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BIOCHEMICAL ANALYSIS OF HIV-1 INTEGRASE INTERACTIONS WITH TRANSPORTIN-SR2 REVEALS FUNCTIONAL PROTEIN-PROTEIN CONTACTS Ross Larue (1), Frauke Christ (2), Christiane Wuensch (1), Eric Danhart (3), Elihu Ihms (3), Mark Foster (3), Zeger Debyser (2), Mamuka Kvaratskhelia (1) (1) Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH 43210; (2) Laboratory of Molecular Virology and Gene Therapy, Division of Molecular Medicine, Katholieke Universiteit Leuven (KULeuven), Leuven, Flanders, Belgium; (3) Department of Biochemistry, College of Arts and Sciences, The Ohio State University, Columbus, OH 43210 Transportin-SR2 (TRN-SR2, TNPO3, Transportin 3) has been shown to be an important factor for the nuclear import of HIV-1 preintegration complex and to affect the integration targeting into the chromosomal sites. However, two separate studies have implicated distinct mechanisms for these observations, either through TRN-SR2 interacting with HIV-1 integrase (IN) or capsid (CA). In the present study, we used purified recombinant proteins to examine TRN-SR2 interactions with either HIV-1 IN or CA. In agreement with one of the previous reports, TRN-SR2 interacted with HIV-1 IN in vitro, but under the same assay conditions we did not detect binding of TRN-SR2 with CA. Next we examined interactions of TRN-SR2 with the stable synaptic complex (SSC), which was formed with recombinant HIV-1 IN and synthetic viral DNA. TRN-SR2 affectively bound to the SSC, but not to naked viral DNA, suggesting that TRN-SR2 can directly engage the HIV-1 IN tetramer prebound to the cognate DNA. To map the protein-protein contacts between HIV-1 IN and TRN-SR2, we used mass spectrometry-based protein footprinting approach. Our results revealed several surface exposed C-terminal domain (CTD) residues of HIV-1 IN that were shielded from modification upon binding to TRN-SR2. Mutation of these residues markedly reduced interactions between HIV-1 IN and cellular TRN-SR2. Analysis of secondary structural elements in TRN-SR2 with circular dichroism demonstrated that the protein is highly alpha helical in nature. Furthermore, molecular shapes of TRN-SR2 and its complex with the CTD were elucidated using small-angle X-ray scattering which confirmed that these two proteins are seen bound in the complex. These experimental findings have been employed to create a molecular model for the SSC interactions with TRN-SR2. We propose that in the context of the SSC, two IN monomers tightly interact with viral DNA, while the other two monomers that are surface exposed readily engage TRN-SR2 to form a ternary complex between viral DNA, IN tetramer and TRN -SR2.