RESUMEN
The fusion peptide of Ebola virus comprises a highly hydrophobic sequence located downstream from the N-terminus of the glycoprotein GP2 responsible for virus-host membrane fusion. The internal fusion peptide of GP2 inserts into membranes of infected cell to mediate the viral and the host cell membrane fusion. Since the sequence length of Ebola fusion peptide is still not clear, we study in the present work the behavior of two fusion peptides of different lengths which were named EBO17 and EBO24 referring to their amino acid length. The secondary structure and orientation of both peptides in lipid model systems made of DMPC:DMPG:cholesterol:DMPE (6:2:5:3) were investigated using PMIRRAS and polarized ATR spectroscopy coupled with Brewster angle microscopy. The infrared results showed a structural flexibility of both fusion peptides which are able to transit reversibly from an α-helix to antiparallel ß-sheets. Ellipsometry results corroborate together with isotherm measurements that EBO peptides interacting with lipid monolayer highly affected the lipid organization. When interacting with a single lipid bilayer, at low peptide content, EBO peptides insert as mostly α-helices mainly perpendicular into the lipid membrane thus tend to organize the lipid acyl chains. Inserted in multilamellar vesicles at higher peptide content, EBO peptides are mostly in ß-sheet structures and induce a disorganization of the lipid chain order. In this paper, we show that the secondary structure of the Ebola fusion peptide is reversibly flexible between α-helical and ß-sheet conformations, this feature being dependent on its concentration in lipids, eventually inducing membrane fusion.
Asunto(s)
Ebolavirus/química , Membrana Dobles de Lípidos/química , Oligopéptidos/química , Proteínas Virales de Fusión/química , Secuencia de Aminoácidos , Colesterol/química , Dimiristoilfosfatidilcolina/química , Interacciones Hidrofóbicas e Hidrofílicas , Fusión de Membrana , Microscopía , Datos de Secuencia Molecular , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Estructura Secundaria de Proteína , Espectrofotometría InfrarrojaRESUMEN
Formation of senile plaques containing amyloid fibrils of Aß (amyloid ß-peptide) is a pathological hallmark of Alzheimer's disease. Unlike globular proteins, which fold into unique structures, the fibrils of Aß and other amyloid proteins often contain multiple polymorphs. Polymorphism of amyloid fibrils leads to different toxicity in amyloid diseases and may be the basis for prion strains, but the structural origin for fibril polymorphism is still elusive. In the present study we investigate the structural origin of two major fibril polymorphs of Aß40: an untwisted polymorph formed under agitated conditions and a twisted polymorph formed under quiescent conditions. Using electron paramagnetic resonance spectroscopy, we studied the inter-strand side-chain interactions at 14 spin-labelled positions in the Aß40 sequence. The results of the present study show that the agitated fibrils have stronger inter-strand spin-spin interactions at most of the residue positions investigated. The two hydrophobic regions at residues 17-20 and 31-36 have the strongest interactions in agitated fibrils. Distance estimates on the basis of the spin exchange frequencies suggest that inter-strand distances at residues 17, 20, 32, 34 and 36 in agitated fibrils are approximately 0.2 Å (1 Å=0.1 nm) closer than in quiescent fibrils. We propose that the strength of inter-strand side-chain interactions determines the degree of ß-sheet twist, which then leads to the different association patterns between different cross ß-units and thus distinct fibril morphologies. Therefore the inter-strand side-chain interaction may be a structural origin for fibril polymorphism in Aß and other amyloid proteins.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Amiloide/química , Amiloide/ultraestructura , Péptidos beta-Amiloides/ultraestructura , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Electrónica de Transmisión , Fragmentos de Péptidos/química , Fragmentos de Péptidos/ultraestructura , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Marcadores de SpinRESUMEN
Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.
