Julia Agapkina

Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor

Integration of the human immunodeficiency virus (HIV‐1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild‐type full‐length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA‐binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral‐induced pathologies.

Probing of HIV-1 integrase/DNA interactions using novel analogs of viral DNA

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2′-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3′-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7–9 affected 3′-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5–8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

Specific features of HIV-1 integrase inhibition by bisphosphonate derivatives

The integration of viral DNA into the cell genome is one of the key steps in the replication cycle of human immunodeficiency virus type 1 (HIV-1). Therefore, the viral enzyme integrase (IN) catalyzing this process is of great interest as a target for new antiviral agents. We performed a structural-functional analysis of five different series of methylenebisphosphonates (BPs), PO3H2-C(R)(X)-PO3H2, as IN inhibitors with the goal of assessing structural elements required for the inhibitory activity. We found that IN is inhibited only by BP bearing a chlorobenzyl substituent R at the bridging carbon of the P-C-P backbone. These BP inhibited both IN-catalyzed reactions with similar efficacies. They were also active toward some INs with mutations characteristic for HIV-1 strains resistant to strand transfer inhibitors. The study of the mechanism of the IN inhibition by various BP showed that it is effected by the nature of the second substituent (X) at the bridging carbon. Among the tested compounds, only the BP with the amino group bound directly to the BP bridging carbon was found to be a noncompetitive inhibitor and, hence, it can be promising for further studies as potential inhibitor of the IN activity within the preintegration complex.

Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells.

Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathways.

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.

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.