ИСТИНА

The human Ku heterodimer consists of two subunits: Ku70 and Ku80. Its main function is the binding of DNA ends produced by double-strand DNA brakes during the first steps of the NHEJ repair process. Ku can also take part in transcription regulation, telomere maintenance, protein turn-over and cytoplasmic DNA-sensing. Reportedly, Ku participates in the HIV-1 replication and favors different stages of the HIV-1 life cycle, such as the formation of 2-LTR circles, integration and transcription of proviral DNA. Viral replication is reduced in cells depleted of either component of Ku and this effect is more pronounced during the early stages of viral replication (Zheng Y., 2011; Manic G., 2013). However, an exact mechanism by which Ku affects the replication of HIV-1 is unclear. It has been proposed, that the binding of Ku70 to HIV-1 integrase (IN) protects the latter from proteasomal degradation. We have shown that a stable complex can be formed between recombinant Ku70 and IN with a Kd ~ 70 nM. Using a set of E. coli expressed deletion mutants of both Ku70 and IN and the GST pull-down assay we localized the binding sites within both proteins. The binding of Ku70 with IN relies at least on two sites in the proteins structure. Specifically, the Ku70(1-250) contacts with an α- helix located in the 160-230 IN region. This observation is supported by the fact that IN with alanine substitutions in positions Q209, E212 and L213 shows a weaker binding with Ku70 and does not bind Ku70(1-250) completely (Fig. 1). The data obtained from experiments on recombinant purified proteins were confirmed by expressing C-terminal HA-tagged fulllength IN and its various deletion mutants in HEK 293T cells with or without a WT Ku70-3FLAG and truncated variants. The coexpression with Ku70 stabilized IN, while the IN expression in cells that were knocked down of Ku70 was reduced. Also, the overexpression of Ku70 diminishes the association of IN with 20S proteasome as shown by immunoprecipitation of transfected cell lysates.