Characterization of HIV-1 integrase interaction with human Ku70 protein and initial implications for drug targeting

Abstract

Human Ku70/Ku80 protein is known to influence HIV-1 replication. One of the possible reasons may be the protection of integrase from proteasomal degradation by Ku70 subunit. We demonstrated that recombinant HIV-1 integrase and Ku70 form a stable complex, while no interaction of Ku70 with integrase from prototype foamy virus was observed. By analyzing protein subdomains we determined two binding sites in the structure of both Ku70 and integrase: the 51–160 a.a. region of integrase interacts with residues 251–438 of Ku70, whereas Ku70 N-terminal domain (1–250 a.a.) contacts an α6-helix in the 200–220 a.a. integrase region. Single substitutions within integrase (E212A or L213A) block the interaction with Ku70 thus indicating that the binding site formed by the 200–220 a.a. integrase region is crucial for complex formation. E212A/L213A substitutions decreased the integrase capacity to bind Ku70 in HEK293T cells. A conjugate of 2′-ОMe-GGUUUUUGUGU oligonucleotide with eosin is shown by molecular modeling to shield integrase residues E212/L213 and is effective in blocking complex formation of Ku70 with integrase what makes the complex between α6-helix and Ku70(1–250) a possible target for drug development.

Targeting the DNA-PK complex: Its rationale use in cancer and HIV-1 infection

“…Ku70 is also implicated in the activation of HIV-1 gene transcription [75]. These latter mechanisms add new insight of additional functions of Ku70 which has been also known to increase HIV-1 replication by other mechanism than retroviral DNA integration [76].…”

Analysis of RNA binding properties of human Ku protein reveals its interactions with 7SK snRNA and protein components of 7SK snRNP complex

“…For the construction of heterodimer Ku expression, vector pET15b_His6_Ku70_ (-1nt) GGTGGCAGCAGCCAACTCGAGTTCCTTTCGGGCTTTGTT-3'). Then, human Ku80 coding sequence (corresponded to NP_066964.1) was amplified from a pCDNA3_Ku80_3xFLAG vector (described in [49]) with primers Ku80-N-BamHI (5'-GGATCCTTAAAAGAGGAATTATAATGGTGCGGTCGGGGAATAA-3') and Ku80-C-XhoI (5'-CTCGAGTTATATCATGTCCAATAAATCGTCCAC -3') and cloned between BamHI and XhoI restriction sites into the vector pET15b-Ku70, obtained at the previous step. The primer Ku80-N-BamHI introduced the Shine-Dalgarno sequence GAGG, the intercistronic sequence AATT, and a stop codon at the 3'-end of the Ku70 gene after the ligation of the PCR product into the vector for the optimal translation efficiency of the Ku80 gene located with -1nt shift relative to the stop codon of the Ku70 gene [50].…”

“…Purification from E.coli cells is mainly used only for truncated Ku proteins [58,59]. Earlier, we designed a method for a recombinant Ku70 subunit production, taking advantages of two-tags (His6-and GST-) protein purification [48,49], and here tried to apply this method to Ku80 production.…”

A Fluorescent Assay to Search for Inhibitors of HIV-1 Integrase Interactions with Human Ku70 Protein, and Its Application for Characterization of Oligonucleotide Inhibitors

“…Another cellular protein Ku70 has been also identified as a host partner for HIV-1 IN [ 15 , 16 , 17 ]. It protects IN from proteasomal degradation by an unknown mechanism [ 15 ] and participates in post-integrational DNA repair [ 17 ].…”

“…Despite the absence of DSB in the HIV-1 integration intermediate (a product of viral DNA insertion into the cell genome produced by IN), the interaction of IN and Ku70 promotes recruitment of the functional DNA-PK complex, the phosphorylation activity of which is necessary for post-integrational DNA repair [ 17 ]. Recently, we have identified point amino acid mutations in IN, which prevent Ku70 binding [ 16 ] and impair HIV-1 replication by affecting post-integrational DNA repair [ 17 ]. Thereby, the search for inhibitors impairing the interaction between IN and Ku70 is a relevant task.…”