Elucidacion de las regiones membranotropicas de la glicoproteina gp41 del hiv1 y su implicacion en el proceso de fusion. Busqueda de inhibidores de la entrada del hiv1

Resumen

In this Thesis we depict a methodology to elucidate the membranotropic regions along viral fusion proteins, we have applied this methodology for the HIV-1 envelope glycoprotein gp41 (Chapter III). Afterwards, two selected membranotropic regions, one pertaining to the initial part of NHR ectodomain and the other one pertaining to the LLP2/3 endodomain, were biophysically characterized (Chapters IV and V). In the last part of the Thesis (chapter VI), we searched for peptide inhibitors for the HIV-1 entry mediated by qp41. Therefore, this Thesis includes biophysical studies as screening and cellular assays. Membrane fusion processes need to overcome several energetic barriers. In the case of HIV-1, the protein that permits the virus to introduce its capside into the host cell is gp41. Nowadays, scientists do not know exactly how several HIV-1 gp41 trimers accomplish the whole viral fusion process. It is postulated that several parts of gp41 are involved in the virus-cell fusion mechanism, in chapter III, different experiments (leakage, lipid mixing and fusion), assaying several lipid compositions, were performed. It was elucidated the different membranotropic regions along the whole HIV-1 envelope gp41 by a pepscan methodology of lipid-peptide interaction. The first region belonged to the fusion peptide (FP), which increased its ability to merge membranes in the presence of anionic phospholipids. The same behaviour was shown for the beginning of the NHR region of gp41. On the other hand, other parts of the NHR region were capable of perturbing the membrane architecture. However, they did not show any fusion properties. The last membranotropic region pertaining to the gp41 ectodomain was the pre-trasmembrane region (PTM), which increased its ability to perturb and merge membranes in the presence of sphingomielin (SM) and cholesterol (Col). We also found peptides belonging to qp41 transmembrane domain (TM) that could perturb and merge membranes, highlighting the last part of it, which increases its effectiveness in the presence of anionic phospholipids. Regarding to the citoplasmatic tail of gp41, it was found several membranotropic regions pertaining mainly to lentivirus litic peptides (LLPs). The region pertaining to the LLP2/3 is the most efficient inducer of membrane perturbation as well as fusion promoting in any lipid composition. Taking together all these results and contrasting them with previous works, it could be postulated that the membrane fusion mediated by the Hiv-1 envelope gp41 is led by synchronized and specific protein-protein and protein-membrane interactions. After the viral attachment to CD4 and CXCR4 or CCR5 and subsequent insertion of the FP into the host plasma membrane to anchor the virus, the other parts of gp41 must help to catalyse the fusion process. Therefore, several proteins of gp41 are able to juxtapose, perturb and merge both viral and cell membranes, even, expand and stabilize the fusogenic pore, which permits the capsid entry into the cell cytoplasm. According to the results, it can be postulated that each part along the sequence of gp41 is thought to interact more properly with specific phospholipids, being this fact important to modify the protein-protein and protein-membrane interactions of the different gp41 regions, which will determine the course of viral fusion. In chapter IV, we studied the peptide-membrane interaction of the N-terminal part of the gp41 ectodomain NHR region and we also tried to interrelate our results with previous studies. We conclude that this region interacts and merges membranes specifically in the presence of anionic phospholipids, i.e., phosphatidilserine (PS) and the conformation could change after interaction with the membrane, being its fusogenic conformation in a-sheet. Our results complement other ones, which show that the NHR region of gp41 ectodomain increases its fusogenic capabilities when exposed to electronegative membranes as it occurs with the FP. On the other hand, other authors have shown that the N36/C34 core(six-helix bundle of gp41) is destabilized by electronegative, but not zwitterionic phospholipids, mainly after the specific N36 interaction with negative phospholipids. The NHR region-PS phospholipids interaction is promoted after the N-terminal FP insertion into the target membrane, because it could cause local membrane perturbations in cell. At the same time, these perturbations could cause the exposure of PS from the membrane inner to membrane outer. Therefore, it implies that inner leaflet phospholipids may serve as a positive regulator of fusion by controlling the kinetics of the ectodomain folding into a six-helix bundle. The six-helix bundle destabilization in presence of PS would prolong the time of the pre-hairpin intermediate life-time, ensuring pore formation and enlargement. The contribution of the ectodomain of gp41 to membrane fusion has been shown by many groups, although a gp41 fusion model has been presented explaining several hypothetical steps of the HIV-1 entry mechanism, very little is known about the roles of the transmembrane domain (TM) and the cytoplasmic tail of gp41. It is postulated that TM is primarily responsible for anchoring the envelope glycoproteins on the lipid bilayer. on the other hand, some works involve TM in the viral fusion process, but controversial results have been shown in this regard. With respect to the endodomain, membranotropic regions along the citoplasmic tail could help to perturb and expand the fusogenic pore durinq the fusion process of HIV-1, acting from inside of the viral envelope. We found three membranotropic regions potentially involved in membrane-interactions, indeed, the most significant region of the whole gp41 belongs to the LLP2/3 region. A biophysical study of this amphipathic region is presented in Chapter V. This work shows the capability of this region to produce high leakage like some antibiotic peptides in any type of membranes. It is also shown the cholesterol dependence to produce membrane fusion, although there was neither specific peptide-cholesterol interaction nor cholesterol segregation promoted by this region. Probably, this cholesterol dependence to merge membranes is due to intrinsic cholesterol properties. Recently, it has been suggested that the cytoplasmatic tail reduces the gp41 fusogenicity through a process that involves conformational changes in the gp41 ectodomain. This findings support a model in which the gp41 endodomain tightly regulates the association with Gag proteins and also the gp41 fusogenic function during its transport to the site of virion assembly. During the last decade, a great number of research have been geared in identifying newer targets for inhibiting the HIV infection as well as understanding the targets for already identified anti-HIV-1 agents. The success in converting a proof-of-concept peptide T-20, from the CHR region of the envelope glycoprotein gp41 of HIV-1, to a drug named enfuvirtide was one of the phenomenal successes in HIV-1 drug discovery research that has been made in recent years, in Chapter VI we performed several strategies for screening (pepscan, phage display and inhibition assays) with the purpose of searching peptide inhibitors against several parts of gp41, preventing the HIV-1 entry. We obtained some peptide inhibitors, although the best one had only micromolar effects. This inhibitory peptide is thousand times less efficient than the known inhibitor peptides C34 or T-20. On the other hand, we also tried to search for possible protein-protein interactions between several membranotropic regions of gp41 with specific parts of gp120 and gp41 by pepscan assays. We can highlight a possible specific interaction between the PTM and the pre-Kennedy region. This interaction could be possible only if the monomer gp41 was a 3- MSD form, where a minor ectodomain adjacent to the main ectodomain would be arranged.