Asunto(s)
Fármacos Anti-VIH/química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/genética , Oligorribonucleótidos/química , Transcripción Reversa , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Células Cultivadas , ADN Complementario/biosíntesis , Transcriptasa Inversa del VIH/genética , VIH-1/fisiología , Humanos , Metilación , Mutación , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction "the dimerization step," followed by conformational changes "the maturation step," yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide P(AW) that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. P(AW) only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, P(AW) prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/enzimología , Péptidos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Replicación Viral/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida/enzimología , Síndrome de Inmunodeficiencia Adquirida/genética , Dimerización , Relación Dosis-Respuesta a Droga , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , Células HeLa , Humanos , Péptidos/química , Péptidos/uso terapéutico , Estructura Terciaria de Proteína , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Replicación Viral/genéticaRESUMEN
Preventing dimerization of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) constitutes an alternative strategy to abolish virus proliferation. We have previously demonstrated that a short peptide derived from the Trp cluster of the connection domain disrupts the RT dimer by interacting with Trp24 and Phe61 in a cleft located between the fingers and the connection domains of p51. Both Trp24 and Phe61 of p51 are essential for the stability of the RT dimer. Here, in order to understand the requirement of Trp24 and Phe61 in the p66 subunit, we have investigated their implication in the formation of RT-primer/template (p/t) complexes and in RT processivity by combining pre-steady-state and steady-state kinetics with site-directed mutagenesis. We demonstrate that both residues are essential for proper binding of the p/t and control conformational changes required for RT ordered mechanism. Trp24 and Phe61 act on p/t binding and remodeling of the catalytic site. Phe61G mutation increases the binding "on" rate of both p/t and mismatched p/t, yielding an unfavorable RT-p/t for polymerase catalysis, unable to pursue mispair extension. Considering the structure of unliganded RT, Phe61 seems to be involved in the dynamics of p66 thumb-finger interactions and in stabilization of the p/t in the catalytic site. In contrast, the p66 Trp24G mutation alters the overall kinetics of p/t binding and is essentially involved in stabilizing the RT-p/t complex by contacting the 5' overhang of the template strand. Mutation of both Trp24 and Phe61 alters mispair extension efficiency, suggesting that disruption of the tight contacts between the fingers domain and the 5' overhang of the template strand increases RT fidelity and reduces RT processivity. Taken together, these studies infer that mutations altering the aromatic nature of Phe61 or Trp24 that may occur to counteract peptide inhibitors targeting this region will generate an unstable RT exhibiting low polymerase activity and higher fidelity. As such, our work suggests that the combined application of peptide-based RT dimerization inhibitors is likely to be highly efficient.
Asunto(s)
Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Fenilalanina/metabolismo , Triptófano/metabolismo , ADN/metabolismo , Transcriptasa Inversa del VIH/química , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Nucleótidos/química , Nucleótidos/metabolismo , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The formulation of human vaccines often includes adjuvants such as aluminum hydroxide that are added to enhance the immune responses to vaccine antigens. However, these adjuvants may also affect the conformation of antigenic proteins. Such structural modifications could lead to changes in antigenicity such that suboptimal protective immune responses could be generated relative to those induced by the vaccine antigens alone. Here, we used attenuated total reflectance infrared spectroscopy (ATR-FTIR) to compare the secondary structures of recombinant HIV-1-gp41 (gp41) in solution or adsorbed to aluminum hydroxide. The gp41 secondary structure content was 72% alpha-helices and 28% beta-sheets in 5 mM formate buffer p(2)H 2.5, while it was 66% beta-sheets and 34% random coil in acetonitril/(2)H(2)O (95/5:v/v). A fully reversible conformational change of gp41 in acetonitril/(2)H(2)O (95/5:v/v) was observed upon addition of either 35 mM formate p(2)H 2.5 or 0.1% (w/v) detergent (Tween 20, Hecameg, Brij 35 or beta-d-octyl-glucopyranoside). When gp41 was adsorbed to aluminum hydroxide in the presence of 0.1% (w/v) detergent, in either formate or in acetonitril/(2)H(2)O (95/5:v/v) its secondary structure remained stable and was identical to that of gp41 in 5 mM formate buffer p(2)H 2.5. The method described here could be applied for the characterization of gp41 conformers for use in immunological screening of antigens, and more generally to all antigenic proteins adsorbed to aluminum hydroxide.