Both ATM and DNA-PK Are the Main Regulators of HIV-1 Post-Integrational DNA Repair

The integration of a DNA copy of an HIV-1 RNA genome into the host genome, carried out by the viral enzyme integrase, results in the formation of single-stranded gaps in cellular DNA that must be repaired. Here, we have analyzed the involvement of the PI3K kinases, ATM, ATR, and DNA-PKcs, which are important players in the DNA damage response (DDR) in HIV-1 post-integrational DNA repair (PIR). The participation of the DNA-PK complex in HIV-1 PIR has been previously shown, and the formation of a complex between the viral integrase and the DNA-PK subunit, Ku70, has been found to be crucial for efficient PIR. Now, we have shown that the inhibition of both DNA-PKcs and ATM, but not ATR, significantly reduces PIR efficiency. The activation of both kinases is a sequential process, where one kinase, being activated, activates the other, and it occurs simultaneously with the integration of viral DNA. This fact suggests that the activation of both kinases triggers PIR. Most interestingly, the activation of not only DNA-PKcs, but also ATM depends on the complex formation between integrase and Ku70. The elucidation of the interactions between viruses and DDR is important both for understanding the modulation of host cell functions by these pathogens and for developing new approaches to combat viral infections

Complex Relationships between HIV-1 Integrase and Its Cellular Partners

RNA viruses, in pursuit of genome miniaturization, tend to employ cellular proteins to facilitate their replication. HIV-1, one of the most well-studied retroviruses, is not an exception. There is numerous evidence that the exploitation of cellular machinery relies on nucleic acid-protein and protein-protein interactions. Apart from Vpr, Vif, and Nef proteins that are known to regulate cellular functioning via interaction with cell components, another viral protein, integrase, appears to be crucial for proper virus-cell dialog at different stages of the viral life cycle. The goal of this review is to summarize and systematize existing data on known cellular partners of HIV-1 integrase and their role in the HIV-1 life cycle

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

The DNA dependent protein kinase (DNA-PK) is a trimeric nuclear complex consisting of a large protein kinase and the Ku heterodimer. The kinase activity of DNA-PK is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). We also showed that the kinase activity of DNA-PK is essential for post-integrational DNA repair in the case of HIV-1 infection. Besides, DNA-PK is known to participate in such cellular processes as protection of mammalian telomeres, transcription, and some others where the need for its phosphorylating activity is not clearly elucidated. We carried out a systematic search and analysis of DNA-PK targets described in the literature and identified 67 unique DNA-PK targets phosphorylated in response to various in vitro and/or in vivo stimuli. A functional enrichment analysis of DNA-PK targets and determination of protein–protein associations among them were performed. For 27 proteins from these 67 DNA-PK targets, their participation in the HIV-1 life cycle was demonstrated. This information may be useful for studying the functioning of DNA-PK in various cellular processes, as well as in various stages of HIV-1 replication

A qPCR assay for measuring the post-integrational DNA repair in HIV-1 replication

Abstract The post-integrational gap repair is a critical and poorly studied stage of the lentiviral life cycle. It might be performed by various cellular DNA repair pathways but the exact mechanism of the repair process has not yet been described. One of the reasons for that is the lack of a functional quantitative assay that could precisely measure the amount of integrated viral DNA that has completed the post-integrational gap repair stage. Here, we present an approach that is based on a widely used Alu-specific PCR for the estimation of integrated viral DNA but includes several steps that allow discrimination between integrated-repaired and integrated-unrepaired viral DNA forms. We used the approach for the estimation of the kinetics of gap repair in a viral vector system and showed that the gap repair process starts at 17 h post infection and lasts 10 more hours. We also showed that the addition of Nu7441 – a small molecule inhibitor of DNA-breaks sensor kinase in the non-homologous end joining DNA repair pathway – specifically inhibits the gap repair process while having no influence on the integration itself

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

The search for compounds that can inhibit the interaction of certain viral proteins with their cellular partners is a promising trend in the development of antiviral drugs. We have previously shown that binding of HIV-1 integrase with human Ku70 protein is essential for viral replication. Here, we present a novel, cheap, and fast assay to search for inhibitors of these proteins’ binding based on the usage of genetically encoded fluorescent tags linked to both integrase and Ku70. Using this approach, we have elucidated structure-activity relationships for a set of oligonucleotide conjugates with eosin and shown that their inhibitory activity is primarily achieved through interactions between the conjugate nucleic bases and integrase. Molecular modeling of HIV-1 integrase in complex with the conjugates suggests that they can shield E212/L213 residues in integrase, which are crucial for its efficient binding to Ku70, in a length-dependent manner. Using the developed system, we have found the 11-mer phosphorothioate bearing 3’-end eosin-Y to be the most efficient inhibitor among the tested conjugates

Complex of HIV-1 Integrase with Cellular Ku Protein: Interaction Interface and Search for Inhibitors

The interaction of HIV-1 integrase and the cellular Ku70 protein is necessary for HIV replication due to its positive effect on post-integration DNA repair. We have previously described in detail the Ku70 binding site within integrase. However, the integrase binding site in Ku70 remained poorly characterized. Here, using a peptide fishing assay and site-directed mutagenesis, we have identified residues I72, S73, and I76 of Ku70 as key for integrase binding. The molecular dynamics studies have revealed a possible way for IN to bind to Ku70, which is consistent with experimental data. According to this model, residues I72 and I76 of Ku70 form a “leucine zipper” with integrase residues, and, therefore, their concealment by low-molecular-weight compounds should impede the Ku70 interaction with integrase. We have identified such compounds by molecular docking and have confirmed their capacity to inhibit the formation of the integrase complex with Ku70. Our data demonstrate that the site of IN binding within Ku70 identified in the present work may be used for further search for inhibitors of the integrase binding to Ku70