E. S. Knyazhanskaya

Modulation of HIV-1 Integrase Activity by Single-Stranded Oligonucleotides and their Conjugates with Eosin

Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates.

Role of DNA-Dependent Protein Kinase in the HIV-1 Replication Cycle

Human immunodeficiency virus type 1 (HIV-1) is among the best-studied viruses, but some aspects of HIV-1 biology remain obscure. The role of cell proteins in virus replication raises especially many questions. One of the proteins is DNA-dependent protein kinase (DNA-PK), which performs crucially important functions in the human body. DNA-PK is known to influence at least two stages in the HIV-1 life cycle, the integration of viral genome in cell DNA and transcription of the integrated provirus. Many details regarding this influence remain unresolved. The review summarizes the known data on the DNA-PK role in the HIV-1 life cycle and its influence on the replication of other members of the family Retroviridae. In the beginning of this review there is a short explanation of the DNA-PK cellular functions that are especially important for understanding its role in the HIV-1 replication.

The search of novel inhibitors of HIV-1 integrase among 5-(4-halogenophenyl)-5-oxopentyl derivatives of nucleic bases

New nucleic base derivatives were obtained by alkylation of uracil, thymine, cytosine, adenine, 6-chloropurine, and 2-amino-6-chloropurine with 5-chloro-1-(4-halogenophenyl)-1-pentanones, and their physical and chemical properties were studied. The influence of the compounds synthesized on the HIV-1 integrase activity was studied.

Synthesis of and HIV-1 Integrase Inhibition by 2-[7-(Fluorobenzyloxy)-4-Oxo-4hchromen-3-Yl]-1-Hydroxyimidazoles

A series of six new 2-[7-(fluorobenzyloxy)-4-oxo-4H-chromen-3-yl]-1-hydroxyimidazoles were synthesized and characterized as potential HIV-1 integrase inhibitors. Prototropic tautomerism of the obtained 1-hydroxyimidazoles was discussed. Their ability to inhibit integrase catalytic activity in 3′-terminal processing and chain transfer reactions was studied. It was shown that these compounds did not exhibit noticeable inhibition.

Approaches to site-directed DNA integration based on transposases and retroviral integrases

The ability of retroviruses and transposons to insert their genome into the host cell makes them attractive objects for constructing gene therapy vectors. However, enzymes that insert genetic material do not possess any selectivity relative to target nucleotide sequences, which results in almost random DNA insertion into the recipient cell genome. This leads to mutations that in turn may cause certain undesirable consequences and sometimes neoplastic cell transformation. For successful functioning, it is a primary necessity to modify a retrovirus and transposon based genetic therapy systems in such a way that the directed vector integration into a target sequence in the human genome can be achieved. In this review, the approaches to date that have been developed for highly specific modification of the genome using fusion protein construction based on retroviral integrases and transposases are discussed, as well as cellular factors interacting with these enzymes.

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

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.

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

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.