RESUMEN
BACKGROUND: Individuals with HIV have â¼2-fold increased risk of developing pulmonary fibrosis. The mechanism(s) by which this occurs has yet to be determined. HIV-1 protein gp120 activates CXCR4 in the lymphocyte, promoting a variety of intracellular signaling pathways including those common to TGFß1 associated with lung fibroblast-to-myofibroblast transdifferentiation. We hypothesized that gp120 promotes pulmonary fibrotic changes via activation of CXCR4 in the lung fibroblast. METHODS: Mouse primary lung fibroblasts (PLFs) were cultured ± gp120, then analyzed for α-SMA expression and stress fiber formation. In parallel, PLFs were cultured ± gp120 ± AMD3100 (a CXCR4 antagonist), and α-SMA, pan and phospho-Akt, and total and phospho-MAPK (or ERK1/2) protein expression was quantified. Finally, lungs and PLFs from wild-type and HIV-1 transgenic mice were analyzed for hydroxyproline and α-SMA content. RESULTS: gp120 treatment increased α-SMA expression and myofibroblast differentiation in PLFs. gp120 treatment activated phosphorylation of ERK1/2, but not PI3K-Akt. Pretreatment with AMD3100 inhibited gp120-induced ERK1/2 phosphorylation and gp120-induced α-SMA expression. In parallel, there was a significant increase in hydroxyproline content in lungs from older HIV-1 transgenic mice and a >3-fold increase in α-SMA expression in PLFs isolated from HIV-1 transgenic mice. CONCLUSIONS: gp120 induces α-SMA expression and fibroblast-to-myofibroblast transdifferentiation by activating the CXCR4-ERK1/2 signaling pathway in mouse PLFs. Lungs of older HIV-1 transgenic mice contain higher hydroxyproline content and their PLFs have a striking increase in α-SMA expression. These results suggest a mechanism by which individuals with HIV are at increased risk of developing pulmonary fibrotic changes as they age.
Asunto(s)
Transdiferenciación Celular , Fibroblastos/metabolismo , Proteína gp120 de Envoltorio del VIH/fisiología , Fibrosis Pulmonar/etiología , Receptores CXCR4/metabolismo , Actinas/metabolismo , Animales , Bencilaminas , Ciclamas , Femenino , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/uso terapéutico , Hidroxiprolina/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones Transgénicos , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/prevención & control , Receptores CXCR4/antagonistas & inhibidoresRESUMEN
The spikes on virus surfaces bind receptors on host cells to propagate infection. High spike densities (SDs) can promote infection, but spikes are also targets of antibody-mediated immune responses. Thus, diverse evolutionary pressures can influence virus SDs. HIV's SD is about two orders of magnitude lower than that of other viruses, a surprising feature of unknown origin. By modeling antibody evolution through affinity maturation, we find that an intermediate SD maximizes the affinity of generated antibodies. We argue that this leads most viruses to evolve high SDs. T helper cells, which are depleted during early HIV infection, play a key role in antibody evolution. We find that T helper cell depletion results in high affinity antibodies when SD is high, but not if SD is low. This special feature of HIV infection may have led to the evolution of a low SD to avoid potent immune responses early in infection.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH/inmunología , Anticuerpos Neutralizantes/inmunología , VIH/patogenicidad , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/fisiología , Infecciones por VIH/virología , Humanos , Linfocitos T Colaboradores-Inductores/inmunología , Estructuras Virales/inmunología , Estructuras Virales/fisiologíaRESUMEN
Over the last decade, multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein (Env) gp120 have been described. Many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of Envs compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid-containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan-dependent epitopes has been challenging. Here, we report the development of a stable suspension-adapted Chinese hamster ovary (CHO) cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1- CHO cell line exhibit improved binding to prototypic glycan-dependent bN-mAbs directed to the V1/V2 domain (e.g., PG9) and the V3 stem (e.g., PGT128 and 10-1074) while preserving the structure of the important glycan-independent epitopes (e.g., VRC01). The ability of the MGAT1- CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway without impairing the doubling time or ability to grow at high cell densities suggests that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.
