Identification of Residues in the C-terminal Domain of HIV-1 Integrase That Mediate Binding to the Transportin-SR2 Protein

• Published in: The Journal of biological chemistry 2012-10, Vol.287 (41), p.34059-34068
• Main Authors: De Houwer, Stephanie, Demeulemeester, Jonas, Thys, Wannes, Taltynov, Oliver, Zmajkovicova, Katarina, Christ, Frauke, Debyser, Zeger
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; beta Karyopherins - chemistry; beta Karyopherins - genetics; beta Karyopherins - metabolism; HIV; HIV Integrase - chemistry; HIV Integrase - genetics; HIV Integrase - metabolism; HIV-1 - enzymology; HIV-1 - genetics; Host-Pathogen Interactions; Humans; Integrase; Lentivirus; Microbiology; Mutagenesis, Site-Directed; Mutation, Missense; Nuclear Transport; Peptide Interactions; Peptide Mapping; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Protein-Protein Interactions; Reverse Transcription - physiology; Viral Protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.387944

Transportin-SR2 (TRN-SR2 and TNPO3) is a cellular cofactor of HIV replication that has been implicated in the nuclear import of HIV. TRN-SR2 was originally identified in a yeast two-hybrid screen as an interaction partner of HIV integrase (IN) and in two independent siRNA screens as a cofactor of viral replication. We have now studied the interaction of TRN-SR2 and HIV IN in molecular detail and identified the TRN-SR2 interacting regions of IN. A weak interaction with the catalytic core domain (CCD) and a strong interaction with the C-terminal domain (CTD) of IN were detected. By dissecting the catalytic core domain (CCD) of IN into short structural fragments, we identified a peptide (INIP1, amino acids 170EHLKTAVQMAVFIHNFKRKGGI191) retaining the ability to interact with TRN-SR2. By dissecting the C-terminal domain (CTD) of IN, we could identify two interacting peptides (amino acids 214QKQITKIQNFRVYYR228 and 262RRKVKIIRDYGK273) that come together in the CTD tertiary structure to form an exposed antiparallel β-sheet. Through site-specific mutagenesis, we defined the following sets of amino acids in IN as important for the interaction with TRN-SR2: Phe-185/Lys-186/Arg-187/Lys-188 in the CCD and Arg-262/Arg-263/Lys-264 and Lys-266/Arg-269 in the CTD. An HIV-1 strain carrying K266A/R269A in IN was replication-defective due to a block in reverse transcription, confounding the study of nuclear import. Insight into the IN/TRN-SR2 interaction interface is necessary to guide drug discovery efforts targeting the nuclear entry step of replication. Background: The molecular details of the TRN-SR2/HIV-1 IN interaction are not known. Results: Crucial amino acids in IN for the interaction with TRN-SR2 are located in the CTD, Arg-262/Arg-263/Lys-264, and Lys-266/Arg-269. Conclusion: TRN-SR2 primarily interacts with the CTD domain of IN. Significance: Understanding of the IN/TRN-SR2 interaction is necessary to guide drug discovery efforts targeting the nuclear entry step of replication.