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

Consensus HIV-1 subtype A integrase and its raltegravir-resistant variants: Design and characterization of the enzymatic properties.

Model studies of the subtype B and non-subtype B integrases are still required to compare their susceptibility to antiretroviral drugs, evaluate the significance of resistance mutations and identify the impact of natural polymorphisms on the level of enzymatic reactivity. We have therefore designed the consensus integrase of the HIV-1 subtype A strain circulating in the former Soviet Union territory (FSU-A) and two of its variants with mutations of resistance to the strand transfer inhibitor raltegravir. Their genes were synthesized, and expressed in E coli; corresponding His-tagged proteins were purified using the affinity chromatography. The enzymatic properties of the consensus integrases and their sensitivity to raltegravir were examined in a series of standard in vitro reactions and compared to the properties of the integrase of HIV-1 subtype B strain HXB2. The consensus enzyme demonstrated similar DNA-binding properties, but was significantly more active than HXB-2 integrase in the reactions of DNA cleavage and integration. All integrases were equally susceptible to inhibition by raltegravir and elvitegravir, indicating that the sporadic polymorphisms inherent to the HXB-2 enzyme have little effect on its susceptibility to drugs. Insensitivity of the mutated enzymes to the inhibitors of strand transfer occurred at a cost of a 30-90% loss of the efficacies of both 3'-processing and strand transfer. This is the first study to describe the enzymatic properties of the consensus integrase of HIV-1 clade A and the effects of the resistance mutations when the complex actions of sporadic sequence polymorphisms are excluded