RESUMEN
In vitro studies have shown that deletion of nef and deleterious mutation in the Nef dimerization interface attenuates HIV replication and associated pathogenesis. Humanized rodents with human immune cells and lymphoid tissues are robust in vivo models for investigating the interactions between HIV and the human immune system. Here, we demonstrate that nef deletion impairs HIV replication and HIV-induced immune dysregulation in the blood and human secondary lymphoid tissue (human spleen) in bone marrow-liver-thymus-spleen (BLTS) humanized mice. Furthermore, we also show that nef defects (via deleterious mutations in the dimerization interface) impair HIV replication and HIV-induced immune dysregulation in the blood and human spleen in BLTS-humanized mice. We demonstrate that the reduced replication of nef-deleted and nef-defective HIV is associated with robust antiviral innate immune response, and T helper 1 response. Our results support the proposition that Nef may be a therapeutic target for adjuvants in HIV cure strategies.
Asunto(s)
Modelos Animales de Enfermedad , Infecciones por VIH , VIH-1 , Hígado , Bazo , Viremia , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Ratones , Humanos , Viremia/inmunología , Bazo/inmunología , Bazo/virología , VIH-1/inmunología , VIH-1/genética , VIH-1/fisiología , Hígado/virología , Hígado/inmunología , Hígado/patología , Médula Ósea/virología , Médula Ósea/inmunología , Timo/inmunología , Timo/virología , Inmunidad InnataRESUMEN
Therapeutic inhibition of the viral protein Nef is an intriguing direction of antiretroviral drug discovery-it may revitalize immune mechanisms to target, and potentially clear, HIV-1-infected cells. Of the many cellular functions of Nef, the most conserved is the downregulation of surface CD4, which takes place through Nef hijacking the clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Our recent crystal structure has unraveled the molecular details of the CD4-Nef-AP2 interaction. Guided by the new structural knowledge, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on CD4. In our assay, AP2 is included along with Nef to facilitate the proper formation of the CD4-binding pocket and a fluorescently labeled CD4 cytoplasmic tail binds competently to the Nef-AP2 complex generating the desired polarization signal. The optimized assay has a good signal-to-noise ratio, excellent tolerance of dimethylsulfoxide and detergent, and the ability to detect competitive binding at the targeted Nef pocket, making it suitable for high-throughput screening.
Asunto(s)
Antígenos CD4 , Regulación hacia Abajo , Polarización de Fluorescencia , VIH-1 , Ensayos Analíticos de Alto Rendimiento , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Antígenos CD4/metabolismo , Antígenos CD4/química , Humanos , Polarización de Fluorescencia/métodos , VIH-1/metabolismo , VIH-1/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Regulación hacia Abajo/efectos de los fármacos , Complejo 2 de Proteína Adaptadora/metabolismo , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/química , Unión ProteicaRESUMEN
The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.
Asunto(s)
Regulación hacia Abajo , Polarización de Fluorescencia , VIH-1 , Ensayos Analíticos de Alto Rendimiento , Antígenos de Histocompatibilidad Clase I , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Humanos , Polarización de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Regulación hacia Abajo/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/metabolismo , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/químicaRESUMEN
Natural Killer (NK) cells have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is tightly regulated by the engagement of its inhibitory and activating receptors. The activating receptor CD16 drives ADCC upon binding to the Fc portion of antibodies; NK cell activation is further sustained by the co-engagement of activating receptors NTB-A and 2B4. During HIV-1 infection, Nef and Vpu accessory proteins contribute to ADCC escape by downregulating the ligands of NTB-A and 2B4. HIV-1 also evades ADCC by keeping its envelope glycoproteins (Env) in a "closed" conformation which effectively masks epitopes recognized by non-neutralizing antibodies (nnAbs) which are abundant in the plasma of people living with HIV. To achieve this, the virus uses its accessory proteins Nef and Vpu to downregulate the CD4 receptor, which otherwise interacts with Env and exposes the epitopes recognized by nnAbs. Small CD4-mimetic compounds (CD4mc) have the capacity to expose these epitopes, thus sensitizing infected cells to ADCC. Given the central role of NK cell co-activating receptors NTB-A and 2B4 in Fc-effector functions, we studied their contribution to CD4mc-mediated ADCC. Despite the fact that their ligands are partially downregulated by HIV-1, we found that both co-activating receptors significantly contribute to CD4mc sensitization of HIV-1-infected cells to ADCC.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Anticuerpos Anti-VIH , Infecciones por VIH , VIH-1 , Células Asesinas Naturales , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Humanos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , VIH-1/inmunología , Células Asesinas Naturales/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/inmunología , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/inmunología , Proteínas Reguladoras y Accesorias Virales/genética , Anticuerpos Neutralizantes/inmunología , Proteínas ViroporinasRESUMEN
