RESUMEN
The human APOBEC3 cytidine deaminases are potent inhibitors of diverse retroviruses, including human immunodeficiency virus-1 (HIV-1). HIV-1 Vif forms an E3 ubiquitin ligase complex with cullin 5 (CUL5), elongin B and elongin C , which promotes the polyubiquitination and degradation of APOBEC3 substrates. Here we demonstrate in human T cells that core binding factor ß (CBF-ß) is a key regulator of the evasion of HIV-1 from the host defence mediated by APOBEC3. CBF-ß, the non-DNA-binding subunit of a heterodimeric transcription factor, regulates the folding and DNA-binding activity of partner RUNX family proteins, which have important roles in the development and differentiation of diverse cell types, including T lymphocytes. In our study, knockdown of endogenous CBF-ß blocked Vif-induced APOBEC3G polyubiquitination and degradation. CBF-ß was not required for the interaction between Vif and APOBEC3G, yet was essential for the assembly of the Vif-CUL5 E3-ubiquitin-ligase complex. CBF-ß proved to be a unique regulator of primate lentiviral Vif and not a general component of the CUL5 E3 ubiquitin ligase. We show that Vif and CBF-ß physically interact, and that the amino-terminal region of Vif is required for this interaction. Furthermore, interactions with Vif required regions in CBF-ß that are not involved in RUNX protein binding. Considering the importance of the interaction between Vif and CBF-ß, disrupting this interaction represents an attractive pharmacological intervention against HIV-1.
Asunto(s)
Diferenciación Celular , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , VIH-1/fisiología , Interacciones Huésped-Patógeno , Evasión Inmune , Linfocitos T/citología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Línea Celular , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/química , Subunidad beta del Factor de Unión al Sitio Principal/deficiencia , Subunidad beta del Factor de Unión al Sitio Principal/genética , Proteínas Cullin/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , VIH-1/genética , VIH-1/inmunología , Humanos , Inmunoprecipitación , Modelos Moleculares , Unión Proteica , Proteolisis , Linfocitos T/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
UNLABELLED: CBFß was recently found to be a key regulator of the ability of human immunodeficiency virus type 1 (HIV-1) Vif to overcome host antiviral APOBEC3 proteins. However, the detailed molecular requirements for the Vif-CBFß interaction are still not clear. Here, we mapped the minimum Vif domain required for CBFß binding. In terms of CBFß binding, the Vif N terminus was very sensitive to deletions. We determined that the Vif fragment from residues 5 to 126 was sufficient to form a stable complex with CBFß in vitro. We also observed that ionic interactions were not the main contributor to the interaction between Vif and CBFß. Instead, hydrophobic interactions were important for maintaining the Vif-CBFß complex, since it could be disrupted by nonionic detergent. Site-directed mutagenesis of conserved hydrophobic amino acids revealed novel residues in Vif that were important for CBFß binding and APOBEC3 inactivation. At least part of the well-characterized HCCH domain (residues 108 to 139) was required to form a stable Vif-CBFß complex. Thus, the HCCH motif may have a dual role in binding both Cul5 and CBFß. Considering the importance of Vif in HIV-1 infection, this unique Vif-CBFß interaction represents an attractive pharmacological intervention target against HIV-1. IMPORTANCE: Vif-induced APOBEC3 protein degradation was the first host antiviral mechanism against HIV-1/simian immunodeficiency virus to be revealed, yet details regarding which proteins are degraded are not fully demonstrated. Recently, host cellular factor CBFß was found to be essential for Vif to function and promote viral infectivity. In this study, we present more critical information on the Vif-CBFß interaction by revealing that hydrophobicity contributes the most to the Vif-CBFß interaction and locating several novel hydrophobic sites (tryptophans and phenylalanines) that are conserved among Vif proteins from different lentiviruses and essential for Vif binding to CBFß. Mutations on these sites result in a reduced/abolished Vif-CBFß interaction, leading to the attenuated potency of Vif on both inducing the degradation of antiviral factors like APOBEC3G and promoting HIV-1 infectivity. Therefore, information from this study will help people to further understand how Vif acts against host antiviral mechanism, which is important for novel anti-HIV-1 drug development.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Citidina Desaminasa/metabolismo , VIH-1/metabolismo , Dominios y Motivos de Interacción de Proteínas , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , Unión Proteica , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
