polymerse chain reac tion(RT-PCR) and inserted into pET28a, then transformed into E. coli BL21(DE3) competent cell. With the indu...Avian influenza virus(AIV) nonstructural 1(NS1) gene was amplified by real-time polymerse chain reac tion(RT-PCR) and inserted into pET28a, then transformed into E. coli BL21(DE3) competent cell. With the induction of isopropyl-β-D-thiogalactoside(IPTG) and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1(NOLC1) and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur ther studying biological function of NS1 protein.展开更多
BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy optio...BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.展开更多
Porcine reproductive and respiratory syndrome virus.(PRRSV) actively induces cell apoptosis both in vitro and in vivo, which can contribute critically to viral pathogenesis. Previous studies have shown that the PRRS...Porcine reproductive and respiratory syndrome virus.(PRRSV) actively induces cell apoptosis both in vitro and in vivo, which can contribute critically to viral pathogenesis. Previous studies have shown that the PRRSV nonstructural protein 4 (nsp4) is an important mediator of this process, but the underlying molecular details remain poorly understood. In this study, we found that the PRRSV nsp4 interacted with the mitochondrial inner membrane protein cytochrome cl (cyto.cl) and induced its proteolytic cleavage. Interestingly, the cleaved N-terminal fragment of cyto.cl was found to exert apoptotic activity, which could cause mitochondrial fragmentation, resulting in apoptotic cell death. And RNA interference (RNAi) silencing experiments further confirmed the crucial role which cyto.cl played in nsp4- and PRRSV-induced cell apoptosis. Thus, our data provide an important piece of mechanistic clues for PRRSV-induced cell apoptosis and also elucidate a novel mechanism for the 3C-like proteases in this finding.展开更多
The nonstructural protein 10(nsp10) of porcine reproductive and respiratory syndrome virus(PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10...The nonstructural protein 10(nsp10) of porcine reproductive and respiratory syndrome virus(PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10 a, was found in PRRSV-infected cells and the production of nsp10 a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10 a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10 a production. Finally, we demonstrated that nsp10 a exerted little influence on the growth kinetics of PRRSV in vitro.展开更多
The outbreak of coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a serious global health threat.This raises an urgent need for the development of effective drug...The outbreak of coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a serious global health threat.This raises an urgent need for the development of effective drugs against the deadly disease.SARS-CoV-2 non-structural protein 14(NSP14)carrying RNA cap guanine N7-methyltransferase and 30-50 exoribonuclease activities could be a potential drug target for intervention.NSP14 of SARS-CoV-2 shares 98.7%of similarity with the one(PDB 5NFY)of acute respiratory syndrome(SARS)by ClustalW.Then,the SARS-CoV-2 NSP14 structures were modelled by Modeller 9.18 using SARS NSP14(PDB 5NFY)as template for virtual screening.Based on the docking score from AutoDock Vina1.1.2,18 small molecule drugs were selected for further evaluation.Based on the 5 ns MD simulation trajectory,binding free energy(DG)was calculated by MM/GBSA method.The calculated binding free energies of Saquinavir,Hypericin,Baicalein and Bromocriptine for the N-terminus of the homology model wereà37.2711±3.2160,à30.1746±3.1914,à23.8953±4.4800,andà34.1350±4.3683 kcal/mol,respectively,while the calculated binding free energies wereà60.2757±4.7708,à30.9955±2.9975,à46.3099±3.5689,andà59.8104±3.5389 kcal/mol,respectively,when binding to the C-terminus.Thus,the compounds including Saquinavir,Hypericin,Baicalein and Bromocriptine could bind to the N-terminus and C-terminus of the homology model of the SARS-CoV-2 NSP14,providing a candidate drug against SARS-CoV-2 for further study.展开更多
Japanese encephalitis (JE) is a central nervous system disease caused by Japanese encephalitis virus (JEV), which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays a...Japanese encephalitis (JE) is a central nervous system disease caused by Japanese encephalitis virus (JEV), which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 ( DE3 ) under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence (IFA). The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.展开更多
Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown...Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.展开更多