Extracellular vesicles (EVs) serve as pivotal mediators of intercellular communication in both health and disease, delivering biologically active molecules from vesicle-producing cells to recipient cells. In the context of HIV infection, EVs have been shown to carry the viral protein Nef, a key pathogenic factor associated with HIV-related co-morbidities. Despite this recognition, the specific localisation of Nef within the vesicles has remained elusive. This study addresses this critical knowledge gap by investigating Nef-containing EVs. Less than 1% of the total released Nef was associated with EVs; most Nef existed as free protein released by damaged cells. Nevertheless, activity of EV-associated Nef in downregulating the major cholesterol transporter ABCA1, a critical aspect linked to the pathogenic effects of Nef, was comparable to that of free Nef present in the supernatant. Through a series of biochemical and microscopic assays, we demonstrate that the majority of EV-associated Nef molecules are localised on the external surface of the vesicles. This distinctive distribution prompts the consideration of Nef-containing EVs as potential targets for immunotherapeutic interventions aimed at preventing or treating HIV-associated co-morbidities. In conclusion, our results shed light on the localisation and functional activity of Nef within EVs, providing valuable insights for the development of targeted immunotherapies to mitigate the impact of HIV-associated co-morbidities.
Asunto(s)
Vesículas Extracelulares , Infecciones por VIH , VIH-1 , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Vesículas Extracelulares/metabolismo , Humanos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/metabolismo , Infecciones por VIH/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismoRESUMEN
Fibrocytes were reported to be host cells for HIV-1, but the immunological recognition of HIV-1-infected fibrocytes has not been studied. Here, we investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific CD8+ T cells. CD8+ T cells specific for five HIV-1 epitopes (HLA-A*24:02-restricted, HLA-B*52:01-restricted, and HLA-C*12:02-restricted epitopes) produced IFN-γ and expressed CD107a after coculture with HIV-1-infected fibrocytes. HIV-1-infected fibrocytes were effectively killed by HIV-1-specific CD8+ T cells. Although it is well known that HIV-1 Nef-mediated downregulation of HLA-A and HLA-B critically affects the T cell recognition of HIV-1-infected CD4+ T cells and HIV-1-infected macrophages, Nef downregulated HLA-A, but not HLA-B, in HIV-1-infected fibrocytes. These findings suggested that HIV-1-specific CD8+ T cells could recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells or HIV-1-infected macrophages. HIV-1-infected fibrocytes were also recognized by HIV-1-specific HLA-DR-restricted T cells, indicating that HIV-1-infected fibrocytes can present HIV-1 epitopes to helper T cells. Collectively, these findings suggest that fibrocytes have an important role as antigen-presenting cells during HIV-1 infection. The present study demonstrates effective recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells and suggests possible roles of fibrocytes in the induction and maintenance of HIV-1-specific T cells. IMPORTANCE: Fibrocytes were identified as unique hematopoietic cells with the features of both macrophages and fibroblasts and were demonstrated to be host cells for HIV-1. However, T cell recognition of HIV-1-infected fibrocytes has not been studied. We investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells. HIV-1-infected fibrocytes were effectively recognized and killed by CD8+ T cells specific for HIV-1 epitopes presented by HLA-A, HLA-B, or HLA-C and were recognized by HIV-1-specific HLA-DR-restricted CD4+ T cells. HIV-1 Nef-mediated downregulation of HLA-A and HLA-B was found in HIV-1-infected CD4+ T cells, whereas Nef did not downregulate HLA-B in HIV-1-infected fibrocytes. These results suggest that HIV-1-specific CD8+ T cells recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells. The present study suggests the importance of fibrocytes in the induction and maintenance of HIV-1-specific T cells.