UNLABELLED: The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-ß) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-ß promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-ß is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-ß from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-ß interfaces. Considering the importance of the interaction between Vif and CBF-ß in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. IMPORTANCE: HIV-1 encodes virion infectivity factor (Vif) to inactivate its host's antiviral APOBEC3 proteins. Vif triggers APOBEC3 degradation by forming Vif-Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-ß) is a novel regulator of Vif-CRL5 function whose mechanism of regulation remains poorly defined. In the present study, we demonstrate that the promotion of Vif-CRL5 assembly by CBF-ß can be separated from its influence on Vif stability. The promotion of Vif-CRL5 assembly, but not the influence on Vif stability, is conserved among primate lentiviral Vif proteins: we found that CBF-ß from diverse vertebrate species supported HIV-1 Vif function. Considering the importance of the interaction between Vif and CBF-ß in viral CRL5 function and HIV-1 replication, disrupting this interaction is an attractive strategy against HIV-1.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Proteínas Cullin/metabolismo , Evolución Molecular , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Subunidad beta del Factor de Unión al Sitio Principal/química , Proteínas Cullin/genética , Elonguina , Productos del Gen vif/química , Productos del Gen vif/genética , Productos del Gen vif/metabolismo , Infecciones por VIH/enzimología , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Virus de la Inmunodeficiencia de los Simios/química , Virus de la Inmunodeficiencia de los Simios/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: HIV-1 Vif promotes the degradation of host anti-retroviral factor family, APOBEC3 proteins via the recruitment of a multi-subunit E3 ubiquitin ligase complex. The complex is composed of a scaffold protein, Cullin 5 (Cul5), RING-box protein (Rbx), a SOCS box binding protein complex, Elongins B/C (Elo B/C), as well as newly identified host co-factor, core binding factor beta (CBF-ß). Cul5 has previously been shown to bind amino acids within an HCCH domain as well as a PPLP motif at the C-terminus of Vif; however, it is unclear whether Cul5 binding requires additional regions of the Vif polypeptide. RESULTS: Here, we provide evidence that an amino terminal region of full length Vif is necessary for the Vif-Cul5 interaction. Single alanine replacement of select amino acids spanning residues 25-30 (25VXHXMY30) reduced the ability for Vif to bind Cul5, but not CBF-ß or Elo B/C in pull-down experiments. In addition, recombinant Vif mutants had a reduced binding affinity for Cul5 compared to wild-type as measured by isothermal titration calorimetry. N-terminal mutants that demonstrated reduced Cul5 binding were also unable to degrade APOBEC3G as well as APOBEC3F and were unable to restore HIV infectivity, in the presence of APOBEC3G. Although the Vif N-terminal amino acids were necessary for Cul5 interaction, the mutation of each residue to alanine induced a change in the secondary structure of the Vif-CBF-ß-Elo B/C complex as suggested by results from circular dichroism spectroscopy and size-exclusion chromatography experiments. Surprisingly, the replacement of His108 to alanine also contributed to the Vif structure. Thus, it is unclear whether the amino acids contribute to a direct interaction with Cul5 or whether the amino acids are responsible for the structural organization of the Vif protein that promotes Cul5 binding. CONCLUSIONS: Taken together, we propose a novel Vif N-terminal motif that is responsible for Vif recruitment of Cul5. Motifs in Vif that are absent from cellular proteins represent attractive targets for future HIV pharmaceutical design.