Asunto(s)
Vacunas contra el SIDA/metabolismo , Células CHO/fisiología , Edición Génica/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/metabolismo , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cricetinae , Cricetulus , Epítopos , Glicosilación , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/fisiología , Seropositividad para VIH , VIH-1/genética , Humanos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/fisiología , Polisacáridos/metabolismo , Ingeniería de Proteínas/métodosRESUMEN
The main goal of this work was to identify molecular signatures in envelope surface glycoprotein that may be correlated with coreceptor usage by different human immunodeficiency virus (HIV)-2 strains. From inspection of aligned HIV-2 sequences, we verified that V1/V2 region showed the highest degree of amino acid sequence heterogeneity, including polymorphisms in N-linked glycosylation sites, sequence, and length. Furthermore, we did not find any correlation between the net charge and specific amino acid positions in V3 region with any particular coreceptor usage pattern. In conclusion, we showed that for HIV-2, the genetic determinants for coreceptor usage are distinct from those of HIV-1. More specifically, we did not identify any molecular signature, based on discrete amino acid positions either in V1/V2 or in V3 regions, which could be assigned to the preferential usage of a specific coreceptor.
Asunto(s)
Secuencia de Aminoácidos/fisiología , Proteína gp120 de Envoltorio del VIH/fisiología , VIH-2/aislamiento & purificación , Glicoproteínas de Membrana/metabolismo , Receptores del VIH/fisiología , Heterogeneidad Genética , Pruebas Genéticas , Glicosilación , Proteína gp120 de Envoltorio del VIH/genética , VIH-2/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMEN
Human immunodeficiency virus (HIV)-associated neuropathic pain is common, and studies have shown that HIV envelope glycoprotein 120 (gp120) can directly stimulate primary sensory afferent neurons causing hyperalgesia. The P2X7 receptor in the dorsal root ganglia (DRG) is involved in pain transmission and is closely related to the inflammatory and immune response. In this study, we aimed to explore the role of the P2X7 receptor in gp120-induced neuropathic pain using a rat model specific for this type of pain. The results showed that mechanical hyperalgesia, thermal hyperalgesia and P2X7 expression levels were increased in rats treated with gp120. The P2X7 antagonist, brilliant blue G (BBG), decreased hyperalgesia and P2X7 expression levels in rats treated with gp120. BBG also decreased IL-1ß and TNF-α receptor expression and ERK1/2 phosphorylation levels and increased IL-10 expression in the gp120-treated rat DRG. In addition, P2X7 agonist (BzATP)-activated currents in DRG neurons cultured with gp120 were larger than those in control neurons, and the inhibitory effect of BBG on BzATP-induced currents in gp120-treated DRG neurons was larger than that in control neurons. Therefore, inhibition of the P2X7 receptor in rat DRG relieved gp120-induced mechanical hyperalgesia and thermal hyperalgesia.
Asunto(s)
Ganglios Espinales/metabolismo , Proteína gp120 de Envoltorio del VIH/fisiología , Hiperalgesia/fisiopatología , Neuralgia/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animales , Ganglios Espinales/fisiopatología , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/fisiopatología , Hiperalgesia/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Masculino , Neuralgia/etiología , Neuronas/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Interferon-inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses, including HIV-1. IFITM proteins deter HIV-1 entry when expressed in target cells and also impair HIV-1 infectivity when expressed in virus producer cells. However, little is known about how viruses resist IFITM inhibition. In this study, we have investigated the susceptibilities of different primary isolates of HIV-1 to the inhibition of viral infectivity by IFITMs. Our results demonstrate that the infectivity of different HIV-1 primary isolates, including transmitted founder viruses, is diminished by IFITM3 to various levels, with strain AD8-1 exhibiting strong resistance. Further mutagenesis studies revealed that HIV-1 Env, and the V3 loop sequence in particular, determines the extent of inhibition of viral infectivity by IFITM3. IFITM3-sensitive Env proteins are also more susceptible to neutralization by soluble CD4 or the 17b antibody than are IFITM3-resistant Env proteins. Together, data from our study suggest that the propensity of HIV-1 Env to sample CD4-bound-like conformations modulates viral sensitivity to IFITM3 inhibition.IMPORTANCE Results of our study have revealed the key features of the HIV-1 envelope protein that are associated with viral resistance to the IFITM3 protein. IFITM proteins are important effectors in interferon-mediated antiviral defense. A variety of viruses are inhibited by IFITMs at the virus entry step. Although it is known that envelope proteins of several different viruses resist IFITM inhibition, the detailed mechanisms are not fully understood. Taking advantage of the fact that envelope proteins of different HIV-1 strains exhibit different degrees of resistance to IFITM3 and that these HIV-1 envelope proteins share the same domain structure and similar sequences, we performed mutagenesis studies and determined the key role of the V3 loop in this viral resistance phenotype. We were also able to associate viral resistance to IFITM3 inhibition with the susceptibility of HIV-1 to inhibition by soluble CD4 and the 17b antibody that recognizes CD4-binding-induced epitopes.