Integration of HIV-1 genome into host cell chromosome is mediated by viral integrase (IN). The IN catalytic core (CC, IN(50-212)) dimerizes through mutual interactions of its alpha1 and alpha5 helices. Peptides INH1 and INH5 reproducing these helix sequences strongly inhibited IN. For instance, an IC(50) of 80 nM was determined for INH5 in integration assays using wild-type IN (wtIN). In size exclusion chromatography, INH1 and INH5 perturbed the association-dissociation equilibrium of both dmIN (IN(1-288)/F185K/C280S) and CC, leading to monomers as surviving species, while in circular dichroism, binding of peptides to dmIN altered the protein conformation. Thus, enzyme deactivation, subunit dissociation, and protein unfolding are events which parallel one another. The target of INH5 in the enzyme was then identified. In fluorescence spectroscopy, C(0.5) values of 168 and 44 nM were determined for the binding affinity of INH5 to IN and CC, respectively, at 115 nM subunit concentration, while interaction of INH5 with INH1 was found stronger than interaction of INH5 with itself (23 times larger in term of dissociation constants). These results strongly suggested that the alpha1 helix is the privileged target of INH5. The latter could serve as a lead for the development of new chemotherapeutic agents against HIV-1.

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The current work concerns the optimization process of phenolic compounds solid liquid extraction from grape byproducts at high temperatures and short incubation times. The effect of five experimental parameters (solidliquid ratio, particle size, time, temperature and solvent mixture) mostly believed to affect the extraction process was undertaken. A first response surface methodology experimental design was used to optimize the solid-liquid ratio and milling time parameters. A second design was used for the optimization of the quantitative and qualitative parameters. The quantitative parameters studied are: total phenolic compounds, flavonoid content, total monomeric anthocyanin composition and tannin concentration. The qualitative parameters analyzed are: antiradical activity and antioxidant capacity. The second design was based on the use of time, temperature and solvent mixture as optimization parameters. The assays were first conducted separately revealing the best experimental conditions for the maximization of each response variable alone. A simultaneous response surface methodology of all the responses taken together was then conducted, showing the optimal extraction conditions to be: 93 minutes at 94˚C and in 66% ethanol/water solvent. The maximal response values obtained for each parameter are: Total Phenolic Compounds yield (5.5 g GAE/100g DM), Flavonoid Content (5.4 g GAE/100g DM), Total Monomeric Anthocyanin yield (70.3 mg/100g DM), Tannin Concentration (12.3 g/L), Antiradical Activity (67.3%) and Total Antioxidant Capacity (393 mgAAE/L). All of the optimal values were acquired at 3 mL/g solid-liquid ratio and 6.8 min milling time. The obtained extracts could be used as natural bioactive compounds in several industrial applications.

The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is sequence-specific, and that of the host DNA does not require particular sequence, although in physicochemical studies IN fails to discriminate between the two interactions. Here, such discrimination was allowed thanks to a model system using designed oligonucleotides and peptides as binding structures. Spectroscopic (circular dichroism, NMR, and fluorescence anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly indicated that the amphipathic helix ␣4, located at the catalytic domain surface, is responsible for the specific high affinity binding of the enzyme to viral DNA. Analogues of the ␣4 peptide having increased helicity and still bearing the biologically relevant lysines 156 and 159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are required to achieve high affinity complexes (K d of 1.5 nM). Data corroborate previous in vivo results obtained with mutated viruses.

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Integration of the human immunodeficiency virus (HIV‐1) DNA into the host genome is catalysed by a virus‐encoded protein integrase. Here, we report some of the structural and functional properties of two synthetic peptides: integrase‐(147–175)‐peptide reproducing the residues 147–175 (SQGVVESMNKELK159KIIGQVRDQAEHLKTAY) of the HIV‐1 integrase, and [Pro159] integrase‐(147–175)‐peptide where the lysine 159 is substituted for a proline. Circular dichroism revealed that both peptides are mostly under unordered conformation in aqueous solution, contrasting with the α‐helix exhibited by residues 147–175 in the protein crystal structure. In a weak α‐helix‐promoting environment, integrase‐(147–175)‐peptide self‐associated into stable coiled‐coil oligomers, while [Pro159] integrase‐(147–175)‐peptide did not. This property was further confirmed by cross‐linking experiments. In our in vitro experiments, only integrase‐(147–175)‐peptide was able to reduce the integration activity of the enzyme. We propose that the inhibitory activity shown by integrase‐(147–175)‐peptide is dependent on its ability to bind to its counterpart in integrase through a peptide‐protein coiled‐coil structure disturbing the catalytic properties of the enzyme.

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