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The human Ku heterodimer is composed of two subunits: Ku70 and Ku80. Its main function is the binding of double-strand DNA breaks during the first steps of the NHEJ repair process. The involvement of the Ku heterodimer in the HIV-1 replication has been shown in various reports. Ku affects different stages of the viral replication cycle particularly the integration and transcription. The integration can be influenced by the functioning of Ku in the DNA repair machinery as well as by a direct stabilization of the viral integrase (IN) by the Ku70 subunit. Earlier we have shown that a stable complex is formed by recombinant HIV-1 IN and Ku70 when both proteins are expressed in bacteria. We performed a mutational analysis and showed that the main binding site within IN and Ku70 is located in the N-terminal domain of Ku70 (a.a. 1-250) and the helix α6 of IN (a.a. 200-220) that links its catalytic and C-terminal domains. Moreover, alanine substitutions of amino acid residues E212 and L213 of IN significantly impede the complex stability not only when it is tested on recombinant proteins but also when the complex is immunoprecipitated from cellular lysates. Additionally we have shown that the cellular stability of HIV-1 IN is indeed positively affected by Ku70 while the influence of Ku80 subunit is rather weak. Furthermore, the integration of a single-round HIV-based luciferase reporter vector in cell lines with a stably reduced Ku70 expression is significantly weakened. Using luciferase reporter assay in human cells that are either depleted of or superexpressing Ku70/Ku80/both we have shown that it is the Ku80 subunit that is essential for positive transcription regulation. Altogether, our results suggest a differential role for the two subunits of the Ku heterodimer in the HIV-1 replication cycle.