Asunto(s)
Linfocitos T CD8-positivos , Regulación hacia Abajo , Infecciones por VIH , VIH-1 , Antígenos HLA-B , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Humanos , VIH-1/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Antígenos HLA-B/inmunología , Antígenos HLA-B/metabolismo , Fibroblastos/virología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Macrófagos/inmunología , Macrófagos/virología , Macrófagos/metabolismoRESUMEN
Bacteria-derived outer membrane vesicles (OMVs) can be engineered to incorporate foreign antigens. This study explored the potential of ClearColi™-derived OMVs as a natural adjuvant and a carrier (recombinant OMVs or rOMVs) for development of an innovative therapeutic vaccine candidate harboring HIV-1 Nef and Nef-Tat antigens. Herein, the rOMVs containing CytolysinA (ClyA)-Nef and ClyA-Nef-Tat fusion proteins were isolated from ClearColi™ strain. The presence of Nef and Nef-Tat proteins on their surface (rOMVNef and rOMVNef-Tat) was confirmed by western blotting after proteinase K treatment. Immune responses induced by Nef and Nef-Tat proteins emulsified with Montanide® ISA720 or mixed with OMVs, and also rOMVNef and rOMVNef-Tat were investigated in BALB/c mice. Additionally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was assessed for the secretion of cytokines in vitro. Our findings showed that the rOMVs as an antigen carrier (rOMVNef and rOMVNef-Tat) induced higher levels of IgG2a, IFN-γ and granzyme B compared to OMVs as an adjuvant (Nef + OMV and Nef-Tat + OMV), and also Montanide® ISA720 (Nef + Montanide and Nef-Tat + Montanide). Moreover, IFN-γ level in splenocytes isolated from mice immunized with rOMVNef-Tat was higher than other regimens after exposure to SCR virions. Generally, ClearColi™-derived rOMVs can serve as potent carriers for developing effective vaccines against HIV-1 infection.
Asunto(s)
Vacunas contra el SIDA , Adyuvantes Inmunológicos , Infecciones por VIH , VIH-1 , Ratones Endogámicos BALB C , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/genética , VIH-1/genética , VIH-1/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Ratones , Adyuvantes Inmunológicos/administración & dosificación , Infecciones por VIH/prevención & control , Infecciones por VIH/inmunología , Femenino , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Citocinas/metabolismo , Inmunoglobulina G/sangre , Anticuerpos Anti-VIH/inmunología , Membrana Externa Bacteriana/metabolismo , Desarrollo de Vacunas , Humanos , Portadores de Fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Bazo/inmunologíaRESUMEN
BACKGROUND: Heterologous combinations in vaccine design are an effective approach to promote T cell activity and antiviral effects. The goal of this study was to compare the homologous and heterologous regimens targeting the Nef-Tat fusion antigen to develop a human immunodeficiency virus-1 (HIV-1) therapeutic vaccine candidate. METHODS: At first, the DNA and protein constructs harboring HIV-1 Nef and the first exon of Tat as linked form (pcDNA-nef-tat and Nef-Tat protein) were prepared in large scale and high purity. The generation of the Nef-Tat protein was performed in the E. coli expression system using an IPTG inducer. Then, we evaluated and compared immune responses of homologous DNA prime/ DNA boost, homologous protein prime/ protein boost, and heterologous DNA prime/protein boost regimens in BALB/c mice. Finally, the ability of mice splenocytes to secret cytokines after exposure to single-cycle replicable (SCR) HIV-1 was compared between immunized and control groups in vitro. RESULTS: The nef-tat gene was successfully subcloned in eukaryotic pcDNA3.1 (-) and prokaryotic pET-24a (+) expression vectors. The recombinant Nef-Tat protein was generated in the E. coli Rosetta strain under optimized conditions as a clear band of ~ 35 kDa detected on SDS-PAGE. Moreover, transfection of pcDNA-nef-tat into HEK-293T cells was successfully performed using Lipofectamine 2000, as confirmed by western blotting. The immunization studies showed that heterologous DNA prime/protein boost regimen could significantly elicit the highest levels of Ig- G2a, IFN-γ, and Granzyme B in mice as compared to homologous DNA/DNA and protein/protein regimens. Moreover, the secretion of IFN-γ was higher in DNA/protein regimens than in DNA/DNA and protein/protein regimens after exposure of mice splenocytes to SCR HIV-1 in vitro. CONCLUSION: The chimeric HIV-1 Nef-Tat antigen was highly immunogenic, especially when applied in a heterologous prime/ boost regimen. This regimen could direct immune response toward cellular immunity (Th1 and CTL activity) and increase IFN-γ secretion after virus exposure.