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/fisiología , Infecciones por VIH/virología , VIH-1/fisiología , Proteínas de la Membrana/fisiología , Proteínas de Unión al ARN/fisiología , Secuencias de Aminoácidos , Células HEK293 , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/inmunología , Humanos , Evasión Inmune , ViriónRESUMEN
In the era of highly active antiretroviral therapy (HAART), HIV-1-associated neurocognitive disorders (HAND) persist in infected individuals with adequate immunological and virological status. Risk factors for cognitive impairment include hepatitis C virus co-infection, host genetic factors predisposing to HAND, the early establishment of the virus in the CNS and its persistence under HAART; thus, the CNS is an important reservoir for HIV. Microglial cells are permissive to HIV-1, and NLRP3 inflammasome-associated genes were found expressed in brains of HIV-1-infected persons, contributing to brain disease. Inflammasomes can be triggered by alarmins or danger-associated molecular patterns (DAMPs), which directly stimulate the production of proinflammatory mediators by glial cells, contribute to blood-brain barrier injury through induction of release of various proteases and allow the passage of infected macrophages, and trigger IL-1ß release from primed cells. Amongst alarmins involved in HIV-1-induced neuropathogenesis, IL-33 and high-mobility group box 1 (HMGB1) are of particular interest. Neurocognitive alterations were recently associated with dysregulation of the IL-33/ST2 axis in the CNS, leading to the induction of neuronal apoptosis, decrease in synaptic function and neuroinflammation. Specific biomarkers, including HMGB1 and anti-HMGB1 antibodies, have been identified in cerebrospinal fluid from patients with HAND, correlated with immune activation and identifying a very early stage of neurocognitive impairment that precedes changes in metabolites detected by magnetic resonance spectroscopy. Moreover, HMGB1 plays a crucial role in HIV-1 persistence in dendritic cells and in the constitution of viral reservoirs. In this review, the mechanisms whereby alarmins contribute to HIV-1-induced CNS inflammation and neuropathogenesis will be discussed.
Asunto(s)
Alarminas/fisiología , Enfermedades del Sistema Nervioso Central/virología , Infecciones por VIH/etiología , VIH-1 , Neuritis/virología , Fármacos Anti-VIH/uso terapéutico , Biomarcadores/metabolismo , Enfermedades del Sistema Nervioso Central/inmunología , Enfermedad Crónica , Reservorios de Enfermedades , Proteína gp120 de Envoltorio del VIH/fisiología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Proteína HMGB1/fisiología , Humanos , Inmunidad Innata/inmunología , Inflamasomas/fisiología , Interleucina-33/fisiología , Neuritis/inmunología , Enfermedades Neurodegenerativas/inmunología , Enfermedades Neurodegenerativas/virología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry into cells by using a spring-loaded mechanism that allows for the controlled insertion of the Env fusion peptide into the target membrane, followed by membrane fusion. Env is the focus of vaccine research aimed at inducing protective immunity by antibodies as well as efforts to develop drugs that inhibit the viral entry process. The molecular factors contributing to Env stability and decay need to be understood better in order to optimally design vaccines and therapeutics. We generated viruses with resistance to VIR165, a peptidic inhibitor that binds the fusion peptide of the gp41 subunit and prevents its insertion into the target membrane. Interestingly, a number of escape viruses acquired substitutions in the C1 domain of the gp120 subunit (A60E, E64K, and H66R) that rendered these viruses dependent on the inhibitor. These viruses could infect target cells only when VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent viruses were resistant to soluble CD4-induced Env destabilization and decay. These data suggest that VIR165-dependent Env proteins are kinetically trapped in the unliganded state and require the drug to negotiate CD4-induced conformational changes. These studies provide mechanistic insight into the action of the gp41 fusion peptide and its inhibitors and provide new ways to stabilize Env trimer vaccines. IMPORTANCE: Because of the rapid development of HIV-1 drug resistance, new drug targets need to be explored continuously. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelope-based HIV vaccines.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/fisiología , Proteína gp41 de Envoltorio del VIH/fisiología , VIH-1/fisiología , Internalización del Virus , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Bencilaminas , Antígenos CD4/metabolismo , Antígenos CD4/farmacología , Ciclamas , Genes env , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Compuestos Heterocíclicos/farmacología , Humanos , Modelos Biológicos , Modelos Moleculares , Mutación , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Internalización del Virus/efectos de los fármacosRESUMEN
A transmission bottleneck occurs during each human immunodeficiency virus (HIV) transmission event, which allows only a few viruses to establish new infection. However, the genetic characteristics of the transmitted viruses that are preferentially selected have not been fully elucidated. Here, we analyzed amino acids changes in the envelope protein during simian immunodeficiency virus (SIV)/HIV deep transmission history and current HIV evolution within the last 15-20 years. Our results confirmed that the V1V2 region of gp120 protein, particularly V1, was preferentially selected. A shorter V1 region was preferred during transmission history, while during epidemic, HIV may evolve to an expanded V1 region gradually and thus escape immune recognition. We then constructed different HIV-1 V1 mutants using different HIV-1 subtypes to elucidate the role of the V1 region in envelope function. We found that the V1 region, although highly variable, was indispensable for virus entry and infection, probably because V1 deletion mutants exhibited impaired processing of gp160 into mature gp120 and gp41. Additionally, the V1 region affected Env incorporation. These results indicated that the V1 region played a critical role in HIV transmission and infection.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/fisiología , Infecciones por VIH/transmisión , VIH-1/fisiología , Glicoproteínas de Membrana/fisiología , Fragmentos de Péptidos/fisiología , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Virus de la Inmunodeficiencia de los Simios/fisiología , Proteínas del Envoltorio Viral/fisiología , Animales , Células HEK293 , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/fisiología , Infecciones por VIH/virología , VIH-1/genética , VIH-2/genética , VIH-2/fisiología , Humanos , Fusión de Membrana/fisiología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Variability in CCR5 levels in the human population is suggested to affect virus evolution, fitness and the course of HIV disease. We previously demonstrated that cell surface CCR5 levels directly affect HIV Envelope mediated bystander apoptosis. In this study, we attempted to understand HIV evolution in the presence of low levels of CCR5, mimicking the limiting CCR5 levels inherent to the host. HIV-1 adaptation in a T cell line expressing low levels of CCR5 resulted in two specific mutations; N302Y and E172K. The N302Y mutation led to accelerated virus replication, increase in Maraviroc IC50 and an increase in Envelope mediated bystander apoptosis in low CCR5 expressing cells. Analysis of subtype B sequences showed that N302Y is over-represented in CXCR4 tropic viruses in comparison to CCR5 tropic isolates. Considering the variability in CCR5 levels between individuals, our findings have implications for virus evolution, MVC susceptibility as well as HIV pathogenesis.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/fisiología , Receptores CCR5/metabolismo , Adaptación Fisiológica , Fármacos Anti-VIH/farmacología , Línea Celular , Ciclohexanos/farmacología , Farmacorresistencia Viral , Proteína gp120 de Envoltorio del VIH/química , VIH-1/genética , VIH-1/patogenicidad , Células HeLa , Humanos , Secuencias Invertidas Repetidas , Maraviroc , Modelos Moleculares , Conformación Proteica , Linfocitos T/metabolismo , Linfocitos T/virología , Triazoles/farmacología , Virulencia , Replicación ViralRESUMEN
HIV-1 entry into host cells is mediated by interactions between the V3-loop of viral glycoprotein gp120 and chemokine receptor CCR5 or CXCR4, collectively known as HIV-1 coreceptors. Accurate genotypic prediction of coreceptor usage is of significant clinical interest and determination of the factors driving tropism has been the focus of extensive study. We have developed a method based on nonlinear support vector machines to elucidate the interacting residue pairs driving coreceptor usage and provide highly accurate coreceptor usage predictions. Our models utilize centroid-centroid interaction energies from computationally derived structures of the V3-loop:coreceptor complexes as primary features, while additional features based on established rules regarding V3-loop sequences are also investigated. We tested our method on 2455 V3-loop sequences of various lengths and subtypes, and produce a median area under the receiver operator curve of 0.977 based on 500 runs of 10-fold cross validation. Our study is the first to elucidate a small set of specific interacting residue pairs between the V3-loop and coreceptors capable of predicting coreceptor usage with high accuracy across major HIV-1 subtypes. The developed method has been implemented as a web tool named CRUSH, CoReceptor USage prediction for HIV-1, which is available at http://ares.tamu.edu/CRUSH/.
