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1.
J Virol ; 85(1): 542-9, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20962083

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) infection is dependent on the proper disassembly of the viral capsid, or "uncoating," in target cells. The HIV-1 capsid consists of a conical multimeric complex of the viral capsid protein (CA) arranged in a hexagonal lattice. Mutations in CA that destabilize the viral capsid result in impaired infection owing to defects in reverse transcription in target cells. We describe here the mechanism of action of a small molecule HIV-1 inhibitor, PF-3450074 (PF74), which targets CA. PF74 acts at an early stage of HIV-1 infection and inhibits reverse transcription in target cells. We show that PF74 binds specifically to HIV-1 particles, and substitutions in CA that confer resistance to the compound prevent binding. A single point mutation in CA that stabilizes the HIV-1 core also conferred strong resistance to the virus without inhibiting compound binding. Treatment of HIV-1 particles or purified cores with PF74 destabilized the viral capsid in vitro. Furthermore, the compound induced the rapid dissolution of the HIV-1 capsid in target cells. PF74 antiviral activity was promoted by binding of the host protein cyclophilin A to the HIV-1 capsid, and PF74 and cyclosporine exhibited mutual antagonism. Our data suggest that PF74 triggers premature HIV-1 uncoating in target cells, thereby mimicking the activity of the retrovirus restriction factor TRIM5α. This study highlights uncoating as a step in the HIV-1 life cycle that is susceptible to small molecule intervention.


Asunto(s)
Fármacos Anti-VIH/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , Cápside/efectos de los fármacos , VIH-1/efectos de los fármacos , Indoles/farmacología , Fenilalanina/análogos & derivados , Animales , Fármacos Anti-VIH/química , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Ciclofilina A/metabolismo , VIH-1/genética , VIH-1/metabolismo , Células HeLa , Humanos , Indoles/química , Fenilalanina/química , Fenilalanina/farmacología , Transcripción Reversa , Virión/metabolismo , Ensamble de Virus/efectos de los fármacos
2.
PLoS Pathog ; 6(12): e1001220, 2010 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-21170360

RESUMEN

Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy.


Asunto(s)
Fármacos Anti-VIH/química , Proteínas de la Cápside/antagonistas & inhibidores , Sustitución de Aminoácidos , Fármacos Anti-VIH/farmacología , Sitios de Unión , Proteínas de la Cápside/genética , Línea Celular , Cristalografía por Rayos X , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Proteínas del Virus de la Inmunodeficiencia Humana , Humanos , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos
3.
J Mol Biol ; 397(2): 600-15, 2010 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-20096702

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) integrase is one of three virally encoded enzymes essential for replication and, therefore, a rational choice as a drug target for the treatment of HIV-1-infected individuals. In 2007, raltegravir became the first integrase inhibitor approved for use in the treatment of HIV-infected patients, more than a decade since the approval of the first protease inhibitor (saquinavir, Hoffman La-Roche, 1995) and two decades since the approval of the first reverse transcriptase inhibitor (retrovir, GlaxoSmithKline, 1987). The slow progress toward a clinically effective HIV-1 integrase inhibitor can at least in part be attributed to a poor structural understanding of this key viral protein. Here we describe the development of a restrained molecular dynamics protocol that produces a more accurate model of the active site of this drug target. This model provides an advance on previously described models as it ensures that the catalytic DDE motif makes correct, monodentate interactions with the two active-site magnesium ions. Dynamic restraints applied to this coordination state create models with the correct solvation sphere for the metal ion complex and highlight the coordination sites available for metal-binding ligands. Application of appropriate dynamic flexibility to the core domain allowed the inclusion of multiple conformational states in subsequent docking studies. These models have allowed us to (1) explore the effects of key drug resistance mutations on the dynamic flexibility and conformational preferences of HIV integrase and to (2) study raltegravir binding in the context of these dynamic models of both wild type and the G140S/Q148H drug-resistant enzyme.