Asunto(s)
Vacunas contra el SIDA , VIH-1 , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión , Vacunas de ADN , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Animales , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/genética , Vacunas de ADN/inmunología , Vacunas de ADN/genética , VIH-1/inmunología , VIH-1/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Ratones , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Humanos , Femenino , Linfocitos T/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Inmunización Secundaria , Citocinas/metabolismo , Escherichia coli/genética , Escherichia coli/inmunologíaRESUMEN
Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.
Asunto(s)
Linfocitos T CD4-Positivos , VIH-1 , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Humanos , VIH-1/genética , VIH-1/fisiología , Replicación Viral/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Células HEK293 , Sistemas CRISPR-Cas , Infecciones por VIH/virología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Internalización del VirusRESUMEN
Antigen presenting cells (APCs)-derived exosomes are nano-vesicles that can induce antigen-specific T cell responses, and possess therapeutic effects in clinical settings. Moreover, dendritic cells (DCs)-based vaccines have been developed to combat human immunodeficiency virus-1 (HIV-1) infection in preclinical and clinical trials. We investigated the immunostimulatory effects (B- and T-cells activities) of DCs- and exosomes-based vaccine constructs harboring HIV-1 Nefmut-Tat fusion protein as an antigen candidate and heat shock protein 70 (Hsp70) as an adjuvant in mice. The modified DCs and engineered exosomes harboring Nefmut-Tat protein or Hsp70 were prepared using lentiviral vectors compared to electroporation, characterized and evaluated by in vitro and in vivo immunological tests. Our data indicated that the engineered exosomes induced high levels of total IgG, IgG2a, IFN-γ, TNF-α and Granzyme B. Moreover, co-injection of exosomes harboring Hsp70 could significantly increase the secretion of antibodies, cytokines and Granzyme B. The highest levels of IFN-γ and TNF-α were observed in exosomes harboring Nefmut-Tat combined with exosomes harboring Hsp70 (Exo-Nefmut-Tat + Exo-Hsp70) regimen after single-cycle replicable (SCR) HIV-1 exposure. Generally, Exo-Nefmut-Tat + Exo-Hsp70 regimen can be considered as a promising safe vaccine candidate due to high T-cells (Th1 and CTL) activity and its maintenance against SCR HIV-1 exposure.
