Nef binds p6* in gagpol during replication of human immunodeficiency virus type 1

The atypical Nef protein (NefF12) from human immunodeficiency virus type 1 strain F12 (HIV-1F12) interferes with virion production and infectivity via a mysterious mechanism. The correlation of these effects with the unusual perinuclear subcellular localization of NefF12 suggested that the wild-type Nef protein could bind to assembly intermediates in late stages of viral replication. To test this hypothesis, Nef from HIV-1NL4-3 was fused to an endoplasmic reticulum (ER) retention signal (NefKKXX). This mutant NefKKXX protein recapitulated fully the effects of NefF12 on Gag processing and virion production, either alone or as a CD8 fusion protein. Importantly, the mutant NefKKXX protein also localized to the intermediate compartment, between the ER and the trans-Golgi network. Furthermore, Nef bound the GagPol polyprotein in vitro and in vivo. This binding mapped to the C-terminal flexible loop in Nef and the transframe p6* protein in GagPol. The significance of this interaction was demonstrated by a genetic assay in which the release of a mutant HIV-1 provirus lacking the PTAP motif in the late domain that no longer binds Tsg101 was rescued by a Nef.Tsg101 chimera. Importantly, this rescue as well as incorporation of Nef into HIV-1 virions correlated with the ability of Nef to interact with GagPol. Our data demonstrate that the retention of Nef in the intermediate compartment interferes with viral replication and suggest a new role for Nef in the production of HIV-1.<br /

Investigating the role of Gag in protease inhibitor susceptibility amongst West African HIV-1 subtypes

HIV-1 Gag contributes to susceptibility of protease inhibitors (PIs) in the absence of known resistance mutations in the protease gene. For the majority of HIV-infected patients worldwide, PIs are the second, and last-line of therapy. Clinically, only around 20% of individuals who fail PI regimen develop major resistance mutations in protease. We previously showed that full-length Gagprotease-derived phenotypic susceptibility to PIs differed between HIV-1 CRF02_AG and subtype G-infected patients who went on to successfully suppress viral replication versus those who experienced virological failure of boosted lopinavir monotherapy as first-line treatment in a clinical trial. We hypothesised therefore that baseline PI susceptibility by Gag-protease phenotyping could be used to predict treatment outcomes for patients on second line, boosted-PI treatment in the real-world clinical setting in Nigeria, where subtypes CRF02_AG/G dominate the epidemic. We used clinical and demographic data; HIV-1subtype, sex, age, viral load, duration of treatment and baseline CD4 count to match individuals who experienced second-line failure with ritonavir-boosted PI-based ART (‘baseline failures’) to those who achieved virological response (‘baseline successes’) with virological failure defined by viral load <400 copies of HIV-1 RNA/mL by week 48. Using a single replication-cycle assay, we carried out in vitro phenotypic susceptibility testing of patient-derived viruses from these two groups. We found no impact of baseline HIV-1 Gagprotease-derived phenotypic susceptibility on outcomes of PI-based second-line ART, treatment outcome could not be predicted using baseline susceptibility alone. Secondly, we sought to explore the role of mutation in Gag-protease genotypic and phenotypic changes within patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. We used longitudinal samples collected at baseline, and at virological failure to explore the role of Gag mutations. Using target enrichment and next-generation sequencing (NGS), followed by haplotype reconstruction and phenotypic drug assays and phylogenetic analysis, we reported for the first time a four-amino acid mutation signature in HIV-1, CRF02_AG matrix (S126del, H127del, T122A and G123E) which confer reduced susceptibility to the PI, lopinavir and atazanavir. Our multi-pronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in HIV- 1 matrix domain reveals the interplay between Gag associated resistance and fitness

Estudios sobre la fosforilación y agregación de la proteína tau y su posible relación con la enfermedad de Alzheimer

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-03-2008The appearance of aberrant structures, in Alzheimer disease, like neurofibrillary tangles, senile plaques or Hirano bodies has been described. Although the etiology of Alzheimer’s disease is still incompletely understood it is thought that oxidative stress could play an important role in the onset and the progression of the disease. In addition to the oxidative stress promoted by amyloid β aggregates, mitochondria generate most of the reactive oxygen species produced during disease. Coenzyme Q is an important cofactor of mitochondrial bioenergetics and it has been directly involved, together with secondary metabolites obtained from xenobiotics metabolism, with aging related energetic changes. Quinones represent a class of intermediary metabolites that may produce cellular damage. The toxicity of quinones may take place through arylation reactions of important biomolecules like DNA or proteins. Alternatively, quinones, depending on the redox state, could exert prooxidant or antioxidant effects. On the other hand, it has been described that compounds like 4-hydroxi-2-nonenal, which are products of lipid peroxidation, could favour the assembly of aberrant structures like paired helical filaments present in neurofibrillary tangles. Compounds that are produced as a consequence of oxidative stress also may affect other microtubular proteins like tubulin. Since it has been described the connection between oxidative stress and the assembly of aberrant structures, we wanted to study if quinones also could have the same effect on protein tau aberrant assembly and Hirano bodies formation. First, we have identified the presence of quinones both in paired helical filaments and Hirano bodies. Once we have stated quinones may be involved in the formation of these aberrant structures that appear in Alzheimer disease, we have studied the effect of this molecules on tau polymerization or aggregation both in vitro and in cell cultures. On the other hand we have also tested if quinones could facilitate the formation of Hirano-like bodies in vitro. Additionally, since microtubule network has been found affected in the onset and progression of Alzheimer disease we have studied the effect of quinones on microtubule network in cell cultures. We have observed that quinones could react with tubulin hampering microtubule polimerization. Thus, we propose that quinones produced by oxidative stress generated during Alzheimer disease, favour polymerization or aggregation of tau protein and prevent microtubule polymerization causing both disturbance on axonal transport and synaptic dysfunction. Other well known inducers of tau aggregation are high molecular weight sulphated glycosaminoglycans, which also are known to favour the Aβ peptide aggregation. Nowadays pharmaceutical companies are developing low molecular weight structural analogs of sulphated glycosaminoglycans to avoid the formation of Aβ aggregates. One of these compounds is Tramiprosate (3-APS). This compound is able to interact with Aβ peptide preventing its aggregation but until now, any research group was studied the effect of this small molecule on tau aggregation. Thus, and finally we have analyzed this interaction demonstrating that 3-APS facilitates aggregation of tau. In this way, also we have tested he effect of its structural analog, taurine, which may promote tau aggregation but in less extent. In summary, our work demonstrates that tau protein modifications provoked by oxidative stress could explain, in part, what is occurring in Alzheimer disease and other tauopathies: its hyperphosphorylation and aggregationIsmael Santa Maria Perez ha recibido financiación del Ministerio de Educación y Ciencia dentro del programa de Formación de Personal Investigador (BES-2004-5505). El trabajo del laboratorio ha sido financiado en parte por el Ministerio de Educación y Ciencia (SAF2003-02697) y por la Fundación LillyPeer reviewe