title: Interaction of the HIV-1 intasome with transportin 3 protein (TNPO3 or TRN-SR2).
abstract: Transportin 3 (TNPO3 or TRN-SR2) has been shown to be an important cellular factor for early steps of lentiviral replication. However, separate studies have implicated distinct mechanisms for TNPO3 either through its interaction with HIV-1 integrase or capsid. Here we have carried out a detailed biophysical characterization of TNPO3 and investigated its interactions with viral proteins. Biophysical analyses including circular dichroism, analytical ultracentrifugation, small-angle x-ray scattering, and homology modeling provide insight into TNPO3 architecture and indicate that it is highly structured and exists in a monomer-dimer equilibrium in solution. In vitro biochemical binding assays argued against meaningful direct interaction between TNPO3 and the capsid cores. Instead, TNPO3 effectively bound to the functional intasome but not to naked viral DNA, suggesting that TNPO3 can directly engage the HIV-1 IN tetramer prebound to the cognate DNA. Mass spectrometry-based protein footprinting and site-directed mutagenesis studies have enabled us to map several interacting amino acids in the HIV-1 IN C-terminal domain and the cargo binding domain of TNPO3. Our findings provide important information for future genetic analysis to better understand the role of TNPO3 and its interacting partners for HIV-1 replication.
authors.affiliation: Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Ross
authors.initials: R
authors.lastname: Larue
authors.forename: Kushol
authors.initials: K
authors.lastname: Gupta
authors.forename: Christiane
authors.initials: C
authors.lastname: Wuensch
authors.forename: Nikolozi
authors.initials: N
authors.lastname: Shkriabai
authors.forename: Jacques J
authors.initials: JJ
authors.lastname: Kessl
authors.forename: Eric
authors.initials: E
authors.lastname: Danhart
authors.forename: Lei
authors.initials: L
authors.lastname: Feng
authors.forename: Oliver
authors.initials: O
authors.lastname: Taltynov
authors.forename: Frauke
authors.initials: F
authors.lastname: Christ
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Van Duyne
authors.forename: Zeger
authors.initials: Z
authors.lastname: Debyser
authors.forename: Mark P
authors.initials: MP
authors.lastname: Foster
authors.forename: Mamuka
authors.initials: M
authors.lastname: Kvaratskhelia
mesh_terms: D002942:Circular Dichroism; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019427:HIV Integrase; D015497:HIV-1 / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D014779:Virus Replication / Q000502:physiology; D014961:X-Ray Diffraction; D028961:beta Karyopherins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004279:DNA, Viral; C546476:TNPO3 protein, human; D028961:beta Karyopherins; D019427:HIV Integrase