Aquareovirus species vary with respect to pathogenicity,and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly,which can form viral inclusion bodie...Aquareovirus species vary with respect to pathogenicity,and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly,which can form viral inclusion bodies(VIBs) and recruit viral proteins to its VIBs in infected cells.NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa.Interestingly,a short specific fragment of NS80 has also been detected in infected cells.In this study,an approximately58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80 C.Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose,suggesting that the fragment is not a transient intermediate of protein degradation.Moreover,another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80 N,indicating that the 58-kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80.Additionally,different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis,implying that the two cleavage fragments of NS80 function differently in the viral life cycle.These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.展开更多
Zika virus(ZIKV),a positive-sense single-stranded RNA virus,causes congenital ZIKV syndrome in children and Guillain-Barre Syndrome(GBS)in adults.ZIKV expresses nonstructural protein 5(NS5),a large protein that is ess...Zika virus(ZIKV),a positive-sense single-stranded RNA virus,causes congenital ZIKV syndrome in children and Guillain-Barre Syndrome(GBS)in adults.ZIKV expresses nonstructural protein 5(NS5),a large protein that is essential for viral replication.ZIKV NS5 confers the ability to evade interferon(IFN)signalling;however,the exact mechanism remains unclear.In this study,we employed affinity pull-down and liquid chromatography-tandem mass spectrometry(LC-MS/MS)analyses and found that splicing factor 3b subunit 3(SF3B3)is associated with the NS5-Flag pull-down complex through interaction with NS5.Functional assays showed that SF3B3 overexpression inhibited ZIKV replication by promoting IFN-stimulated gene(ISG)expression whereas silencing of SF3B3 inhibited expression of ISGs to promote ZIKV replication.GTP cyclohydrolase I(GCH1)is the first and ratelimiting enzyme in tetrahydrobiopterin(BH4)biosynthesis.NS5 upregulates the expression of GCH1 during ZIKV infection.And GCH1 marginally promoted ZIKV replication via the IFN pathway.Additionally,GCH1 expression is related to the regulation of SF3B3.Overexpression of the SF3B3 protein effectively reduced GCH1 protein levels,whereas SF3B3 knockdown increased its levels.These findings indicated that ZIKV NS5 binding protein SF3B3 contributed to the host immune response against ZIKV replication by modulating the expression of GCH1.展开更多
Objective:To explore potential natural products against severe acute respiratory syndrome coronavirus(SARS-CoV-2)via the study of structural and non-structural proteins of human coronaviruses.Methods:In this study,we ...Objective:To explore potential natural products against severe acute respiratory syndrome coronavirus(SARS-CoV-2)via the study of structural and non-structural proteins of human coronaviruses.Methods:In this study,we performed an in-silico survey of 25 potential natural compounds acting against SARS-CoV-2.Molecular docking studies were carried out using compounds against 3-chymotrypsin-like protease(3 CL^(PHO)),papain-like protease(PL^(PRO)),RNA-dependent RNA polymerase(RdRp),non-structural protein(nsp),human angiotensin converting enzyme 2 receptor(hACE2 R),sapike glycoprotein(S protein),abelson murine leukemia viral oncogene homolog 1(ABL1),calcineurin-nuclear factor of activated T-cells(NFAT)and transmembrane protease serine 2.Results:Among the screened compounds,amentoflavone showed the best binding affinity with the 3 CL^(PRO),RdRp,nsp13,nsp15,hACE2 R,ABL1 and calcineurin-NFAT;berbamine with hACE2 R and ABL1;cepharanthine with nsp10,nsp14,nsp16,S protein and ABL1;glucogallin with nsp15;and papyriflavonol A with PL^(PRO)protein.Other good interacting compounds were juglanin,betulinic acid,betulonic acid,broussooflavan A,tomentin A,B and E,7-methoxycryptopleurine,aloe emodin,quercetin,tanshinone I,tylophorine and furruginol,which also showed excellent binding affinity towards a number of target proteins.Most of these compounds showed better binding affinities towards the target proteins than the standard drugs used in this study.Conclusion:Natural products or their derivatives may be one of the potential targets to fight against SARS-CoV-2.展开更多