Asunto(s)
Vacunas contra el SIDA , Células Dendríticas , Exosomas , VIH-1 , Proteínas HSP70 de Choque Térmico , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Exosomas/inmunología , Exosomas/metabolismo , Células Dendríticas/inmunología , Animales , VIH-1/inmunología , VIH-1/genética , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/genética , Vacunas contra el SIDA/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Ratones , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Humanos , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Citocinas/metabolismoRESUMEN
Escape from cytotoxic T lymphocyte (CTL) responses toward HIV-1 Gag and Nef has been associated with reduced control of HIV-1 replication in adults. However, less is known about CTL-driven immune selection in infants as longitudinal studies of infants are limited. Here, 1,210 gag and 1,264 nef sequences longitudinally collected within 15 months after birth from 14 HIV-1 perinatally infected infants and their mothers were analyzed. The number of transmitted founder (T/F) viruses and associations between virus evolution, selection, CTL escape, and disease progression were determined. The analyses indicated that a paraphyletic-monophyletic relationship between the mother-infant sequences was common (80%), and that the HIV-1 infection was established by a single T/F virus in 10 of the 12 analyzed infants (83%). Furthermore, most HIV-1 CTL escape mutations among infants were transmitted from the mothers and did not revert during the first year of infection. Still, immune-driven selection was observed at approximately 3 months after HIV-1 infection in infants. Moreover, virus populations with CTL escape mutations in gag evolved faster than those without, independently of disease progression rate. These findings expand the current knowledge of HIV-1 transmission, evolution, and CTL escape in infant HIV-1 infection and are relevant for the development of immune-directed interventions in infants.IMPORTANCEDespite increased coverage in antiretroviral therapy for the prevention of perinatal transmission, paediatric HIV-1 infection remains a significant public health concern, especially in areas of high HIV-1 prevalence. Understanding HIV-1 transmission and the subsequent virus adaptation from the mother to the infant's host environment, as well as the viral factors that affect disease outcome, is important for the development of early immune-directed interventions for infants. This study advances our understanding of vertical HIV-1 transmission, and how infant immune selection pressure is shaping the intra-host evolutionary dynamics of HIV-1.
Asunto(s)
Evolución Molecular , Infecciones por VIH , VIH-1 , Transmisión Vertical de Enfermedad Infecciosa , Mutación , Linfocitos T Citotóxicos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Humanos , VIH-1/genética , VIH-1/inmunología , Linfocitos T Citotóxicos/inmunología , Infecciones por VIH/virología , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , Lactante , Femenino , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Evasión Inmune/genética , Recién Nacido , Filogenia , Masculino , Estudios Longitudinales , Embarazo , AdultoRESUMEN
HIV-associated neurocognitive disorders (HAND) are a spectrum of cognitive impairments that continue to affect approximately half of all HIV-positive individuals despite effective viral suppression through antiretroviral therapy (ART). White matter pathologies have persisted in the ART era, and the degree of white matter damage correlates with the degree of neurocognitive impairment in patients with HAND. The HIV protein Nef has been implicated in HAND pathogenesis, but its effect on white matter damage has not been well characterized. Here, utilizing in vivo, ex vivo, and in vitro methods, we demonstrate that Nef-containing extracellular vesicles (Nef EVs) disrupt myelin sheaths and inflict damage upon oligodendrocytes within the murine central nervous system. Intracranial injection of Nef EVs leads to reduced myelin basic protein (MBP) staining and a decreased number of CC1 + oligodendrocytes in the corpus callosum. Moreover, cerebellar slice cultures treated with Nef EVs exhibit diminished MBP expression and increased presence of unmyelinated axons. Primary mixed brain cultures and enriched oligodendrocyte precursor cell cultures exposed to Nef EVs display a decreased number of O4 + cells, indicative of oligodendrocyte impairment. These findings underscore the potential contribution of Nef EV-mediated damage to oligodendrocytes and myelin maintenance in the pathogenesis of HAND.
Asunto(s)
Vesículas Extracelulares , VIH-1 , Oligodendroglía , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Ratones , Células Cultivadas , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Sistema Nervioso Central/virología , Vesículas Extracelulares/metabolismo , VIH-1/metabolismo , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Oligodendroglía/virologíaAsunto(s)
VIH-1 , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Humanos , VIH-1/efectos de los fármacos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Infecciones por VIH/tratamiento farmacológico , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Proteolisis/efectos de los fármacosRESUMEN
The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.
Asunto(s)
Infecciones por VIH , VIH-1 , ATPasas de Translocación de Protón Vacuolares , Humanos , VIH-1/fisiología , Evasión Inmune , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Lisosomas/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Members of the serine incorporator (SERINC) protein family exert broad antiviral activity, and many viruses encode SERINC antagonists to circumvent these restrictions. Significant new insight was recently gained into the mechanisms that mediate restriction and antagonism. In this review, we summarize our current understanding of the mode of action and relevance of SERINC proteins in HIV-1 infection. Particular focus will be placed on recent findings that provided important new mechanistic insights into the restriction of HIV-1 virion infectivity, including the discovery of SERINC's lipid scramblase activity and its antagonism by the HIV-1 pathogenesis factor Nef. We also discuss the identification and implications of several additional antiviral activities by which SERINC proteins enhance pro-inflammatory signaling and reduce viral gene expression in myeloid cells. SERINC proteins emerge as versatile and multifunctional regulators of cell-intrinsic immunity against HIV-1 infection.
