Role of I182, R187, and K188 Amino Acid Residues in the Catalytic Domain of HIV-1 Integrase in the Processes of Reverse Transcription and Integration.

Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3'-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of the newly formed virions. It has been suggested that HIV-1 integrase is a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain, I182, R187, and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in formation of the integrase structure, which is important for the effective reverse transcription.

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.

KuINins as a New Class of HIV-1 Inhibitors That Block Post-Integration DNA Repair.

Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins.

DNA sequence-specific ligands. XVIII. Synthesis, physico-chemical properties; genetic, virological, and biochemical studies of fluorescent dimeric bisbenzimidazoles DBPA(n).

A set of AT-specific fluorescent dimeric bisbenzimidazoles DBPA(n) with linkers of different lengths bound to DNA in the minor groove were synthesized and their genetic, virological, and biochemical studies were performed. The DBPA(n) were shown to be effective inhibitors of the histon-like protein H-NS, a regulator of the DNA transcription factor, as well as of the Aliivibrio logei Quorum Sensing regulatory system in E. coli cells. Their antiviral activity was tested in model cell lines infected with herpes simplex virus type I. Also, it was found that DBPA(n) could inhibit catalytic activities of HIV-1 integrase at low micromolar concentrations. All of the dimeric bisbenzimidazoles DBPA(n) manifested fluorescent properties, were well soluble in water, nontoxic up to concentrations of 200 µM, and could penetrate into nuclei followed by binding to DNA.

The cellular SFPQ protein as a positive factor in the HIV-1 integration.

The cellular SFPQ protein is involved in several stages of the HIV-1 life cycle, but the detailed mechanism of its involvement is not yet fully understood. Here, the role of SFPQ in the early stages of HIV-1 replication has been studied. It is found that changes in the intracellular level of SFPQ affect the integration of viral DNA, but not reverse transcription, and SFPQ is a positive factor of integration. A study of the SFPQ interaction with HIV-1 integrase (IN) has revealed two diRGGX1-4 motifs in the N-terminal region of SFPQ, which are involved in IN binding. Substitution of a single amino acid residue in any of these regions led to a decrease in binding efficiency, while mutations in both motifs almost completely disrupted the SFPQ interaction with IN. The effect of the SFPQ mutants with impaired ability to bind IN on viral replication has been analyzed. Unlike the wild-type protein, the SFPQ mutants did not affect viral integration. This confirms that SFPQ influences the integration stage through direct interaction with IN. Our results indicate that the SFPQ/IN complex can be considered as a potential therapeutic target for the development of new inhibitors of HIV replication.

Cellular Immune Response Induced by DNA Immunization of Mice with Drug Resistant Integrases of HIV-1 Clade A Offers Partial Protection against Growth and Metastatic Activity of Integrase-Expressing Adenocarcinoma Cells.

Therapeutic DNA-vaccination against drug-resistant HIV-1 may hinder emergence and spread of drug-resistant HIV-1, allowing for longer successful antiretroviral treatment (ART) up-to relief of ART. We designed DNA-vaccines against drug-resistant HIV-1 based on consensus clade A integrase (IN) resistant to raltegravir: IN_in_r1 (L74M/E92Q/V151I/N155H/G163R) or IN_in_r2 (E138K/G140S/Q148K) carrying D64V abrogating IN activity. INs, overexpressed in mammalian cells from synthetic genes, were assessed for stability, route of proteolytic degradation, and ability to induce oxidative stress. Both were found safe in immunotoxicity tests in mice, with no inherent carcinogenicity: their expression did not enhance tumorigenic or metastatic potential of adenocarcinoma 4T1 cells. DNA-immunization of mice with INs induced potent multicytokine T-cell response mainly against aa 209-239, and moderate IgG response cross-recognizing diverse IN variants. DNA-immunization with IN_in_r1 protected 60% of mice from challenge with 4Tlluc2 cells expressing non-mutated IN, while DNA-immunization with IN_in_r2 protected only 20% of mice, although tumor cells expressed IN matching the immunogen. Tumor size inversely correlated with IN-specific IFN-γ/IL-2 T-cell response. IN-expressing tumors displayed compromised metastatic activity restricted to lungs with reduced metastases size. Protective potential of IN immunogens relied on their immunogenicity for CD8+ T-cells, dependent on proteasomal processing and low level of oxidative stress.