RESUMEN
Elucidation of mechanisms underlying the establishment, maintenance of and reactivation from HIV-1 latency is essential for the development of therapeutic strategies aimed at eliminating HIV-1 reservoirs. Microbial translocation, as a consequence of HIV-1-induced deterioration of host immune system, is known to result in a systemic immune activation and transient outbursts of HIV-1 viremia in chronic HIV-1 infection. How these microbes cause the robust HIV-1 reactivation remains elusive. Dendritic cells (DCs) have previously been shown to reactivate HIV-1 from latency; however, the precise role of DCs in reactivating HIV-1 from latently infected T-cell remains obscure. In this study, by using HIV-1 latently infected Jurkat T cells, we demonstrated that AIDS-associated pathogens as represented by Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) and bacterial component lipopolysaccharide (LPS) were unable to directly reactivate HIV-1 from Jurkat T cells; instead, they mature DCs to secrete TNF-α to accomplish this goal. Moreover, we found that HIV-1 latently infected Jurkat T cells could also mature DCs and enhance their TNF-α production during co-culture in a CD40-CD40L-signaling-dependent manner. This in turn led to viral reactivation from Jurkat T cells. Our results reveal how DCs help AIDS-associated pathogens to trigger HIV-1 reactivation from latency.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , Células Dendríticas/inmunología , VIH-1/fisiología , Células Jurkat/virología , Factor de Necrosis Tumoral alfa/farmacología , Latencia del Virus , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/virología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Técnicas de Cocultivo , Células Dendríticas/citología , VIH-1/genética , Humanos , Células Jurkat/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Activación ViralRESUMEN
HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating LTR activity contribute to HIV-1 latency, and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleocytoplasmic shuttling protein that regulates multiple cellular signaling pathways and HIV-1 production. We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production. Here we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both latently HIV-1-infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo Our findings provide new insights for a better understanding of HIV-1 latency and suggest that inhibition of Naf1 activity to activate latently HIV-1-infected cells may be a potential therapeutic strategy. IMPORTANCE: HIV-1 latency is characterized mainly by a reversible silencing of LTR promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that the host protein Naf1 inhibited HIV-1 LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into the effects of host modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting the Naf1 protein.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Regulación Viral de la Expresión Génica , Infecciones por VIH/genética , VIH-1/genética , Ribonucleoproteínas/genética , Latencia del Virus/genética , Linfocitos T CD4-Positivos/virología , Núcleo Celular/metabolismo , Núcleo Celular/virología , Silenciador del Gen , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , FN-kappa B/genética , FN-kappa B/metabolismo , Cultivo Primario de Células , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Ribonucleoproteínas/antagonistas & inhibidores , Ribonucleoproteínas/metabolismo , Transducción de Señal , Transcripción Genética , Activación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.
Asunto(s)
Proteínas Portadoras/metabolismo , Carioferinas/metabolismo , Precursores del ARN/metabolismo , Transporte de ARN , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Proteínas Portadoras/genética , Supervivencia Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , VIH-1/fisiología , Humanos , Inmunoprecipitación , Carioferinas/antagonistas & inhibidores , Carioferinas/genética , Microscopía Confocal , Señales de Exportación Nuclear , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Mutación Puntual , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Proteína Exportina 1RESUMEN
P-selectin glycoprotein ligand-1 (PSGL-1) has been proved to serve as the functional receptor for enterovirus 71 (EV71). We found the abundant expression of PSGL-1 on monocyte-derived dendritic cells (MDDCs). However, we have previously demonstrated that MDDCs did not support efficient replication of EV71. Dendritic cells (DCs) have been described to be subverted by various viruses including EV71 for viral dissemination, we thus explore the potential contribution of PSGL-1 on DC-mediated EV71 transmission. We found that the cell-surface-expressing PSGL-1 on MDDCs mediated EV71 binding, and intriguingly, these loaded-viruses on MDDCs could be transferred to encountered target cells; Prior-treatment with PSGL-1 antibodies or interference with PSGL-1 expression diminished MDDC-mediated EV71 transfer and rescued virus-induced cell death. Our data uncover a novel role of PSGL-1 in DC-mediated EV71 spread, and provide an insight into blocking primary EV71 infection.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/virología , Enterovirus Humano A/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Anticuerpos Monoclonales/inmunología , Muerte Celular/inmunología , Línea Celular , Células Dendríticas/inmunología , Enterovirus Humano A/metabolismo , Citometría de Flujo , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , Glicoproteínas de Membrana/metabolismo , ARN Interferente Pequeño , Receptores Virales/metabolismo , Acoplamiento Viral/efectos de los fármacosRESUMEN
BACKGROUND: Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease that occurs in young children. Neither antiviral agents nor vaccines are available for efficiently combating viral infection. Study of EV71-host interplay is important for understanding viral infection and developing strategies for prevention and therapy. Here the interactions of EV71 with human dendritic cells were analyzed. METHODS: EV71 capture, endocytosis, infection, and degradation in monocyte-derived dendritic cells (MDDCs) were detected by Flow cytometry or real-time (RT-) PCR, and MDDCs-mediated EV71 trans-infection of RD cells was determined via coculture system. Cell morphology or viability was monitored with microscopy or flow cytometry. SiRNA interference was used to knock down gene expression. RESULTS: MDDCs can bind EV71, but these loaded-EV71 particles in MDDCs underwent a rapid degradation in the absence of efficient replication; once the captured EV71 encountered susceptible cells, MDDCs efficiently transferred surface-bound viruses to target cells. The molecule of DC-SIGN (DC-specific intercellular adhesion molecule-3 grabbing nonintegrin) mediated viral binding and transfer, because interference of DC-SIGN expression with specific siRNAs reduced EV71 binding and impaired MDDC-mediated viral trans-infection, and exogenous expression of DC-SIGN molecule on Raji cell initiated viral binding and subsequent transmission. CONCLUSION: MDDCs could bind efficiently EV71 viruses through viral binding to DC-SIGN molecule, and these captured-viruses could be transferred to susceptible cells for robust infection. The novel finding of DC-mediated EV71 dissemination might facilitate elucidation of EV71 primary infection and benefit searching for new clues for preventing viruses from initial infection.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/virología , Enterovirus Humano A/inmunología , Enterovirus Humano A/fisiología , Interacciones Huésped-Patógeno , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Acoplamiento Viral , Moléculas de Adhesión Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Humanos , Lectinas Tipo C/genética , Receptores de Superficie Celular/genéticaRESUMEN
Altered cytokine profiles and imbalanced frequencies of CD4(+) T helper cell subsets are thought to be linked with HIV-1/AIDS pathogenesis, but the causes need to be further clarified. Histamine, a biogenic amine with many functions, shows enhancement in HIV-1 infected individuals, which are considered to link with disease progression, but is poorly understood. This study investigated histamine-assisted HIV-1 modulation of dendritic cell (DC) functions. Histamine and HIV-1 showed a synergistic role in induction of interleukin-10; histamine inhibited HIV-1-induced IL-12 production from MDDCs (monocyte-derived DCs); notably, histamine augmented HIV-1-induced MDDC functional polarization and skewed naïve T cell differentiation toward regulatory T cells (Tregs). The results indicate the novel role of histamine in HIV-1-induced DC functional regulation, which promoted Treg cell differentiation and up-regulated immunosuppressive factors. These findings help to bridge the correlation between elevated histamine and increased Treg cell frequency in HIV-1 infected individuals, and add to our understanding of HIV-1-induced immunosuppression.