title: Development of viable TAP-tagged dengue virus for investigation of host-virus interactions in viral replication.
abstract: Dengue virus (DENV) is a mosquito-borne flavivirus responsible for life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The viral replication machinery containing the core non-structural protein 5 (NS5) is implicated in severe dengue symptoms but molecular details remain obscure. To date, studies seeking to catalogue and characterize interaction networks between viral NS5 and host proteins have been limited to the yeast two-hybrid system, computational prediction and co-immunoprecipitation (IP) of ectopically expressed NS5. However, these traditional approaches do not reproduce a natural course of infection in which a number of DENV NS proteins colocalize and tightly associate during the replication process. Here, we demonstrate the development of a recombinant DENV that harbours a TAP tag in NS5 to study host-virus interactions in vivo. We show that our engineered DENV was infective in several human cell lines and that the tags were stable over multiple viral passages, suggesting negligible structural and functional disturbance of NS5. We further provide proof-of-concept for the use of rationally tagged virus by revealing a high confidence NS5 interaction network in human hepatic cells. Our analysis uncovered previously unrecognized hnRNP complexes and several low-abundance fatty acid metabolism genes, which have been implicated in the viral life cycle. This study sets a new standard for investigation of host-flavivirus interactions.
authors.affiliation: Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Teera
authors.initials: T
authors.lastname: Poyomtip
authors.affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Phutthamonthon, Nakhon Pathom 73170, Thailand.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Hodge
authors.affiliation: Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.|Systems Biology of Diseases Research Unit, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Ponpan
authors.initials: P
authors.lastname: Matangkasombut
authors.affiliation: Systems Biology of Diseases Research Unit, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.|Functional Genetics of Infectious Diseases Unit, Institute Pasteur, Paris, France.|Centre National de la Recherche Scientifique (CNRS), URA3012, F-75015 Paris, France.
authors.forename: Anavaj
authors.initials: A
authors.lastname: Sakuntabhai
authors.affiliation: Systems Biology Center, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.|Systems Biology of Diseases Research Unit, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Siwanon
authors.initials: S
authors.lastname: Jirawatnotai
authors.affiliation: Systems Biology of Diseases Research Unit, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.|Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Phutthamonthon, Nakhon Pathom 73170, Thailand.
authors.forename: Sarin
authors.initials: S
authors.lastname: Chimnaronk
mesh_terms: D002846:Chromatography, Affinity; D003715:Dengue / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins