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Background & Aims: Micro-elimination of hepatitis C virus (HCV) in high-risk populations is a feasible approach towards achieving the World Health Organization's targets for viral hepatitis elimination by 2030. Prisons represent an area of high HCV prevalence and so initiatives that improve testing and treatment of residents are needed to eliminate HCV from prisons. This initiative aimed to improve the HCV screening and treatment rates of new residents arriving at prisons in England. Methods: A rapid test and treat pathway was developed and implemented in 47 prisons in England between May 2019 and October 2021 as a healthcare service improvement initiative. Prison healthcare staff performed opt-out HCV testing for all new residents at each prison within 7 days of arrival, and those who were positive for HCV RNA were offered treatment with direct-acting antivirals (DAAs). The Hepatitis C Trust provided peer support for all residents on treatment and those who were released into the community. Results: Of 107,260 new arrivals, 98,882 (92.2%) were offered HCV antibody testing, 63,137 (63.9%) were tested and 1,848 were treated. Testing rates increased from 53.7% in Year 1 to 86.0% in Year 3. Between May 2020 and October 2021, 40,727 residents were tested, 2,286 residents were positive for HCV antibodies and 940 residents were HCV RNA positive, giving an antibody prevalence of 5.6% and an RNA prevalence of 2.3%. A total of 921 residents were referred for treatment and 915 initiated DAA treatment (97.3% of whom were HCV RNA positive). Conclusions: This initiative showed that an opt-out HCV test and treat initiative in prison receptions is feasible and can be adapted to the needs of individual prisons as a viable way to achieve HCV micro-elimination. Impact and implications: Prisons represent an area of high HCV prevalence and so initiatives that improve testing and treatment of residents are needed to eliminate HCV from prisons. The reception testing protocol improved HCV screening in new arrivals across 47 prisons in England and could be a viable way for countries to achieve HCV micro-elimination in their prison systems. The reception testing protocol presented here can be adapted to the individual needs of prisons, globally, to improve HCV screening and treatment in this setting.
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Background and Aim: Prison residents are at high risk for hepatitis C virus (HCV) infection. HCV test-and-treat initiatives within prisons provide an opportunity to engage with prison residents and achieve HCV micro-elimination. The aim of the prison HCV-intensive test and treat initiative was to screen over 95% of all prison residents for HCV infection within a defined number of days determined by the size of the prison population and to initiate treatment within 7-14 days of a positive HCV RNA diagnosis. Methods: An HCV-intensive test and treat toolkit was developed based on learnings from pilot HCV-intensive test and treat events. From January 2020 to September 2021, 13 HCV-intensive test and treat events took place at prisons in England selected based on high levels of reception blood-borne virus testing and good access to peers from The Hepatitis C Trust. Results: Among a total of 8487 residents, 8139 (95.9%) underwent testing for HCV. Across the 13 prisons included, HCV antibody and RNA prevalence was 8.2% and 1.5%, respectively. The treatment initiation rate among HCV RNA-positive individuals (n = 124) was 79.0%. Conclusion: The HCV-intensive test and treat initiative presented here provides a feasible and rapid test-and-treat process to achieve HCV elimination within individual prisons. The HCV-intensive test and treat toolkit can be adapted for rapid HCV testing and treatment events at other prisons in the United Kingdom and worldwide.
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Filibuvir (PF-00868554) is an investigational nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural 5B (NS5B) RNA-dependent RNA polymerase currently in development for treating chronic HCV infection. The aim of this study was to characterize the selection of filibuvir-resistant variants in HCV-infected individuals receiving filibuvir as short (3- to 10-day) monotherapy. We identified amino acid M423 as the primary site of mutation arising upon filibuvir dosing. Through bulk cloning of clinical NS5B sequences into a transient-replicon system, and supported by site-directed mutagenesis of the Con1 replicon, we confirmed that mutations M423I/T/V mediate phenotypic resistance. Selection in patients of an NS5B mutation at M423 was associated with a reduced replicative capacity in vitro relative to the pretherapy sequence; consistent with this, reversion to wild-type M423 was observed in the majority of patients following therapy cessation. Mutations at NS5B residues R422 and M426 were detected in a small number of patients at baseline or the end of therapy and also mediate reductions in filibuvir susceptibility, suggesting these are rare but clinically relevant alternative resistance pathways. Amino acid variants at position M423 in HCV NS5B polymerase are the preferred pathway for selection of viral resistance to filibuvir in vivo.
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Antivirales/administración & dosificación , Farmacorresistencia Viral/genética , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Pironas/administración & dosificación , Triazoles/administración & dosificación , Proteínas no Estructurales Virales/genética , Secuencia de Bases , Línea Celular Tumoral , Clonación Molecular , Farmacorresistencia Viral/efectos de los fármacos , Hepacivirus/fisiología , Hepatitis C Crónica/virología , Humanos , Concentración 50 Inhibidora , Metionina/genética , Metionina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Replicón/genética , Transfección , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
UNLABELLED: More effective and better-tolerated therapies are needed for chronic hepatitis C virus (HCV) infection. Among the direct-acting anti-HCV agents in development is the nonstructural 5B protein (NS5B polymerase) non-nucleoside inhibitor filibuvir. We investigated the antiviral activity, pharmacokinetics, safety, and tolerability of multiple doses of filibuvir in treatment-naive and treatment-experienced patients who were chronically infected with HCV genotype 1 in two phase 1b clinical studies (study 1 was a randomized, placebo-controlled dose escalation study and study 2 was a nonrandomized, open-label study). The filibuvir doses evaluated ranged from 200-1400 mg daily, and the duration of dosing ranged from 3-10 days. Genotypic changes in the NS5B nucleotide sequence following short-term filibuvir therapy were also assessed. Filibuvir potently inhibited viral replication in a dose-dependent manner. Mean maximum HCV RNA change from baseline ranged from -0.97 log(10) IU/mL with filibuvir given at 100 mg twice daily to -2.30 log(10) IU/mL with filibuvir given at 700 mg twice daily in treatment-naive patients. In treatment-experienced patients, an HCV RNA reduction of 2.20 log(10) IU/mL was achieved with filibuvir given at 450 mg twice daily. Filibuvir was well tolerated in both studies. Adverse events were mild or moderate in severity. No discontinuations, serious adverse events, or deaths were reported. NS5B sequencing identified residue 423 as the predominant site of mutation after filibuvir dosing. CONCLUSION: Filibuvir administration resulted in significant reductions in HCV RNA concentrations at doses that were well tolerated in patients infected with HCV genotype 1. Filibuvir is currently being evaluated in combination with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients.
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Antivirales/uso terapéutico , Hepacivirus/inmunología , Hepatitis C/tratamiento farmacológico , Pironas/uso terapéutico , Triazoles/uso terapéutico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Adulto , Antivirales/efectos adversos , Antivirales/farmacocinética , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Quimioterapia Combinada , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C/genética , Hepatitis C/metabolismo , Humanos , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Masculino , Persona de Mediana Edad , Polietilenglicoles/uso terapéutico , Pironas/efectos adversos , Pironas/farmacocinética , ARN Viral/metabolismo , Proteínas Recombinantes , Ribavirina/uso terapéutico , Resultado del Tratamiento , Triazoles/efectos adversos , Triazoles/farmacocinética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
The product of the SALL2 protein p150(Sal2) is a multi-zinc finger transcription factor with growth arrest and proapoptotic functions that overlap those of p53. Its DNA-binding properties are unknown. We have used a modified SELEX procedure with purified p150(Sal2) and a pool of oligonucleotides of random sequence to identify those that are bound preferentially by p150(Sal2). The consensus sequence for optimal binding in vitro is GGG(T/C)GGG, placing p150(Sal2) among a large group of GC box-binding proteins including the Sp1 family of transcription factors. A triple zinc finger motif in p150(Sal2) similar to that in Sp1 is required for DNA binding. p150(Sal2) and Sp1 show evidence of co-operative binding in vitro and of interaction in vivo. p150(Sal2), a known activator of the CDK inhibitor p21(Cip1/Waf1) (p21), binds to regions of the human p21 promoter that contain variations of the consensus sequence in multiple copies. p150(Sal2) is also shown to bind to the BAX promoter with similar elements and to activate its expression following an apoptotic stimulus. These results demonstrate binding of p150(Sal2) to two natural promoters with GC elements related to the optimal binding sequence defined in vitro and whose regulation is important for suppression of tumor growth.
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Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ADN/genética , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnica SELEX de Producción de Aptámeros , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.