Zika virus(ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells(NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have be...Zika virus(ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells(NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection, yet the detailed mechanism is not well understood. In the present study, we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that, among the ten viral proteins,the nonstructural protein 5(NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach, we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition, the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore, lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of h NPCs.Taken together, these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs, which may contribute to the ZIKV-caused abnormal neurodevelopment.展开更多
Protein degradation technology,which is one of the most direct and effective ways to regulate the life activities of cells,is expected to be applied to the treatment of various diseases.However,current protein degrada...Protein degradation technology,which is one of the most direct and effective ways to regulate the life activities of cells,is expected to be applied to the treatment of various diseases.However,current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation,making them difficult to transform into clinical applications.In this article,an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation.This system worked via two interconnected parts:1)the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure;2)the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation.Experimental results based on model protein(Green Fluorescent Protein,GFP)validated that this system could fulfill protein degradation both in vitro(both Hela and 293T cells)and in vivo(by upconversion optogenetic nanosystem),and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time(0–25 h)and the illumination frequency(the illuminating frequency of 0–30 s every 1 min).We further took another functional protein(The Nonstructural Protein 9,NSP9)into experiment.Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus(PRRSV)was inhibited by degrading the NSP9 in this light-induced system,and PRRSV proliferation was affected by different light conditions(illumination time varies from 0–24 h).We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.展开更多
基金Supported by the National Natural Science Foundation of China(No.30671852)the Open Research Fund Program of the State Key Laboratory of Virology of China(Nos.2010009, 2007007) the Research Fund of the Key Laboratory of Department of Education of Liaoning Province, China(No.2009S043)
文摘Avian influenza virus(AIV) nonstructural 1(NS1) gene was amplified by real-time polymerse chain reac tion(RT-PCR) and inserted into pET28a, then transformed into E. coli BL21(DE3) competent cell. With the induction of isopropyl-β-D-thiogalactoside(IPTG) and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1(NOLC1) and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur ther studying biological function of NS1 protein.
基金the National Key Research and Development Program of China,No.2017YFC0908104National Science and Technology Projects,No.2017ZX10203201,No.2017ZX10201201,and No.2017ZX10202202.
文摘BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.
基金supported by the National 973 Program of China(2014CB542700)the National Natural Science Foundation of China(31330077,31540004)the earmarked fund for China Agriculture Research System(CARS-36)
文摘Porcine reproductive and respiratory syndrome virus.(PRRSV) actively induces cell apoptosis both in vitro and in vivo, which can contribute critically to viral pathogenesis. Previous studies have shown that the PRRSV nonstructural protein 4 (nsp4) is an important mediator of this process, but the underlying molecular details remain poorly understood. In this study, we found that the PRRSV nsp4 interacted with the mitochondrial inner membrane protein cytochrome cl (cyto.cl) and induced its proteolytic cleavage. Interestingly, the cleaved N-terminal fragment of cyto.cl was found to exert apoptotic activity, which could cause mitochondrial fragmentation, resulting in apoptotic cell death. And RNA interference (RNAi) silencing experiments further confirmed the crucial role which cyto.cl played in nsp4- and PRRSV-induced cell apoptosis. Thus, our data provide an important piece of mechanistic clues for PRRSV-induced cell apoptosis and also elucidate a novel mechanism for the 3C-like proteases in this finding.
基金supported by the National Key Technology R&D Program of China (2015BAD12B01-2)the Major Program of National Natural Science Foundation of China (31490603)the earmarked fund for Modern Agroindustry Technology Research System of China (CARS-36)
文摘The nonstructural protein 10(nsp10) of porcine reproductive and respiratory syndrome virus(PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10 a, was found in PRRSV-infected cells and the production of nsp10 a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10 a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10 a production. Finally, we demonstrated that nsp10 a exerted little influence on the growth kinetics of PRRSV in vitro.