Asunto(s)
Dominio Catalítico , Farmacorresistencia Viral , Integrasa de VIH/química , Integrasa de VIH/metabolismo , VIH-1/química , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Pirrolidinonas/metabolismo , Raltegravir Potásico
4.
J Virol ; 80(14): 7009-19, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16809306

RESUMEN

West Nile virus (WNV) is a neurotropic, mosquito-borne flavivirus that can cause lethal meningoencephalitis. Type I interferon (IFN) plays a critical role in controlling WNV replication, spread, and tropism. In this study, we begin to examine the effector mechanisms by which type I IFN inhibits WNV infection. Mice lacking both the interferon-induced, double-stranded-RNA-activated protein kinase (PKR) and the endoribonuclease of the 2',5'-oligoadenylate synthetase-RNase L system (PKR(-/-) x RL(-/-)) were highly susceptible to subcutaneous WNV infection, with a 90% mortality rate compared to the 30% mortality rate observed in congenic wild-type mice. PKR(-/-) x RL(-/-) mice had increased viral loads in their draining lymph nodes, sera, and spleens, which led to early viral entry into the central nervous system (CNS) and higher viral burden in neuronal tissues. Although mice lacking RNase L showed a higher CNS viral burden and an increased mortality, they were less susceptible than the PKR(-/-) x RL(-/-) mice; thus, we also infer an antiviral role for PKR in the control of WNV infection. Notably, a deficiency in both PKR and RNase L resulted in a decreased ability of type I IFN to inhibit WNV in primary macrophages and cortical neurons. In contrast, the peripheral neurons of the superior cervical ganglia of PKR(-/-) x RL(-/-) mice showed no deficiency in the IFN-mediated inhibition of WNV. Our data suggest that PKR and RNase L contribute to IFN-mediated protection in a cell-restricted manner and control WNV infection in peripheral tissues and some neuronal subtypes.


Asunto(s)
Endorribonucleasas/metabolismo , Meningoencefalitis/enzimología , Neuronas/enzimología , Replicación Viral , Fiebre del Nilo Occidental/enzimología , Virus del Nilo Occidental/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Corteza Cerebelosa/enzimología , Corteza Cerebelosa/virología , Endorribonucleasas/deficiencia , Interferón gamma/metabolismo , Macrófagos/enzimología , Macrófagos/virología , Meningoencefalitis/genética , Meningoencefalitis/virología , Ratones , Ratones Noqueados , Neuronas/virología , Especificidad de Órganos , Ganglio Cervical Superior/enzimología , Ganglio Cervical Superior/virología , Replicación Viral/genética , Fiebre del Nilo Occidental/genética , Fiebre del Nilo Occidental/virología , eIF-2 Quinasa/deficiencia
5.
J Virol ; 80(11): 5338-48, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16699014

RESUMEN

West Nile virus (WNV) causes a severe central nervous system (CNS) infection in humans, primarily in the elderly and immunocompromised. Prior studies have established an essential protective role of several innate immune response elements, including alpha/beta interferon (IFN-alpha/beta), immunoglobulin M, gammadelta T cells, and complement against WNV infection. In this study, we demonstrate that a lack of IFN-gamma production or signaling results in increased vulnerability to lethal WNV infection by a subcutaneous route in mice, with a rise in mortality from 30% (wild-type mice) to 90% (IFN-gamma(-/-) or IFN-gammaR(-/-) mice) and a decrease in the average survival time. This survival pattern in IFN-gamma(-/-) and IFN-gammaR(-/-) mice correlated with higher viremia and greater viral replication in lymphoid tissues. The increase in peripheral infection led to early CNS seeding since infectious WNV was detected several days earlier in the brains and spinal cords of IFN-gamma(-/-) or IFN-gammaR(-/-) mice. Bone marrow reconstitution experiments showed that gammadelta T cells require IFN-gamma to limit dissemination by WNV. Moreover, treatment of primary dendritic cells with IFN-gamma reduced WNV production by 130-fold. Collectively, our experiments suggest that the dominant protective role of IFN-gamma against WNV is antiviral in nature, occurs in peripheral lymphoid tissues, and prevents viral dissemination to the CNS.


