title: Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication.
abstract: Influenza A virus (IAV) poses significant threats to public health because of the recent emergence of highly pathogenic strains and wide-spread resistance to available anti-influenza drugs. Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RNA transcription/replication represents a promising target for antiviral drug development, no assay ideal for high-throughput screening (HTS) application is currently available to identify inhibitors targeting these processes. In this work, we developed a novel HTS assay to analyze the transcription and replication of IAV RNA using an A549 cell line stably expressing IAV RNA-dependent RNA polymerase (RdRp) complex, NP and a viral mini-genomic RNA. Both secreted Gaussia luciferase (Gluc) and blasticidin resistance gene (Bsd) were encoded in the viral minigenome and expressed under the control of IAV RdRp. Gluc serves as a reporter to monitor the activity of IAV RdRp, and Bsd is used to maintain the expression of all foreign genes. Biochemical studies and the statistical analysis presented herein demonstrate the high specificity, sensitivity and reproducibility of the assay. This work provides an ideal HTS assay for the identification of inhibitors targeting the function of IAV RdRp and a convenient reporting system for mechanism study of IAV RNA transcription / replication.
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Zhao
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Gao
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Zhenlong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Li
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Yuhuan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biochemistry, Guangdong Medical College, Zhanjiang, Guangdong, China.
authors.forename: Weilie
authors.initials: W
authors.lastname: Ma
authors.affiliation: Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Deng
authors.affiliation: Department of Biochemistry, Guangdong Medical College, Zhanjiang, Guangdong, China.
authors.forename: Zhizhen
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Pekin Union Medical College, Beijing, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Cen
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D024882:Drug Resistance, Viral / Q000235:genetics; D017930:Genes, Reporter; D016678:Genome; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000502:physiology; D007251:Influenza, Human / Q000821:virology; D008156:Luciferases / Q000378:metabolism; D011760:Pyrrolidinones; D034741:RNA, Small Interfering / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D015203:Reproducibility of Results; D014158:Transcription, Genetic; D014779:Virus Replication / Q000187:drug effects
chemical_list: D011760:Pyrrolidinones; D034741:RNA, Small Interfering; D012367:RNA, Viral; C420726:blasticidin A; D008156:Luciferases; D012324:RNA-Dependent RNA Polymerase