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A Convenient and Biosafe replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery

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Virologica Sinica 2021年第5期36卷 913-923页

作者： Yun-Yun Jin Hanwen Lin Liu Cao Wei-Chen Wu Yanxi Ji Liubing Du Yiling Jiang Yanchun Xie Kuijie Tong Fan Xing Fuxiang Zheng Mang Shi Ji-An Pan Xiaoxue Peng Deyin Guo The Center for Infection and Immunity Study School of MedicineSun Yat-sen UniversityGuangming Science CityShenzhen 518107China

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far.Due to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined in the biosafety level 3(BSL3)laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious.In this study,we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome(BAC)vector with deletion of the spike(S)gene.Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein.Therefore,such a replicon system is not infectious and can be used in ordinary biological laboratories.We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus.By mutational analysis of nsp12 and nsp14,we showed that the RNA polymerase,exonuclease,and cap N7 methyltransferase play essential roles in genome replication and sgRNA production.We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors.Thus,such a one-plasmid system is biosafe and convenient to use,which will benefit both fundamental research and development of antiviral drugs.

关键词： SARS-CoV-2 Reverse genetics replicon Antiviral drug screening

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A novel package system based on an EBV replicon vector for producing high titer recombinant adeno-associated virus vector

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Chinese Science Bulletin 1997年第20期42卷 1741-1744页

作者： Yan, ZY Yao, EM Zhang, T Shu, YL Hou, YD Chinese Acad Prevent Med Inst Virol State Key Lab Mol Virol & Genet Engn Beijing 100052 Peoples R China

ADENO-ASSOCIATED virus (AAV) was a human parvovirus considered as a gene delivery vehiclefor human gene therapy. Recombinant AAV vector (rAAV) could mediate the foreign DNAintegration into chromosome and establish a stable expression in the infected cells. As a viraltransduction vector, rAAV was superior to the traditional retrovirus vector for its high safety.no pathogenicity, and capacity of infecting postmitosis cells. The current strategy for produc-

关键词： recombinant AAV vector EBV replicon vector package cell line.

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Comparison of viral propagation and drug response among SARS-CoV-2 VOCs using replicons capable of recapitulating virion assembly and release

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Virologica Sinica 2022年第5期37卷 695-703页

作者： Lingqian Tian Qiuhong Liu Rongjuan Pei Yingshan Chen Chonghui Xu Jielin Tang Hao Sun Kunpeng Liu Qi Yang Lei Yang Leshan Li Yongli Zhang Yuan Zhou Chao Shan Xue Hu Xinwen Chen Yun Wang State Key Laboratory of Virology Wuhan Institute of VirologyCenter for Biosafety Mega-ScienceChinese Academy of SciencesWuhan430071China University of Chinese Academy of Sciences Beijing100049China Department of Gastroenterology Guangzhou Women and Children's Medical CenterGuangzhou510623China Guangzhou Institutes of Biomedicine and Health Chinese Academy of SciencesGuangzhou510530China Innovation Center for Pathogen Research Guangzhou LaboratoryGuangzhou510320China

Several variants of concern(VOCs)have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was first isolated in January 2020.Due to mutations in the spike(S)protein,these VOCs have evolved to enhance viral infectivity and immune evasion.However,whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure.The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle.Although several SARS-CoV-2 replicons have been developed,none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA(sgRNA)transcription.In this study,SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs(the Beta and Delta variants)were prepared by removing the S gene from their genomes,while other structural genes remained untouched.These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles,as manifested by electron microscopic assays.Thus,the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs,including inhibitors of virion assembly and release.Through the quantification of replicon RNA released into the supernatant,we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.

关键词： SARS-CoV-2 Variants of concern(VOC) Viral-like particle(VLP) replicon Remdesivir

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Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes

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World Journal of Gastroenterology 2014年第48期20卷 18284-18295页

作者： Xin Cheng Xiang-Cui Gao Jun-Ping Wang Xin-Ying Yang Yan Wang Bao-Sheng Li Fu-Biao Kang Hai-Jun Li Yue-Min Nan Dian-Xing Sun The Liver Disease Diagnosis and Treatment Center of PLA Bethune International Peace Hospital Department of Traditional and Western MedicalHepatology Third Hospital of Hebei Medical University

