Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form.

There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme.

Impact of immigration on HIV-1 molecular epidemiology in West Africa, Maghreb and Southern Europe.

There is global concern about the relation between international migration and the course of the AIDS epidemic. Maghreb is a North African region, which lies between sub-Saharan Africa and Europe. It has been turned recently into a region of immigration, since there are more and more flows of West African migrants hoping to reach European countries. Here we provide an overview on HIV-1 molecular epidemiology particularly in West African countries, Maghreb (Morocco, Algeria, Tunisia) and southern European countries (Spain, France, and Italy). The studies conducted in several countries of the region revealed different features of HIV-1 molecular epidemiology, especially for the distribution of viral subtypes and for transmitted drug resistance profiles. Furthermore, migration from West Africa to Europe seems to be a potential source of non-B subtype mobility to Maghreb and eventually to southern Europe, where HIV-1 non-B variants significantly increased in the last 10 to 15 years. As genetic differences between subtypes might impact the drug resistance pathways, it is important to provide continuous surveillance programs for the early detection of new variants spreading in the population before they become more prevalent, and to identify resistance profiles in different infected populations, especially migrants.

Stabilization of the integrase-DNA complex by Mg2+ ions and prediction of key residues for binding HIV-1 integrase inhibitors.

The HIV-1 integrase is an attractive target for the therapeutics development against AIDS, as no host homologue of this protein has been identified. The integrase strand transfer inhibitors (INSTIs), including raltegravir, specifically target the second catalytic step of the integration process by binding to the DDE motif of the catalytic site and coordinating Mg(2+) ions. Recent X-ray crystallographic structures of the integrase/DNA complex from prototype foamy virus allowed to investigate the role of the different partners (integrase, DNA, Mg(2+) ions, raltegravir) in the complex stability using molecular dynamics (MD) simulations. The presence of Mg(2+) ions is found to be essential for the stability, whereas the simultaneous presence of raltegravir and Mg(2+) ions has a destabilizing influence. A homology model of HIV-1 integrase was built on the basis of the X-ray crystallographic information, and protein marker residues for the ligand binding were detected by clustering the docking poses of known HIV-1 integrase inhibitors on the model. Interestingly, we had already identified some of these residues to be involved in HIV-1 resistance mutations and in the stabilization of the catalytic site during the MD simulations. Classification of protein conformations along MD simulations, as well as of ligand docking poses, was performed by using an original learning method, based on self-organizing maps. This allows us to perform a more in-depth investigation of the free-energy basins populated by the complex in MD simulations on the one hand, and a straightforward classification of ligands according to their binding residues on the other hand.

Characterization of protease resistance-associated mutations in HIV type 1 drug-naive patients following the increasing prevalence of the CRF02_AG strain in Morocco.

The purpose of this study was to investigate the amino acid substitutions in the protease of HIV-1 B and non-B subtypes and evaluate whether the emergence of resistance-associated mutations (RAMs) could have a significant correlation with the increasing prevalence of CRF02_AG strains in Morocco. A total of 162 protease gene sequences were successfully amplified from drug-naive HIV-1-infected individuals. We identified eight (sub)subtypes and CRFs: B(66%), A1(3.7%), C(1.2%), F1(0.6%), F2(0.6%), G(1.2%), CRF02_AG(25.3%), and CRF01_AE(1.2%). Phylogenetic analysis of CRF02_AG strains showed that 9.8% of isolates had a closer connection with reference strains from Morocco and 15.4% clustered with reference strains from eight West African and three European countries. When compared to the B subtype, patients with the CRF02_AG strain had a significantly higher prevalence of mutations associated with resistance to some antiprotease drugs, mainly tipranavir (TPV): H69K (97% vs. 5%; p<0.0001), L89M (95% vs. 1%; p<0.0001), and M36I/L (93% vs. 44%; p<0.0001). Most of the CRF02_AG strains (97%) significantly showed at least two TPV-RAMs (p=0.002) compared to the B subtype (7%). Multivariate analysis revealed that CRF02_AG infection was the only factor highly associated with the occurrence of more than two TPV-RAMs (C=0.42; p<0.0001). These results support the importance of transmitted drug resistance mutations (M36I/L, H69K, and L89M) in the protease gene of HIV-1 CRF02_AG isolates. This HIV drug resistance transmission before protease inhibitor (PI) exposure raises concern about its influence on the susceptibility of CRF02_AG strains to some PIs, especially tipranavir, which will soon be introduced as part of the second line therapeutic regimens in Morocco.