title: In vitro and in vivo analysis of the interaction between RNA helicase A and HIV-1 RNA.
abstract: RNA helicase A (RHA) promotes multiple steps of HIV-1 RNA metabolism during viral replication, including transcription, translation, and the annealing of primer tRNA(3)(Lys) to the viral RNA. RHA is a member of the DExH subclass of RNA helicases that uniquely contains two double-stranded RNA binding domains (dsRBDs) at its N terminus. Here, we performed a genome-wide analysis of the interaction of RHA with HIV-1 RNA both in vitro, using fluorescence polarization, and during viral replication, using an RNA-protein coprecipitation assay. In vitro, RHA binds to all the isolated regions of the HIV-1 RNA genome tested, with K(d) (equilibrium dissociation constant) values ranging from 44 to 178 nM. In contrast, during viral replication, RNA-protein coprecipitation assays detected only a major interaction of RHA with the 5'-untranslated region (5'-UTR) and a minor interaction with the Rev response element (RRE) of HIV-1 RNA. Since RHA does not associate well with all the highly structured regions of HIV-1 RNA tested in vivo, the results suggest that other viral or cellular factors not present in vitro may modulate the direct interaction of RHA with HIV-1 RNA during virus replication. Nevertheless, a role for duplex RNA as a target for RHA binding in vivo is suggested by the fact that the deletion of either one or both dsRBDs eliminates the in vivo interaction of RHA with HIV-1 RNA. Furthermore, these mutant RHAs do not promote the in vivo annealing of tRNA(3)(Lys) to viral RNA, nor are they packaged into virions, demonstrating that the dsRBDs are essential for the role of RHA in HIV-1 replication.
authors.affiliation: Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Li
authors.initials: L
authors.lastname: Xing
authors.forename: Meijuan
authors.initials: M
authors.lastname: Niu
authors.forename: Lawrence
authors.initials: L
authors.lastname: Kleiman
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005454:Fluorescence Polarization; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D020365:RNA Helicases / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D020365:RNA Helicases