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.31870135,31600116)the“1000 Talent Plan”of Sichuan Province(No.980)。
文摘The outbreak of coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a serious global health threat.This raises an urgent need for the development of effective drugs against the deadly disease.SARS-CoV-2 non-structural protein 14(NSP14)carrying RNA cap guanine N7-methyltransferase and 30-50 exoribonuclease activities could be a potential drug target for intervention.NSP14 of SARS-CoV-2 shares 98.7%of similarity with the one(PDB 5NFY)of acute respiratory syndrome(SARS)by ClustalW.Then,the SARS-CoV-2 NSP14 structures were modelled by Modeller 9.18 using SARS NSP14(PDB 5NFY)as template for virtual screening.Based on the docking score from AutoDock Vina1.1.2,18 small molecule drugs were selected for further evaluation.Based on the 5 ns MD simulation trajectory,binding free energy(DG)was calculated by MM/GBSA method.The calculated binding free energies of Saquinavir,Hypericin,Baicalein and Bromocriptine for the N-terminus of the homology model wereà37.2711±3.2160,à30.1746±3.1914,à23.8953±4.4800,andà34.1350±4.3683 kcal/mol,respectively,while the calculated binding free energies wereà60.2757±4.7708,à30.9955±2.9975,à46.3099±3.5689,andà59.8104±3.5389 kcal/mol,respectively,when binding to the C-terminus.Thus,the compounds including Saquinavir,Hypericin,Baicalein and Bromocriptine could bind to the N-terminus and C-terminus of the homology model of the SARS-CoV-2 NSP14,providing a candidate drug against SARS-CoV-2 for further study.
基金Supported by Youth Fund of Hubei Academy of Agricultural Sciences(2013NKYJJ12)
文摘Japanese encephalitis (JE) is a central nervous system disease caused by Japanese encephalitis virus (JEV), which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 ( DE3 ) under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence (IFA). The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.
基金supported by grants from the National Natural Science Foundation of China(31900146)the key Biosafety Science and Technology Program of Hubei Jiangxia Laboratory(JXBS001)+1 种基金the Hubei Natural Science Foundation for Distinguished Young Scholars(2021CFA050)the Creative Research Group Program of Natural Science Foundation of Hubei Province(2022CFA021).
文摘Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.
基金supported by funding from the National Natural Science Foundation of China (NO.31372565,31402340,31400139 and 31370190)
文摘Aquareovirus species vary with respect to pathogenicity,and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly,which can form viral inclusion bodies(VIBs) and recruit viral proteins to its VIBs in infected cells.NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa.Interestingly,a short specific fragment of NS80 has also been detected in infected cells.In this study,an approximately58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80 C.Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose,suggesting that the fragment is not a transient intermediate of protein degradation.Moreover,another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80 N,indicating that the 58-kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80.Additionally,different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis,implying that the two cleavage fragments of NS80 function differently in the viral life cycle.These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.
基金supported by the National Key R&D Project of China(2021YFC230170402)CAMS Innovation Fund for Medical Sciences(2021-1-I2M-038).
文摘Zika virus(ZIKV),a positive-sense single-stranded RNA virus,causes congenital ZIKV syndrome in children and Guillain-Barre Syndrome(GBS)in adults.ZIKV expresses nonstructural protein 5(NS5),a large protein that is essential for viral replication.ZIKV NS5 confers the ability to evade interferon(IFN)signalling;however,the exact mechanism remains unclear.In this study,we employed affinity pull-down and liquid chromatography-tandem mass spectrometry(LC-MS/MS)analyses and found that splicing factor 3b subunit 3(SF3B3)is associated with the NS5-Flag pull-down complex through interaction with NS5.Functional assays showed that SF3B3 overexpression inhibited ZIKV replication by promoting IFN-stimulated gene(ISG)expression whereas silencing of SF3B3 inhibited expression of ISGs to promote ZIKV replication.GTP cyclohydrolase I(GCH1)is the first and ratelimiting enzyme in tetrahydrobiopterin(BH4)biosynthesis.NS5 upregulates the expression of GCH1 during ZIKV infection.And GCH1 marginally promoted ZIKV replication via the IFN pathway.Additionally,GCH1 expression is related to the regulation of SF3B3.Overexpression of the SF3B3 protein effectively reduced GCH1 protein levels,whereas SF3B3 knockdown increased its levels.These findings indicated that ZIKV NS5 binding protein SF3B3 contributed to the host immune response against ZIKV replication by modulating the expression of GCH1.
