NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

Abstract

BackgroundHIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process.ResultsWe engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs.ConclusionsOur data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

Phosphorylation Targets of DNA-PK and Their Role in HIV-1 Replication

“…The dependence of the HIV-1 replication on the DNA-PK-complex components has been previously shown by different groups [ 10 , 101 , 102 , 103 , 104 , 105 , 106 , 107 ]. We previously showed that the phosphorylating activity of DNA-PKcs was necessary for HIV-1 PIR [ 10 ]. To search for possible downstream proteins involved in this process, we analyzed the data on the role of DNA-PK targets in Table 1 in HIV-1 replication.…”

“…One more example of the non-canonical activation of DNA-PKcs has been discovered by studying how the DNA-PK complex is involved in HIV-1 post-integrational repair (PIR) [ 10 ]. The integration of HIV-1 genome is vital for efficient production of new viral particles.…”

“…Using a modified variant of gag-alu specific PCR [ 12 ], we found that DNA-PK components, Ku70, Ku80, and DNA-PKs, are involved in PIR of the integration intermediate [ 10 ]. Moreover, the recruitment of DNA-PK to viral DNA integration sites and the activation of its catalytic subunit is mediated by the direct interaction of HIV-1 integrase (IN) (the enzyme accomplishing the integration of viral DNA into the cell’s genome) and Ku70, a part of DNA-PK [ 10 ] ( Figure 1 ). The impaired interaction between these proteins decreases the infectivity of VSV-G-pseudotyped HIV-like particles.…”

“…The impaired interaction between these proteins decreases the infectivity of VSV-G-pseudotyped HIV-like particles. We also showed that the phosphorylating activity of DNA-PKcs is crucial for the efficient accomplishment of PIR, since the low-molecular specific inhibitor of DNA-PKcs kinase activity Nu7441 decreases both the PIR efficiency and the pseudovirus infectivity [ 10 ]. Thus, it is obvious, that DNA-PK as well as its targets, phosphorylated during PIR, are necessary for the efficient completion of this process.…”

“…Importantly, there are no double-stranded DNA breaks in the product of viral DNA integration. In this case, DNA-PK activation is triggered by IN binding to Ku70, since IN mutations disturbing this binding significantly reduce the PIR efficiency and disrupt the pseudovirus sensitivity to Nu7441 [ 10 ]. Therefore, this is an example of non-canonical activation of DNA-PK due to protein–protein binding.…”