AIM:To construct a tricistronic hepatitis C virus(HCV)replicon with double internal ribosome entry sites(IRESes)of only 22 nucleotides for each,substituting the encephalomyocarditis virus(EMCV)IRESes,which are most often used as the translation initiation element to form HCV replicons.METHODS:The alternative 22-nucleotide IRES,RNA-binding motif protein 3 IRES(Rbm3 IRES),was used to form a tricistronic HCV replicon,to facilitate constructing HCV-harboring stable cell lines andsuccessive antiviral screening using a luciferase marker.Briefly,two sequential Rbm3 IRESes were inserted into bicistronic p UC19-HCV plasmid,consequently forming a tricistronic HCV replicon(p HCV-rep-Neo R-h Rluc),initiating the translation of humanized Renilla luciferase and HCV non-structural gene,along with HCV authentic IRES initiating the translation of neomycin resistance gene.The s H7 cell lines,in which the novel replicon RNA stably replicated,were constructed by neomycin and luciferase activity screening.The intracellular HCV replicon RNA,expression of inserted foreign genes and HCV non-structural gene,as well as response to anti-HCV agents,were measured in s H7 cells and cells transiently transfected with tricistronic replicon RNA.RESULTS:The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV nonstructural gene in s H7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons.The average relative light unit in p HCV-rep-Neo R-h Rluc group was approximately 2-fold of those in the p UC19-HCV-h RLuc and Tri-JFH1 groups(1.049×108±2.747×107 vs 5.368×107±1.016×107,P<0.05;1.049×108±2.747×107 vs 5.243×107±1.194×107,P<0.05),suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES.The fold changes of 72 h/4 h relative light units in the p HCV-rep-Neo R-h Rluc and p UC19-HCV-h RLuc groups w

关键词： Hepacivirus replicon Internal ribosome entry site

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Identification of HCV Inhibitors from a Cell-Based Sub-Genomic replicon Screen

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Open Journal of Medicinal Chemistry 2013年第1期3卷 16-25页

作者： David C. Pryde Thien-Duc Tran Mark Gardner Chris Pickford Stephen M. Shaw Mike Westby Tanya Parkinson Caroline Smith-Burchnell Rob Webster Satish Dayal Anti-Infectives Research Unit Pfizer Global Research and Develoapment Sandwich UK Anti-Infectives Research Unit Pfizer Global Research and Development Sandwich UK Pharmacokinetics Dynamics and Metabolism Pfizer Global Research and Development Sandwich UK Worldwide Medicinal Chemistry Pfizer Global Research and Development Sandwich UK

A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity without overt cytotoxicity were further evaluated, leading to the identification of a series of pyrrolopyridines with excellent antiviral activity in a fully infectious HCV cell-based assay and pharmacokinetic properties.

关键词： HCV replicon Antiviral Cell-Based Screen

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The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10

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Virologica Sinica 2020年第4期35卷 426-435页

作者： Min Wang Jingjing Yan Liuyao Zhu Meng Wang Lizhen Liu Rui Yu Ming Chen Jingna Xun Yuling Zhang Zhigang Yi Shuye Zhang Shanghai Public Health Clinical Center and Institute of Biomedical Sciences Fudan UniversityShanghai 201508China

Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth disease.There are no vaccine and antiviral drugs for controlling CVA10 infection.Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals.Here,two infectious clones for the prototype and a Myc-tagged CVA10 were constructed.Viable CVA10 viruses were harvested by transfecting the viral m RNA into human rhabdomyosarcoma(RD)cells.Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally.We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter,respectively.Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293 T(HEK293 T)cells led to the production of single round infectious particles(SRIPs).Based on CVA10 replicon RNA,SRIPs with either the enterovirus A71(EVA71)capsid or the CVA10 capsid were generated.Infection by EVA71 SRIPs required SCARB2,while CVA10 SRIPs did not.Finally,we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme(HHRib)before the 50-untranslated region(UTR).In summary,reverse genetic tools for prototype strain of CVA10,including both the infectious clone and the SRIPs system,were successfully established.These tools will facilitate the basic and translational study of CVA10.