Asunto(s)
VIH-1/fisiología , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Metabolismo Energético , Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/crecimiento & desarrollo , Humanos , Modelos Biológicos , Dominios y Motivos de Interacción de Proteínas/fisiología , Relación Estructura-Actividad , TropismoRESUMEN
The ER stress-mediated apoptosis has been implicated in several neurodegenerative diseases; however, its role in HIV/neuroAIDS remains largely unexplored. The present study was undertaken to assess the involvement and detailed mechanism of IRE1α pathway in HIV-1 gp120-mediated ER stress and its possible involvement in cell death. Various signaling molecules for IRE1α pathway were assessed using SVGA cells, primary astrocytes and gp120 transgenic mice, which demonstrated gp120-mediated increase in phosphorylated JNK, XBP-1 and AP-1 leading to upregulation of CHOP. Furthermore, HIV-1 gp120-mediated activation of IRE1α also increased XBP-1 splicing. The functional consequence of gp120-mediated ER stress was determined via assessment of gp120-mediated cell death using PI staining and MTT assay. The gp120-mediated cell death also involved caspase-9/caspase-3-mediated apoptosis. These findings were confirmed with the help of specific siRNA for IRE1α, JNK, AP-1, BiP and CHOP showing significant reduction in gp120-mediated CHOP expression. Additionally, silencing all the intermediates also reduced the gp120-mediated cell death and caspase-9/caspase-3 activation at differential levels. This study provides ER-stress as a novel therapeutic target in the management of gp120-mediated cell death and possibly in the treatment of neuroAIDS.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , Endorribonucleasas/metabolismo , Proteína gp120 de Envoltorio del VIH/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Astrocitos/fisiología , Secuencia de Bases , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Empalme del ARN , ARN Interferente Pequeño/genética , Factor de Transcripción CHOP/metabolismo , Proteína 1 de Unión a la X-BoxRESUMEN
Proline oxidase (POX) catalytically converts proline to pyrroline-5-carboxylate. This catabolic conversion generates reactive oxygen species (ROS) that triggers cellular signaling cascades including autophagy and apoptosis. This study for the first time demonstrates a role of POX in HIV-1 envelope glycoprotein (gp120)-induced neuronal autophagy. HIV-1 gp120 is a neurotoxic factor and is involved in HIV-1-associated neurological disorders. However, the mechanism of gp120-mediated neurotoxicity remains unclear. Using SH-SY5Y neuroblastoma cells as a model, this study demonstrates that gp120 treatment induced POX expression and catalytic activity. Concurrently, gp120 also increased intracellular ROS levels. However, increased ROS had a minimal effect on neuronal apoptosis. Further investigation indicated that the immediate cellular response to increased ROS paralleled with induction of autophagy markers, beclin-1 and LC3-II. These data lead to the hypothesis that neuronal autophagy is activated as a cellular protective response to the toxic effects of gp120. A direct and functional role of POX in gp120-mediated neuronal autophagy was examined by inhibition and overexpression studies. Inhibition of POX activity by a competitive inhibitor "dehydroproline" decreased ROS levels concomitant with reduced neuronal autophagy. Conversely, overexpression of POX in neuronal cells increased ROS levels and activated ROS-dependent autophagy. Mechanistic studies suggest that gp120 induces POX by targeting p53. Luciferase reporter assays confirm that p53 drives POX transcription. Furthermore, data demonstrate that gp120 induces p53 via binding to the CXCR4 co-receptor. Collectively, these results demonstrate a novel role of POX as a stress response metabolic regulator in HIV-1 gp120-associated neuronal autophagy.