Asunto(s)
Proteínas Cullin/metabolismo , Citidina Desaminasa/antagonistas & inhibidores , Citosina Desaminasa/antagonistas & inhibidores , VIH-1/inmunología , VIH-1/fisiología , Evasión Inmune , Desaminasa APOBEC-3G , Citidina Desaminasa/metabolismo , Citosina Desaminasa/metabolismo , Humanos , Mapeo de Interacción de Proteínas , Proteolisis , Productos del Gen vif del Virus de la Inmunodeficiencia HumanaRESUMEN
The use of antimicrobials in commercial broiler poultry production results in the presence of drug-resistant bacteria shed in the excreta of these birds. Because these wastes are largely land-disposed these pathogens can affect the surrounding environment and population. In this analysis, we characterized the survival of antimicrobial-resistant enterococci and staphylococci and resistance genes in poultry litter. Temperature, moisture, and pH were measured in the litter over a 120-day period from storage sheds at three conventional US broiler chicken farms, as well as colony-forming units of Enterococcus spp. and Staphylococcus spp. Selected isolates from each sampling event were tested for resistance to eight antimicrobials used in poultry feeds as well as the presence of resistance genes and mobile genetic elements. Temperatures greater than 60 degrees C were only intermittently observed in the core of the litter piles. Both antimicrobial-resistant enterococci and staphylococci, as well as resistance genes persisted throughout the 120-day study period. Resistance genes identified in the study include: erm(A), erm(B), erm (C), msr(A/B), msr(C), and vat(E). This study indicates that typical storage practices of poultry litter are insufficient for eliminating drug-resistant enterococci and staphylococci, which may then be released into the environment through land disposal.
Asunto(s)
Antiinfecciosos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Enterococcus/aislamiento & purificación , Estiércol/microbiología , Aves de Corral/crecimiento & desarrollo , Staphylococcus/aislamiento & purificación , Agricultura/normas , Animales , Química Física , Delaware , Farmacorresistencia Bacteriana/genética , Enterococcus/efectos de los fármacos , Enterococcus/genética , Enterococcus/crecimiento & desarrollo , Estiércol/análisis , Staphylococcus/efectos de los fármacos , Staphylococcus/genética , Staphylococcus/crecimiento & desarrolloRESUMEN
Use of antibiotics as feed additives in poultry production has been linked to the presence of antibiotic resistant bacteria in farm workers, consumer poultry products and the environs of confined poultry operations. There are concerns that these resistant bacteria may be transferred to communities near these operations; however, environmental pathways of exposure are not well documented. We assessed the prevalence of antibiotic resistant enterococci and staphylococci in stored poultry litter and flies collected near broiler chicken houses. Drug resistant enterococci and staphylococci were isolated from flies caught near confined poultry feeding operations in the summer of 2006. Susceptibility testing was conducted on isolates using antibiotics selected on the basis of their importance to human medicine and use in poultry production. Resistant isolates were then screened for genetic determinants of antibiotic resistance. A total of 142 enterococcal isolates and 144 staphylococcal isolates from both fly and poultry litter samples were identified. Resistance genes erm(B), erm(A), msr(C), msr(A/B) and mobile genetic elements associated with the conjugative transposon Tn916, were found in isolates recovered from both poultry litter and flies. Erm(B) was the most common resistance gene in enterococci, while erm(A) was the most common in staphylococci. We report that flies collected near broiler poultry operations may be involved in the spread of drug resistant bacteria from these operations and may increase the potential for human exposure to drug resistant bacteria.
Asunto(s)
Agricultura , Alimentación Animal , Antibacterianos/farmacología , Dípteros/microbiología , Farmacorresistencia Bacteriana/genética , Enterococcus/efectos de los fármacos , Aves de Corral/microbiología , Staphylococcus/efectos de los fármacos , Animales , Proteínas Bacterianas/genética , Elementos Transponibles de ADN , Enterococcus/genética , Enterococcus/aislamiento & purificación , Metiltransferasas/genética , Pruebas de Sensibilidad Microbiana , Staphylococcus/genética , Staphylococcus/aislamiento & purificación , Estados UnidosRESUMEN
The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-ß1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.