文摘Objective:To explore potential natural products against severe acute respiratory syndrome coronavirus(SARS-CoV-2)via the study of structural and non-structural proteins of human coronaviruses.Methods:In this study,we performed an in-silico survey of 25 potential natural compounds acting against SARS-CoV-2.Molecular docking studies were carried out using compounds against 3-chymotrypsin-like protease(3 CL^(PHO)),papain-like protease(PL^(PRO)),RNA-dependent RNA polymerase(RdRp),non-structural protein(nsp),human angiotensin converting enzyme 2 receptor(hACE2 R),sapike glycoprotein(S protein),abelson murine leukemia viral oncogene homolog 1(ABL1),calcineurin-nuclear factor of activated T-cells(NFAT)and transmembrane protease serine 2.Results:Among the screened compounds,amentoflavone showed the best binding affinity with the 3 CL^(PRO),RdRp,nsp13,nsp15,hACE2 R,ABL1 and calcineurin-NFAT;berbamine with hACE2 R and ABL1;cepharanthine with nsp10,nsp14,nsp16,S protein and ABL1;glucogallin with nsp15;and papyriflavonol A with PL^(PRO)protein.Other good interacting compounds were juglanin,betulinic acid,betulonic acid,broussooflavan A,tomentin A,B and E,7-methoxycryptopleurine,aloe emodin,quercetin,tanshinone I,tylophorine and furruginol,which also showed excellent binding affinity towards a number of target proteins.Most of these compounds showed better binding affinities towards the target proteins than the standard drugs used in this study.Conclusion:Natural products or their derivatives may be one of the potential targets to fight against SARS-CoV-2.
基金The work is supported by the National Natural Science Foundation of China[NSFC Grant#81620108020 and#32041002,to D.G.,Grant#31800151,to J.W.]Guangdong Zhujiang Talents Program(to D.G.)+2 种基金Shenzhen Science and Technology Program[Grant#KQTD20180411143323605 and#JSGG20200225150431472 to D.G.]National Ten-thousand Talents Program(to D.G.)Guangdong Province “Pearl River Talent Plan” Innovation and Entrepreneurship Team Project(Grant #2019ZT08Y464 to Li,C.M)。
文摘Zika virus(ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells(NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection, yet the detailed mechanism is not well understood. In the present study, we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that, among the ten viral proteins,the nonstructural protein 5(NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach, we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition, the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore, lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of h NPCs.Taken together, these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs, which may contribute to the ZIKV-caused abnormal neurodevelopment.
基金This work was sponsored by the National Key Research and Development Program of China(Nos.2019YFA0906500 and 2017YFA0205104)the National Natural Science Foundation of China(Nos.31971300,817719709,51873150 and 51573128)Tianjin Natural Science Foundation(No.19JCYBJC28800)and Young Elite Scientists Sponsorship Program by Tianjin.
文摘Protein degradation technology,which is one of the most direct and effective ways to regulate the life activities of cells,is expected to be applied to the treatment of various diseases.However,current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation,making them difficult to transform into clinical applications.In this article,an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation.This system worked via two interconnected parts:1)the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure;2)the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation.Experimental results based on model protein(Green Fluorescent Protein,GFP)validated that this system could fulfill protein degradation both in vitro(both Hela and 293T cells)and in vivo(by upconversion optogenetic nanosystem),and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time(0–25 h)and the illumination frequency(the illuminating frequency of 0–30 s every 1 min).We further took another functional protein(The Nonstructural Protein 9,NSP9)into experiment.Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus(PRRSV)was inhibited by degrading the NSP9 in this light-induced system,and PRRSV proliferation was affected by different light conditions(illumination time varies from 0–24 h).We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.