ИСТИНА

Ku is a heterodimer complex composed of two DNA-binding subunits, Ku70 and Ku80, displaying essential functions for human cell survival. At the same time, Ku has been identified as a cellular factor important for HIV-1 replication. It is known that the levels of viral replication are significantly lowered in cells depleted of Ku70. Reportedly, Ku complex may act at different stages of the HIV-1 cycle, such as the formation of 2-LTR circles, integration and transcription of the integrated provirus. Remarkably, Ku may also be incorporated into virions during viral propagation. However, the precise impact of Ku on HIV-1 expression remains unclear and requires further examinations. A putative role of Ku in the HIV-1 integration is that its Ku70 subunit protects viral integrase (IN) from degradation by a direct interaction. Thus, the inhibition of Ku70-IN interaction might affect viral replication. A detailed structure of Ku70/IN complex would greatly facilitate the inhibitor design. In our work we have proven the existence of a stable complex between purified Ku70 and HIV-1 IN with a Kd ~ 70 nM. To gain insights on the structure of Ku70/IN complex, we performed a systematic analysis of subdomains within IN that are required for the complex formation. We used both pull-down and SPR-technique to elucidate interactions of full-length Ku70 with IN fragments. N-His6-tagged HIV-1 IN separate domains (N-terminal (1-50 aa), catalytic (51-220 aa) and C-terminal (220-270 aa)) were expressed in E. coli. Several truncated IN variants containing amino acids 1-160, 1-220, 51-160 and 51-280 were also prepared. A full-size Ku70 with a GST-tag on its N-terminus was purified from E. coli. All the experiments performed showed that neither N-terminal nor C-terminal domains of HIV-1 IN are essential for its binding with Ku70 despite a weak binding capacity retaining to the C-terminal domain. The catalytic core (51-220 aa) as well as the mutant lacking C-terminal domain (1-220) both demonstrated affinity to Ku70 comparable to the affinity of the full-size IN, whereas its truncated variant (51-160 aa) bound to Ku70 protein only weakly. We also expressed a C-terminal HA-tagged full-length IN and its 1-220 variant in HEK 293T cells together with a WT Ku70-3FLAG and showed that both IN variants are stabilized by co-expression with Ku70 by approx. twofold. We hypothesize that the binding surface within IN lies in the region from 160 to 230 a.a. that is a long α-helix. We have shown that a homologous integrase from prototype foamy virus that lacks this structural element does not bind to Ku70. It is worth noting that Ku70 does not affect the interaction of IN with its major cellular partner – LEDGF/p75 as well as its interaction with the DNA substrate. This work was supported by an RFBR grant 14-04-00833 and by an RSCF grant 14-14-00489.