Asunto(s)
Infecciones por VIH , Proteínas de la Membrana , Humanos , Proteínas de la Membrana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Interacciones Huésped-Patógeno , Virión/metabolismo , AntiviralesRESUMEN
The HIV-1 virus has been regarded as a catastrophe for human well-being. The global incidence of HIV-1-infected individuals is increasing. Hence, development of effective immunostimulatory molecules has recently attracted an increasing attention in the field of vaccine design against HIV-1 infection. In this study, we explored the impacts of CD40L and IFN-γ as immunostimulatory adjuvants for our candidate HIV-1 Nef vaccine in human and mouse using immunoinformatics analyses. Overall, 18 IFN-γ-based vaccine constructs (9 constructs in human and 9 constructs in mouse), and 18 CD40L-based vaccine constructs (9 constructs in human and 9 constructs in mouse) were designed. To find immunogenic epitopes, important characteristics of each component (e.g., MHC-I and MHC-II binding, and peptide-MHC-I/MHC-II molecular docking) were determined. Then, the selected epitopes were applied to create multiepitope constructs. Finally, the physicochemical properties, linear and discontinuous B cell epitopes, and molecular interaction between the 3D structure of each construct and CD40, IFN-γ receptor or toll-like receptors (TLRs) were predicted. Our data showed that the full-length CD40L and IFN-γ linked to the N-terminal region of Nef were capable of inducing more effective immune response than multiepitope vaccine constructs. Moreover, molecular docking of the non-allergenic full-length- and epitope-based CD40L and IFN-γ constructs to their cognate receptors, CD40 and IFN-γ receptors, and TLRs 4 and 5 in mouse were more potent than in human. Generally, these findings suggest that the full forms of these adjuvants could be more efficient for improvement of HIV-1 Nef vaccine candidate compared to the designed multiepitope-based constructs.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , Interferón gamma , Vacunas de Subunidades Proteicas , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Humanos , Ratones , Adyuvantes Inmunológicos/farmacología , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/química , Ligando de CD40/inmunología , Ligando de CD40/química , Simulación por Computador , Epítopos/inmunología , Epítopos/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/química , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1 , Interferón gamma/metabolismo , Interferón gamma/inmunología , Simulación del Acoplamiento Molecular , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Vacunas de Subunidades Proteicas/química , Vacunas de Subunidades Proteicas/inmunologíaRESUMEN
The Human Immunodeficiency Virus-1 (HIV-1) tends to activate cellular promoters driving expression of pro-viral genes by complex host-virus interactions for productive infection. We have previously demonstrated that expression of such a positive host factor HSF1 (heat shock factor 1) is elevated during HIV-1 infection; however, the mechanism remains to be elucidated. In the present study, we therefore examined whether HSF1 promoter is induced during HIV-1 infection leading to up-regulation of hsf1 gene expression. We mapped the putative transcription start site (TSS) predicted by Eukaryotic promoter database and deletion constructs of the predicted promoter region were tested through luciferase assay to identify the active promoter. The 347 bp upstream to 153 bp downstream region around the putative TSS displayed the highest activity and both Sp1 (stimulating protein 1) and HSF1 itself were identified to be important for its basal activation. Activity of HSF1 promoter was further stimulated during HIV-1 infection in CD4+ T cells, where interestingly the HSF1-site itself seems to play a major role. In addition, HIV-1 protein Nef (negative factor) was also observed to be responsible for the virus-mediated induction of hsf1 gene expression. Chromatin-immunoprecipitation assays further demonstrate that Nef and HSF1 are co-recruited to the HSF1-binding site and cooperatively act on this promoter. The interplay between host HSF1 and viral Nef on HSF1 promoter eventually leads to increase in HSF1 expression during HIV-1 infection. Understanding the mechanism of HSF1 up-regulation during HIV-1 infection might contribute to future antiviral strategies as HSF1 is a positive regulator of virus replication.