Asunto(s)
Autofagia/fisiología , Proteína gp120 de Envoltorio del VIH/fisiología , Neuronas/fisiología , Prolina Oxidasa/metabolismo , Línea Celular Tumoral , VIH-1 , Humanos , Mitocondrias/enzimología , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: The structure of HIV-1 envelope glycoprotein (Env) is flexible and heterogeneous on whole virions. Although functional Env complexes are thought to require trimerization of cleaved gp41/gp120 heterodimers, variable processing can result in the potential incorporation of non-functional uncleaved proteins (gp160), non-trimeric arrangements of gp41/gp120 heterodimers, and gp120 depleted gp41 stumps. The potential distribution of functional and non-functional Env forms across replication-competent viral populations may have important implications for neutralizing and non-neutralizing antibody functions. This study applied an immuno-bead viral capture assay (VCA) to interrogate the potential distribution (heterologous vs homologous) of functional and non-functional forms of virion associated Env. RESULTS: The VCA revealed a significant association between depletion of infectious virions and virion Env incorporation, but not between infectivity and p24-gag. Three distinct subpopulations of virions were identified within pools of genetically homogenous viral particles. Critically, a significant subpopulation of infectious virions were exclusively captured by neutralizing antibodies (nAbs) indicative of a homologous distribution of functional trimeric Env forms. A second infectious subpopulation bound both neutralizing and non-neutralizing antibodies (nnAbs) representative of a heterologous distribution of Env forms, while a third non-infectious subpopulation was predominantly bound by nnAbs recognizing gp41 stumps. CONCLUSIONS: The observation that a distinct and significant subpopulation of infectious virions is exclusively captured by neutralizing antibodies has important implications for understanding antibody binding and neutralization, as well as other antibody effector functions.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/fisiología , Proteínas gp160 de Envoltorio del VIH/fisiología , VIH-1/fisiología , Virión/aislamiento & purificación , Virión/fisiología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/fisiología , VIH-1/inmunología , Humanos , Inmunoensayo , Unión Proteica , Virión/inmunologíaRESUMEN
Neuronal cell dysfunction and apoptosis are the main causes of the invasion of the central nervous system by human immunodeficiency virus type 1 (HIV-1), although the underlying mechanism has not been well understood. Recent research has shown that curcumin might play an important role in regulating HIV-1 development. Heat shock protein 70 (HSP70), a protein induced by heat, was reported to inhibit apoptosis through various cell signaling pathways in brain. Overexpression of HSP70 could effectively protected neurons in many animal and cellular models of dementia. In the present study, the expression of HSP70 in the gp120 V3 loop peptide-induced neuronal apoptosis was investigated. Our results demonstrated that gp120 V3 loop peptide could induce primary rat cortical neuronal apoptosis. We also found that curcumin could increase HSP70 expression. In addition, the expression level of both HSP70 mRNA and HSP70 protein were dependent on the curcumin dose in the rat cortical neurons. Curcumin could improve HSP70 expression in gp120 V3 loop peptide-induced primary rat cortical neuronal apoptosis. In general, our results indicated that curcumin played an important role in the gp120 V3 loop peptide induced neuronal apoptosis by regulating HSP70.
