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

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 

机构地区：[1]The Center for Infection and Immunity Study,School of Medicine,Sun Yat-sen University,Guangming Science City,Shenzhen 518107,China

出　　处：《Virologica Sinica》2021年第5期913-923,共11页中国病毒学（英文版）

基　　金：supported by Grants(the National Natural Science Foundation of China#32041002,#31971161,#31900546 and#81620108020);the Guangdong Science and Technology Department(#2019A1515011332);the Shenzhen Science and Technology Innovation Program(JSGG20200225150431472,JCYJ20190807160615255,JCYJ20190807153203560,and KQTD20180411143323605);supported by the Guangdong Zhujiang Leading Talents Programme and the National Tenthousand Talents Program。

摘　　要：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 

分 类 号：R978.7[医药卫生—药品]