Asunto(s)
Núcleo Celular/genética , Proteínas de Unión al GTP Monoméricas/genética , Señales de Localización Nuclear/genética , Proteínas Reguladoras y Accesorias Virales/genética , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Células HEK293 , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Señales de Localización Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte de Proteínas , Proteolisis , Proteína 1 que Contiene Dominios SAM y HD , Proteínas Reguladoras y Accesorias Virales/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
Long interspersed elements 1 (LINE-1) occupy at least 17% of the human genome and are its only active autonomous retrotransposons. However, the host factors that regulate LINE-1 retrotransposition are not fully understood. Here, we demonstrate that the Aicardi-Goutières syndrome gene product SAMHD1, recently revealed to be an inhibitor of HIV/simian immunodeficiency virus (SIV) infectivity and neutralized by the viral Vpx protein, is also a potent regulator of LINE-1 and LINE-1-mediated Alu/SVA retrotransposition. We also found that mutant SAMHD1s of Aicardi-Goutières syndrome patients are defective in LINE-1 inhibition. Several domains of SAMHD1 are critical for LINE-1 regulation. SAMHD1 inhibits LINE-1 retrotransposition in dividing cells. An enzymatic active site mutant SAMHD1 maintained substantial anti-LINE-1 activity. SAMHD1 inhibits ORF2p-mediated LINE-1 reverse transcription in isolated LINE-1 ribonucleoproteins by reducing ORF2p level. Thus, SAMHD1 may be a cellular regulator of LINE-1 activity that is conserved in mammals.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/genética , Elementos de Nucleótido Esparcido Largo , Proteínas de Unión al GTP Monoméricas/genética , Malformaciones del Sistema Nervioso/genética , Elementos Alu , Células HEK293 , Células HeLa , Humanos , Sistemas de Lectura Abierta , Ribonucleoproteínas/genética , Proteína 1 que Contiene Dominios SAM y HD , Alineación de Secuencia , TransfecciónRESUMEN
Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFß), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFß interaction. Furthermore, association of Vif with CBFß, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFß-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFß-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Factores de Transcripción/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Animales , Subunidad beta del Factor de Unión al Sitio Principal/genética , Proteínas Cullin/metabolismo , Elonguina , Expresión Génica , Humanos , Datos de Secuencia Molecular , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Factores de Transcripción/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: The human immune response to mercury is not well characterized despite the body of evidence that suggests that Hg can modulate immune responses, including the induction of autoimmune disease in some mouse models. Dysregulation of cytokine signaling appears to play an important role in the etiology of Hg-induced autoimmunity in animal models. OBJECTIVES: In this study, we systematically investigated the human immune response to Hg in vitro in terms of cytokine release. METHODS: Human peripheral blood mononuclear cells (PBMCs) were isolated from 20 volunteers who donated blood six separate times. PBMCs were cultured with lipopolysaccharide and concentrations of mercuric chloride (HgCl(2)) up to 200 nM. Seven cytokines representing important pathways in physiologic and pathologic immune responses were measured in supernatants. We used multilevel models to account for the intrinsic clustering in the cytokine data due to experimental design. RESULTS: We found a consistent increase in the release of the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha, and concurrent decrease in release of the antiinflammatory cytokines interleukin 1-receptor antagonist (IL-1Ra) and IL-10 in human PBMCs treated with subcytotoxic concentrations of HgCl(2). IL-4, IL-17, and interferon-gamma increased in a concentration-response manner. These results were replicated in a second, independently recruited population of 20 different volunteers. CONCLUSIONS: Low concentrations of HgCl(2) affect immune function in human cells by dysregulation of cytokine signaling pathways, with the potential to influence diverse health outcomes such as susceptibility to infectious disease or risk of autoimmunity.
Asunto(s)
Inflamación/inducido químicamente , Leucocitos Mononucleares/efectos de los fármacos , Mercurio/toxicidad , Adulto , Donantes de Sangre , Células Cultivadas , Citocinas/biosíntesis , Femenino , Humanos , Lipopolisacáridos/toxicidad , MasculinoRESUMEN
Use of antimicrobial feed additives in food animal production is associated with selection for drug resistance in bacterial pathogens, which can then be released into the environment through occupational exposures, high volume ventilation of animal houses, and land application of animal wastes. We tested the hypothesis that current methods of transporting food animals from farms to slaughterhouses may result in pathogen releases and potential exposures of persons in vehicles traveling on the same road. Air and surface samples were taken from cars driving behind poultry trucks for 17 miles. Air conditioners and fans were turned off and windows fully opened. Background and blank samples were used for quality control. Samples were analyzed for susceptible and drug-resistant strains. Results indicate an increase in the number of total aerobic bacteria including both susceptible and drug-resistant enterococci isolated from air and surface samples, and suggest that food animal transport in open crates introduces a novel route of exposure to harmful microorganisms and may disseminate these pathogens into the general environment. These findings support the need for further exposure characterization, and attention to improving methods of food animal transport, especially in highly trafficked regions of high density farming such as the Delmarva Peninsula.