Asunto(s)
Interferón gamma/fisiología , Fiebre del Nilo Occidental/prevención & control , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/patogenicidad , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/crecimiento & desarrollo , Virus del Nilo Occidental/inmunología
6.
J Virol ; 79(14): 8698-706, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15994763

RESUMEN

Previous studies have suggested that alpha-glucosidase inhibitors such as castanospermine and deoxynojirimycin inhibit dengue virus type 1 infection by disrupting the folding of the structural proteins prM and E, a step crucial to viral secretion. We extend these studies by evaluating the inhibitory activity of castanospermine against a panel of clinically important flaviviruses including all four serotypes of dengue virus, yellow fever virus, and West Nile virus. Using in vitro assays we demonstrated that infections by all serotypes of dengue virus were inhibited by castanospermine. In contrast, yellow fever virus and West Nile virus were partially and almost completely resistant to the effects of the drug, respectively. Castanospermine inhibited dengue virus infection at the level of secretion and infectivity of viral particles. Importantly, castanospermine prevented mortality in a mouse model of dengue virus infection, with doses of 10, 50, and 250 mg/kg of body weight per day being highly effective at promoting survival (P < or = 0.0001). Correspondingly, castanospermine had no adverse or protective effect on West Nile virus mortality in an analogous mouse model. Overall, our data suggest that castanospermine has a strong antiviral effect on dengue virus infection and warrants further development as a possible treatment in humans.


Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Indolizinas/farmacología , Animales , Dengue/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , ARN Viral/biosíntesis , Virión/efectos de los fármacos , Fiebre del Nilo Occidental/tratamiento farmacológico , Virus del Nilo Occidental/efectos de los fármacos , Virus de la Fiebre Amarilla/efectos de los fármacos
7.
J Virol ; 79(12): 7466-77, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15919902

RESUMEN

Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.


Asunto(s)
Anticuerpos Antivirales/sangre , Activación de Complemento , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Virus del Nilo Occidental/patogenicidad , Animales , Anticuerpos Antivirales/inmunología , Encéfalo/inmunología , Encéfalo/virología , Línea Celular , Complemento C3/genética , Cricetinae , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Ratones , Pruebas de Neutralización , Receptores de Complemento/genética , Fiebre del Nilo Occidental/virología
8.
Antivir Chem Chemother ; 15(3): 141-51, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15266896

RESUMEN

Alpha-glucosidase I inhibitors have been shown to inhibit the replication of a broad range of enveloped viruses by preventing the correct folding of their envelope glycoproteins. This study assesses the potential of 6 O-butanoyl castanospermine (celgosivir) as a treatment for hepatitis C virus (HCV). In the absence of an adequate culture system for HCV, the closely related virus, bovine viral diarrhoea virus (BVDV), was used as a surrogate model. Using both a plaque assay and a cytopathic effect assay, celgosivir (IC50 16 and 47 microM respectively) was shown to be more potent than N-nonyl DNJ (105 and 74 microM), castanospermine (110 and 367 microM) and N-butyl DNJ (> 250 and 550 microM). Of the alpha-glucosidase inhibitors tested, only N-nonyl DNJ showed evidence of toxicity (CC50 > or = 120 microM). Two-way combinations of interferon-alpha, ribavirin and either celgosivir or castanospermine demonstrated that each could enhance the antiviral efficacy of the others, either additively or synergistically. The observation that the number of viral genomes released from BVDV-infected cells was inhibited by either castanospermine or celgosivir in parallel with the number of infectious units was taken as confirmation that these alpha-glucosidase I inhibitors block the production or release of flavivirus particles.


Asunto(s)
Antivirales/farmacología , Diarrea Mucosa Bovina Viral/tratamiento farmacológico , Virus de la Diarrea Viral Bovina/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hepatitis C/tratamiento farmacológico , Indolizinas/farmacología , Animales , Antivirales/uso terapéutico , Bovinos , Línea Celular , Efecto Citopatogénico Viral , Virus de la Diarrea Viral Bovina/enzimología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de Glicósido Hidrolasas , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Humanos , Indolizinas/uso terapéutico , Concentración 50 Inhibidora , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , ARN Viral/química , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribavirina/farmacología , Ribavirina/uso terapéutico , Proteínas del Envoltorio Viral/metabolismo , Ensayo de Placa Viral , alfa-Glucosidasas
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