关键词： Coxsackievirus A10(CVA10) Reverse genetics Enterovirus Single round infectious particles(SRIPs) replicon

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Construction of Non-infectious SARS-CoV-2 replicons and Their Application in Drug Evaluation

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Virologica Sinica 2021年第5期36卷 890-900页

作者： Bei Wang Chongyang Zhang Xiaobo Lei Lili Ren He Huang Jianwei Wang Zhendong Zhao NHC Key Laboratory of Systems Biology of Pathogens Institute of Pathogen BiologyChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100176China NHC Key Laboratory of Systems Biology of Pathogens and Christophe Me´rieux Laboratory Institute of Pathogen BiologyChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100176China

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a devastating pandemic worldwide.Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic,and scientists all over the world have made great efforts to this end.However,manipulation of the SARS-CoV-2 should be performed in the biosafety level3 laboratory.This makes experiments complicated and time-consuming.Therefore,a safer system for working with this virus is urgently needed.Here,we report the construction of plasmid-based,non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae.Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes.Using SARS-CoV-2 replicons,the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed,and the halfmaximal effective concentration(EC50)value of remdesivir and E64-D was estimated by different quantification methods respectively,indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation.

关键词： SARS-CoV-2 Reverse genetics replicon Antiviral drugs Drug evaluation

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Assembly and release of infectious hepatitis C virus involving unusual organization of the secretory pathway

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World Journal of Hepatology 2016年第19期8卷 796-814页

作者： Miriam Triyatni Edward A Berger Bertrand Saunier Roche Innovation Center Roche Pharma Research and Early Development F. Hoffmann-La Roche Ltd. Molecular Structure Section Laboratory of Viral Diseases NIAID NIH Unité de Virologie Structurale Institut Pasteur and CNRS UMR 3569

AIM: To determine if calnexin(CANX), RAB1 and alphatubulin were involved in the production of hepatitis C virus(HCV) particles by baby hamster kidney-West Nile virus(BHK-WNV) cells. METHODS: Using a si RNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observedin thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model.RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome.CONCLUSION: Prior activity

关键词： Membrane rearrangements Hepatitis C virus Flavivirus replicon Virus assembly and secretion Host cell

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A Streptomyces PLASMID pHZ65 AND ITS DEVELOPMENT INTO CLONING VECTORS

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Science China Chemistry 1992年第11期35卷 1315-1322页

作者：邓子新周秀芬 Laboratory of Agricultural Antibiotics Huazhong Agricultural University Wuhan 430070 PRC

FR--0001 and FR--005 are two fusants obtained through protoplast fusion between differ-ently marked derivatives of Streptomyces hygroscopicus 5102, which produce three agricul-turally important antibiotics. Although they show distinct differences in morphologicaland cultural characteristics, antimicrobial activity and metabolites, they all carry a 20.1 kbplasmid (pHZ65) with complete DNA homology. A detailed restriction map with 29 sitesfor restriction enzymes BamHI, BclI, EcoRV, KpnI, PstI, SphI, SstI and XhoI has been deter-mined. Four Strepiomyces-E. coli bifunctional plasmids, pHZ200, pHZ201, pHZ206 andpHZ207, have been constructed by the in vitro insertion of an E. coli plasmid pIJ2703 whichcarries the tsr gene (expressible in Streptomyces) and amp gene (expressible in E. coli)into the BclI or BglII sites of pHZ65. The replicability of a series of plasmids obtainedby the cloning of DNA fragments from pHZ65 into the BclI site of pIJ2703 in S. lividansshows that the essential replication region of

关键词： replicon restriction map Streptomyces

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A novel liver-directed gene delivery system using an autonomously replicating vector specifically expressed in AFP positive hepatoma cells

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Science China(Life Sciences) 1998年第1期41卷 80-86页

作者：颜子颖乔健贡惠宇侯云德

A composite EBV based expression vector, pEBAF, was constructed with the 5′ flanking sequence of human α fetoprotein gene as a promoter. Complexed to galactosylated histone, this vector with a foreign DNA insertion could be transferred into hepatic cells via the asialoglycoprotein receptor mediated endocytosis pathway. It was replicated as an episome, and its expression was restricted to the AFP positive hepatoma cells. This new gene delivery method has the following advantages: (i) absence of a potential toxicity is related to viral vectors; (ii) the targeting is specific to AFP positive hepatoma cells; (iii) the introduction of the EBV replicon into this system results in the high level expression and long term maintenance of the transferred gene. This novel gene delivery system has potential applications in gene therapy for the treatment of hepatocellular carcinoma.

关键词： EBV replicon vector AFP promoter receptor mediated gene transfer hepatoma cell.

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