Asunto(s)
Infecciones por VIH , VIH-1 , Factores de Transcripción del Choque Térmico , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Humanos , VIH-1/fisiología , Regiones Promotoras Genéticas , Activación Transcripcional , Proteínas Virales/genética , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Infecciones por VIH/metabolismo , Regulación hacia ArribaRESUMEN
The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, using quantitative live-cell microscopy in genome-edited Jurkat cells, we investigate the impact of Nef on clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment is associated with an increase in the recruitment and lifetime of the CME coat protein AP-2 and the late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2 and transferrin, suggesting that Nef recruitment to CME sites promotes site maturation to ensure high efficiency in host protein downregulation. Implications of these observations for HIV-1 infection are discussed.
Asunto(s)
Clatrina , Endocitosis , VIH-1 , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Humanos , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis/fisiología , VIH-1/metabolismo , Células Jurkat , Proteínas de la Membrana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-associated neurocognitive disorders (HAND) is a term describing a complex set of cognitive impairments accompanying HIV infection. Successful antiretroviral therapy (ART) reduces the most severe forms of HAND, but milder forms affect over 50% of people living with HIV (PLWH). Pathogenesis of HAND in the ART era remains unknown. A variety of pathogenic factors, such as persistent HIV replication in the brain reservoir, HIV proteins released from infected brain cells, HIV-induced neuroinflammation, and some components of ART, have been implicated in driving HAND pathogenesis in ART-treated individuals. Here, we propose another factor-impairment of cholesterol homeostasis and lipid rafts by HIV-1 protein Nef-as a possible contributor to HAND pathogenesis. These effects of Nef on cholesterol may also underlie the effects of other pathogenic factors that constitute the multifactorial nature of HAND pathogenesis. The proposed Nef- and cholesterol-focused mechanism may provide a long-sought unified explanation of HAND pathogenesis that takes into account all contributing factors. Evidence for the impairment by Nef of cellular cholesterol balance, potential effects of this impairment on brain cells, and opportunities to therapeutically target this element of HAND pathogenesis are discussed.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/patología , VIH-1/metabolismo , Encéfalo/metabolismo , Colesterol/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/uso terapéuticoRESUMEN
BACKGROUND: HIV subtype is associated with varied rates of disease progression. The HIV accessory protein, Nef, continues to be present during antiretroviral therapy (ART) where it has numerous immunoregulatory effects. In this study, we analyzed Nef sequences from HIV subtypes A1, B, C, and D using a machine learning approach that integrates functional amino acid information to identify if unique physicochemical features are associated with Nef functional/structural domains in a subtype-specific manner. METHODS: 2253 sequences representing subtypes A1, B, C, and D were aligned and domains with known functional properties were scored based on amino acid physicochemical properties. Following feature generation, we used statistical pruning and evolved neural networks (ENNs) to determine if we could successfully classify subtypes. Next, we used ENNs to identify the top five key Nef physicochemical features applied to specific immunoregulatory domains that differentiated subtypes. A signature pattern analysis was performed to the assess amino acid diversity in sub-domains that differentiated each subtype. RESULTS: In validation studies, ENNs successfully differentiated each subtype at A1 (87.2%), subtype B (89.5%), subtype C (91.7%), and subtype D (85.1%). Our feature-based domain scoring, followed by t-tests, and a similar ENN identified subtype-specific domain-associated features. Subtype A1 was associated with alterations in Nef CD4 binding domain; subtype B was associated with alterations with the AP-2 Binding domain; subtype C was associated with alterations in a structural Alpha Helix domain; and, subtype D was associated with alterations in a Beta-Sheet domain. CONCLUSIONS: Recent studies have focused on HIV Nef as a driver of immunoregulatory disease in those HIV infected and on ART. Nef acts through a complex mixture of interactions that are directly linked to the key features of the subtype-specific domains we identified with the ENN. The study supports the hypothesis that varied Nef subtypes contribute to subtype-specific disease progression.