title: Construction of a non-infectious SARS coronavirus replicon for application in drug screening and analysis of viral protein function.
abstract: Severe acute respiratory syndrome virus (SARS-CoV) was the causative agent of the SARS outbreaks in 2002-2003. A safer in vitro system is desirable for conducting research on SARS-CoV and to screen for antiviral drugs against the virus. Based on the infectious cDNA clone of rSARS-CoV-DeltaE, in which the E gene has been deleted, a safe non-infectious replicon was constructed by replacing the S gene with the enhanced green fluorescent protein (eGFP) gene. Successful replication was achieved as evident from continuous expression of eGFP detected by both fluorescence and Western blot. Treatment with antiviral drugs demonstrated that the replication could be significantly inhibited by 0.4 mg/ml of cysteine proteinase inhibitor E-64D, but not by ribavirin. The same replicons containing further deletion of the coding regions for non-structural proteins (nsp) 1, 2 or 16 confirmed previous observation that nsp16, but not nsp1 or nsp2, was essential for efficient viral replication or transcription.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, 44 Xiao Hong Shan, Wuhan, Hubei 430071, PR China.
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Wang
authors.forename: Lin-Fa
authors.initials: LF
authors.lastname: Wang
authors.forename: Zheng-Li
authors.initials: ZL
authors.lastname: Shi
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D045473:Severe acute respiratory syndrome-related coronavirus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D017361:Viral Nonstructural Proteins; D049452:Green Fluorescent Proteins; C521592:Nsp1 protein, SARS coronavirus; D012324:RNA-Dependent RNA Polymerase