Asunto(s)
Apoptosis/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Curcumina/farmacología , Proteína gp120 de Envoltorio del VIH/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Neuronas/efectos de los fármacos , Animales , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Approximately 25 % of HIV patients use marijuana for its putative therapeutic benefit; however, it is unknown how cannabinoids affect the immune status of HIV patients. Previously, a surrogate in vitro mouse model was established, which induced CD8(+) T cell proliferation and gp120-specific IFNγ production. ∆(9)-Tetrahydrocannabinol (THC), the predominant psychoactive compound in marijuana, suppressed or enhanced the responses depending on the magnitude of cellular activation. The purpose of the current study was to investigate whether THC produced similar effects in vivo and therefore a mouse model to induce HIVgp120-specific immune responses was established. A gp120-expressing plasmid, pVRCgp120, or a vector plasmid, pVRC2000, was injected intramuscularly into mice, which were also dosed with THC orally. The gp120-specific IFNγ and IL-2 responses were detected when splenocytes were restimulated with gp120-derived peptide 81 (IIGDIRQAHCNISRA), which was identified as being immunodominant. Various cellular populations were activated in response to pVRCgp120 stimulation followed by peptide restimulation, as evidenced by increased expression levels of activation markers (e.g., CD69, CD80, and major histocompatibility complex II [MHC II]). The IFNγ response and cellular activation were enhanced by THC in C57Bl/6 wild type (WT) mice but suppressed or not affected by THC in cannabinoid receptor 1 (CB1) and 2 (CB2) knockout (CB1 (-/-)CB2 (-/-)) mice. Furthermore, CB1 (-/-)CB2 (-/-) mice exhibited augmented IFNγ production when compared to WT mice in the absence of THC. Collectively, our findings demonstrate that under certain conditions, THC enhances HIV antigen-specific immune responses, which occurs through CB1/CB2-dependent and -independent mechanisms.
Asunto(s)
Dronabinol/farmacología , Proteína gp120 de Envoltorio del VIH/fisiología , Antígenos de Histocompatibilidad Clase II/inmunología , Animales , Femenino , Proteína gp120 de Envoltorio del VIH/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/deficiencia , Receptor Cannabinoide CB1/inmunología , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/deficiencia , Receptor Cannabinoide CB2/inmunologíaRESUMEN
In 2014, 3.2 million children (< 15 years of age) were estimated to be living with HIV and AIDS worldwide, with the 240,000 newly infected children in the past year, i.e., another child infected approximately every two minutes [1]. The primary mode of HIV infection is through mother-to-child transmission (MTCT), occurring either in utero, intrapartum, or during breastfeeding. The effects of HIV-1 on the central nervous system (CNS) are putatively accepted to be mediated, in part, via viral proteins, such as Tat and gp120. The current review focuses on the targets of HIV-1 proteins during the development of the dopamine (DA) system, which appears to be specifically susceptible in HIV-1-infected children. Collectively, the data suggest that the DA system is a clinically relevant target in chronic HIV-1 infection, is one of the major targets in pediatric HIV-1 CNS infection, and may be specifically susceptible during development. The present review discusses the development of the DA system, follows the possible targets of the HIV-1 proteins during the development of the DA system, and suggests potential therapeutic approaches. By coupling our growing understanding of the development of the CNS with the pronounced age-related differences in disease progression, new light may be shed on the neurological and neurocognitive deficits that follow HIV-1 infection.
Asunto(s)
Sistema Nervioso Central/fisiología , Desarrollo Infantil/fisiología , Dopamina/fisiología , Proteína gp120 de Envoltorio del VIH/fisiología , Infecciones por VIH/fisiopatología , VIH-1 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/fisiología , Niño , Infecciones por VIH/transmisión , Humanos , Transmisión Sináptica/fisiologíaRESUMEN
Antibodies with modest neutralizing activity and narrow breadth are commonly elicited in HIV-1. Here, we evaluated the complementary and synergistic activities of a set of monoclonal antibodies (MAb) isolated from a single patient, directed to V3, CD4 binding site (CD4bs), and CD4 induced (CD4i) epitopes. Despite low somatic hypermutation percentages in the variable regions, these MAbs covered viral strains from subtypes B, C, A and CRF01_AE and transmitted/founder viruses in terms of binding, neutralizing and antibody-dependent cell-mediated cytotoxicity (ADCC) activities. In addition, a combination of the anti-V3 and CD4bs MAbs showed a synergistic effect over the neutralization of HIV-1JR-FL. A humoral response from a single patient covered a wide range of viruses by complementary and synergistic activities of antibodies with different specificities. Inducing a set of narrow neutralizing antibodies, easier to induce than the broadly neutralizing antibodies, could be a strategy for developing an effective vaccine against HIV-1.
Asunto(s)
Antígenos CD4/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/clasificación , VIH-1/inmunología , Fragmentos de Péptidos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos/inmunología , Epítopos/inmunología , Anticuerpos Anti-VIH/fisiología , Proteína gp120 de Envoltorio del VIH/fisiología , Infecciones por VIH/virología , HumanosRESUMEN
Genotype-based algorithms are valuable tools for the identification of patients eligible for CCR5 inhibitors administration in clinical practice. Among the available methods, geno2pheno[coreceptor] (G2P) is the most used online tool for tropism prediction. This study was conceived to assess if the combination of G2P prediction with V3 peptide net charge (NC) value could improve the accuracy of tropism prediction. A total of 172 V3 bulk sequences from 143 patients were analyzed by G2P and NC values. A phenotypic assay was performed by cloning the complete env gene and tropism determination was assessed on U87_CCR5(+)/CXCR4(+) cells. Sequences were stratified according to the agreement between NC values and G2P results. Of sequences predicted as X4 by G2P, 61% showed NC values higher than 5; similarly, 76% of sequences predicted as R5 by G2P had NC values below 4. Sequences with NC values between 4 and 5 were associated with different G2P predictions: 65% of samples were predicted as R5-tropic and 35% of sequences as X4-tropic. Sequences identified as X4 by NC value had at least one positive residue at positions known to be involved in tropism prediction and positive residues in position 32. These data supported the hypothesis that NC values between 4 and 5 could be associated with the presence of dual/mixed-tropic (DM) variants. The phenotypic assay performed on a subset of sequences confirmed the tropism prediction for concordant sequences and showed that NC values between 4 and 5 are associated with DM tropism. These results suggest that the combination of G2P and NC could increase the accuracy of tropism prediction. A more reliable identification of X4 variants would be useful for better selecting candidates for Maraviroc (MVC) administration, but also as a predictive marker in coreceptor switching, strongly associated with the phase of infection.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/fisiología , Fragmentos de Péptidos/fisiología , Tropismo Viral , Antagonistas de los Receptores CCR5/uso terapéutico , Genotipo , Proteína gp120 de Envoltorio del VIH/genética , Inhibidores de Fusión de VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , Humanos , Técnicas de Amplificación de Ácido Nucleico , Fragmentos de Péptidos/genética , Valor Predictivo de las Pruebas , Receptores del VIH/genética , Receptores del VIH/fisiología , Alineación de Secuencia , Resultado del Tratamiento , Tropismo Viral/fisiologíaRESUMEN
BACKGROUND: Variable loops 1 and 2 (V1V2) of the HIV-1 envelope glycoprotein gp120 perform two key functions: ensuring envelope trimer entry competence and shielding against neutralizing antibodies. While preserving entry functionality would suggest a high need for V1V2 sequence optimization and conservation, shielding efficacy is known to depend on a high flexibility of V1V2 giving rise to its substantial sequence variability. How entry competence of the trimer is maintained despite the continuous emergence of antibody escape mutations within V1V2 has not been resolved. Since HIV cell-cell transmission is considered a highly effective means of virus dissemination, we investigated whether cell-cell transmission may serve to enhance infectivity of V1V2 variants with debilitated free virus entry. RESULTS: In a detailed comparison of wt and V1V2 mutant envelopes, V1V2 proved to be a key factor in ascertaining free virus infectivity, with V1V2 mutants displaying significantly reduced trimer integrity. Despite these defects, cell-cell transmission was able to partially rescue infectivity of V1V2 mutant viruses. We identified two regions, encompassing amino acids 156 to 160 (targeted by broadly neutralizing antibodies) and 175 to 180 (encompassing the α4ß7 binding site) which were particularly prone to free virus infectivity loss upon mutation but maintained infectivity in cell-cell transmission. Of note, V1V2 antibody shielding proved important during both free virus infection and cell-cell transmission. CONCLUSIONS: Based on our data we propose a model for V1V2 evolution that centers on cell-cell transmission as a salvage pathway for virus replication. Escape from antibody neutralization may frequently result in V1V2 mutations that reduce free virus infectivity. Cell-cell transmission could provide these escape viruses with sufficiently high replication levels that enable selection of compensatory mutations, thereby restoring free virus infectivity while ensuring antibody escape. Thus, our study highlights the need to factor in cell-cell transmission when considering neutralization escape pathways of HIV-1.