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1-Fosfatidilinositol 4-Quinasa/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hígado/virología , Replicación Viral , 1-Fosfatidilinositol 4-Quinasa/química , Antivirales/farmacología , Unión Competitiva , Línea Celular , Silenciador del Gen , Genotipo , Humanos , Concentración 50 Inhibidora , Espectrometría de Masas/métodos , Proteómica/métodos , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiazoles/farmacologíaRESUMEN
MOZ-TIF2 and MOZ-CBP are leukemogenic fusion proteins associated with therapy-induced acute myeloid leukemia. These proteins are thought to subvert normal gene expression in differentiating hematopoietic progenitor cells. We have previously shown that MOZ-TIF2 inhibits transcription by CREB-binding protein (CBP)/p300-dependent activators such as nuclear receptors and p53. Here we have shown that MOZ-TIF2 associates with the RARbeta2 promoter in vivo, resulting in altered recruitment of CBP/p300, aberrant histone modification, and down-regulation of the RARbeta2 gene. In contrast, MOZ-TIF2 up-regulated transcription mediated by the MOZ/MYST3-dependent activator AML1/RUNX1. Both wild type MOZ and MOZ-TIF2 were found to colocalize with AML1, and MOZ-TIF2 was recruited to an AML1 target promoter. A MOZ-CBP fusion protein showed similar functions to MOZ-TIF2 in that it inhibited retinoic acid receptor-mediated transcription but enhanced AML1 reporter activation. Although it contains almost the entire CBP sequence, MOZ-CBP does not appear to associate with PML bodies. In summary, our results indicate that leukemogenic MOZ fusion proteins have differential effects on the activities of CBP-dependent and MOZ-dependent activators because of their ability to alter cofactor recruitment and chromatin modification at target promoters.
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Regulación Neoplásica de la Expresión Génica , Histonas/química , Proteínas de Fusión Oncogénica/fisiología , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/genética , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Proteínas de Fusión Oncogénica/química , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/química , Factores de Transcripción p300-CBP/químicaRESUMEN
MOZ (monocytic leukaemia zinc finger protein; also known as ZNF220 or MYST3) is a member of the MYST family of protein acetyltransferases. Chromosomal translocations involving the MOZ gene are associated with AML (acute myeloid leukaemia), suggesting that it has a role in haematopoiesis. Recurrent reciprocal translocations fuse the MOZ gene [or the gene encoding MORF (MOZ-related factor); also known as MYST4] to genes encoding the nuclear receptor co-activators CBP [CREB (cAMP response element-binding protein)-binding protein], p300 or the p160 protein TIF2 (transcription intermediary factor 2). The resulting fusion proteins can transform haematopoietic progenitors in vitro, and induce myeloproliferative disease in mice. Recent insights into the molecular mechanisms underlying these effects indicate that MOZ fusion proteins interfere with the activities of transcription factors such as nuclear receptors, p53 and Runx proteins. Our studies suggest that subverting the function of cellular CBP and p300 proteins may play a key role in this process. Here we review the recent progress in understanding the role of MOZ fusion proteins in the aetiology of AML.
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Histona Acetiltransferasas/metabolismo , Leucemia Mieloide Aguda/etiología , Proteínas de Fusión Oncogénica/metabolismo , Animales , Proteína de Unión a CREB/química , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Transformación Celular Neoplásica , Fusión Génica , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Coactivador 2 del Receptor Nuclear/química , Coactivador 2 del Receptor Nuclear/genética , Coactivador 2 del Receptor Nuclear/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Estructura Terciaria de Proteína , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Dedos de Zinc/genéticaRESUMEN
Chromosomal rearrangements associated with acute myeloid leukemia (AML) include fusions of the genes encoding the acetyltransferase MOZ or MORF with genes encoding the nuclear receptor coactivator TIF2, p300, or CBP. Here we show that MOZ-TIF2 acts as a dominant inhibitor of the transcriptional activities of CBP-dependent activators such as nuclear receptors and p53. The dominant negative property of MOZ-TIF2 requires the CBP-binding domain (activation domain 1 [AD1]), and coimmunoprecipitation and fluorescent resonance energy transfer experiments show that MOZ-TIF2 interacts with CBP directly in vivo. The CBP-binding domain is also required for the ability of MOZ-TIF2 to extend the proliferative potential of murine bone marrow lineage-negative cells in vitro. We show that MOZ-TIF2 displays an aberrant nuclear distribution and that cells expressing this protein have reduced levels of cellular CBP, leading to depletion of CBP from PML bodies. In summary, our results indicate that disruption of the normal function of CBP and CBP-dependent activators is an important feature of MOZ-TIF2 action in AML.
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Acetiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Regulación Neoplásica de la Expresión Génica/genética , Células HL-60 , Histona Acetiltransferasas , Humanos , Leucemia Mieloide Aguda/metabolismo , Coactivador 2 del Receptor Nuclear , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the alpha-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ERalpha/SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by co-transfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the alpha-helix Aalpha1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic alpha-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP.