RESUMO
Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.
Assuntos
Antibacterianos , Lipopolissacarídeos , Proteínas de Membrana Transportadoras , Animais , Humanos , Camundongos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/classificação , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico/efeitos dos fármacos , Modelos Animais de Doenças , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Desenvolvimento de MedicamentosRESUMO
The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.
Assuntos
Esclerose Lateral Amiotrófica/genética , Influenza Humana/imunologia , Orthomyxoviridae/fisiologia , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Degenerações Espinocerebelares/genética , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/fisiologia , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Citocinas/metabolismo , DNA Helicases , Cães , Regulação para Baixo , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Análise em Microsséries , Enzimas Multifuncionais , RNA Helicases/genética , RNA Polimerase II/genética , RNA Interferente Pequeno/genética , Ataxias Espinocerebelares/congênito , Células Vero , Replicação Viral/genéticaRESUMO
BACKGROUND & AIMS: The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. METHODS: A phenotypic assay with HBV-infected primary human hepatocytes (PHHs) was employed to screen for novel cccDNA inhibitors. A HBVcircle mouse model and a uPA-SCID (urokinase-type plasminogen activator-severe combined immunodeficiency) humanized liver mouse model were used to evaluate the anti-HBV efficacy of the discovered cccDNA inhibitors. RESULTS: Potent and dose-dependent reductions in extracellular HBV DNA, HBsAg, and HBeAg levels were achieved upon the initiation of ccc_R08 treatment two days after the HBV infection of PHHs. More importantly, the level of cccDNA was specifically reduced by ccc_R08, while it did not obviously affect mitochondrial DNA. Additionally, ccc_R08 showed no significant cytotoxicity in PHHs or in multiple proliferating cell lines. The twice daily oral administration of ccc_R08 to HBVcircle model mice, which contained surrogate cccDNA molecules, significantly decreased the serum levels of HBV DNA and antigens, and these effects were sustained during the off-treatment follow-up period. Moreover, at the end of follow-up, the levels of surrogate cccDNA molecules in the livers of ccc_R08-treated HBVcircle mice were reduced to below the lower limit of quantification. CONCLUSIONS: We have discovered a small-molecule cccDNA inhibitor that reduces HBV cccDNA levels. cccDNA inhibitors potentially represent a new approach to completely cure patients chronically infected with HBV. IMPACT AND IMPLICATIONS: Covalently closed circular DNA (cccDNA) persistence in HBV-infected hepatocytes is the root cause of chronic hepatitis B. We discovered a novel small-molecule cccDNA inhibitor that can specifically reduce cccDNA levels in HBV-infected hepatocytes. This type of molecule could offer a new approach to completely cure patients chronically infected with HBV.
Assuntos
Hepatite B Crônica , Humanos , Animais , Camundongos , Hepatite B Crônica/tratamento farmacológico , Vírus da Hepatite B , DNA Circular/uso terapêutico , DNA Viral/genética , Replicação Viral , Camundongos SCID , Antivirais/farmacologia , Antivirais/uso terapêuticoRESUMO
BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is ranked among the top health priorities worldwide. Accumulating evidence suggests that HBV infection and replication are closely associated with liver metabolism. The liver X receptors (LXRs), which belong to the superfamily of nuclear hormone receptors, are important physiological regulators of lipid and cholesterol metabolism. However, the association between the LXR pathway and HBV infection remains largely unclear. APPROACH AND RESULTS: In this study, the antiviral activity of LXR agonists was investigated using multiple HBV cellular models. We observed that in HBV-infected primary human hepatocytes (PHHs), synthetic LXR agonists (T0901317, GW3965, and LXR-623), but not an LXR antagonist (SR9238), potently inhibited HBV replication and gene expression, as demonstrated by substantial reductions in viral RNA, DNA, and antigen production following agonist treatment. However, covalently closed circular DNA (cccDNA) levels were not significantly reduced by the agonists. In addition, no rebound in viral replication was observed after treatment withdrawal, indicating a long-lasting inhibitory effect. These results suggest that LXR agonists decrease the transcriptional activity of cccDNA. In contrast, no significant anti-HBV effect was observed in HepG2-derived cell lines. Interestingly, LXR agonist treatment strongly reduced cholesterol 7α-hydroxylase 1 (CYP7A1) mRNA levels. Knockdown of CYP7A1 gene expression with small interfering RNA inhibited HBV activity in PHHs, suggesting CYP7A1 as a potential factor contributing to the antiviral effects of LXR agonists. CONCLUSIONS: We found that activation of the LXR pathway with synthetic LXR agonists could elicit potent anti-HBV activity in PHHs, possibly through sustained suppression of cccDNA transcription. Our work highlights the therapeutic potential of targeting the LXR pathway for the treatment of chronic HBV infection.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Receptores X do Fígado/agonistas , Fígado/metabolismo , Antígenos Virais/genética , Antígenos Virais/isolamento & purificação , Antivirais/uso terapêutico , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Benzilaminas/farmacologia , Benzilaminas/uso terapêutico , Células Cultivadas , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , DNA Viral/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos , Técnicas de Silenciamento de Genes , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Hepatócitos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Hidrocarbonetos Fluorados/farmacologia , Hidrocarbonetos Fluorados/uso terapêutico , Indazóis/farmacologia , Indazóis/uso terapêutico , Fígado/citologia , Receptores X do Fígado/antagonistas & inibidores , Receptores X do Fígado/metabolismo , Cultura Primária de Células , RNA Viral/isolamento & purificação , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
RG7834 is a potent, orally bioavailable small-molecule inhibitor of hepatitis B virus (HBV) gene expression that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three-hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerase associated domain containing proteins 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies that phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down, suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. In addition, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusion: We identify PAPD5 and PAPD7 as cellular host factors required for HBV RNA stabilization and as therapeutic targets for the HBV cure.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/fisiologia , Terapia de Alvo Molecular , RNA Nucleotidiltransferases/fisiologia , Hepatite B/tratamento farmacológico , Humanos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Hepatitis B e antigen (HBeAg) is an important immunomodulator for promoting host immune tolerance during chronic hepatitis B (CHB) infection. In patients with CHB, HBeAg loss and seroconversion represent partial immune control of CHB infection and are regarded as valuable endpoints. However, the current approved treatments have only a limited efficacy in achieving HBeAg seroconversion in HBeAg-positive patients. Hepatitis B virus (HBV) core protein has been recognized as an attractive antiviral target, and two classes of core protein allosteric modulator (CpAM) have been discovered: the phenylpropenamides (PPAs) and the heteroaryldihydropyrimidines (HAPs). However, their differentiation and potential therapeutic benefit beyond HBV DNA inhibition remain to be seen. Here, we show that in contrast to PPA series compound AT-130, a HAP CpAM, HAP_R01, reduced HBeAg levels in multiple in vitro and in vivo HBV experimental models. Mechanistically, we found that HAP_R01 treatment caused the misassembly of capsids formed by purified HBeAg in vitro. In addition, HAP_R01 directly reduces HBeAg levels by inducing intracellular precore protein misassembly and aggregation. Using a HAP_R01-resistant mutant, we found that HAP_R01-mediated HBeAg and core protein reductions were mediated through the same mechanism. Furthermore, HAP_R01 treatment substantially reduced serum HBeAg levels in an HBV mouse model. Conclusion: Unlike PPA series compound AT-130, HAP_R01 not only inhibits HBV DNA levels but also directly reduces HBeAg through induction of its misassembly. HAP_R01, as well as other similar CpAMs, has the potential to achieve higher anti-HBeAg seroconversion rates than currently approved therapies for patients with CHB. Our findings also provide guidance for dose selection when designing clinical trials with molecules from HAP series.
Assuntos
Antígenos E da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Pirimidinas/farmacologia , Regulação Alostérica , Avaliação Pré-Clínica de Medicamentos , Células Hep G2 , Humanos , Terapia de Alvo Molecular , Pirimidinas/uso terapêuticoRESUMO
BACKGROUND & AIMS: The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834). METHODS: RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir. RESULTS: Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09â¯log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice. CONCLUSION: We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues. LAY SUMMARY: We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.
Assuntos
Antivirais , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Vírus da Hepatite B , Hepatite B Crônica , Bibliotecas de Moléculas Pequenas , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Antivirais/farmacocinética , Disponibilidade Biológica , DNA Viral/isolamento & purificação , Modelos Animais de Doenças , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Camundongos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/efeitos adversos , Bibliotecas de Moléculas Pequenas/farmacocinética , Resultado do Tratamento , Carga Viral/efeitos dos fármacosRESUMO
A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs.IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , HIV-1/efeitos dos fármacos , Quinases Lim/antagonistas & inibidores , Liberação de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , Antivirais/isolamento & purificação , Células Cultivadas , Ebolavirus/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/isolamento & purificação , HIV-1/fisiologia , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Vírus da Febre do Vale do Rift/efeitos dos fármacosRESUMO
BACKGROUND & AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as a stable episome in infected hepatocytes and serves as a template for the transcription of all viral genes. Due to the narrow host range of HBV, the development of a robust mouse model that supports cccDNA-dependent viral replication is a key hurdle in the development of novel HBV therapeutics. This study aimed to develop a novel tool to investigate HBV cccDNA. METHODS: Through minicircle technology, HBVcircle, a recombinant cccDNA, was easily generated and extracted from a genetically engineered E. coli strain. We characterized the performance of HBVcircle in cell culture by transfection and in immunocompetent mice by hydrodynamic injection (HDI). RESULTS: We demonstrated that HBVcircle formed authentic cccDNA-like molecules in vitro in transiently transfected hepatic cells and in vivo in mouse liver after HDI. HBVcircle supported high levels and persistent HBV replication. In addition, we investigated different factors affecting HBV in vivo replication and persistence, including the host genetic background, vector design and dosage, viral genes and genotypes, and immune activation status. Furthermore, different classes of anti-HBV drugs were also assessed with the HBVcircle system. CONCLUSION: Compared with previous reported HBV mouse models which employ other viral vectors to introduce overlength HBV genomes, viral gene expression and associated phenotypes are entirely driven by cccDNA-like viral genomes in the HBVcircle mouse model. Therefore, the HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery. LAY SUMMARY: To establish a mouse model that supports cccDNA-dependent transcription, a novel tool named HBVcircle, was developed with minicircle technology. HBVcircle formed authentic cccDNA-like molecules in hepatocytes, and supported high levels and persistent HBV replication in vivo. The HBVcircle is a close mimic of cccDNA, and it represents a novel tool for addressing HBV cccDNA related biological questions and for anti-HBV drug discovery.
Assuntos
DNA Circular/genética , DNA Viral/genética , Técnicas Genéticas , Vírus da Hepatite B/genética , Imunidade Adaptativa , Animais , Linhagem Celular , DNA Circular/biossíntese , DNA Circular/imunologia , DNA Viral/biossíntese , DNA Viral/imunologia , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Genes Virais , Engenharia Genética , Células Hep G2 , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Modelos Genéticos , Transcrição Gênica , Transfecção , Replicação Viral/genéticaRESUMO
BACKGROUND: The cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases, which sulfonate proteins, glycoproteins, glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs), which sulfonate various small molecules including hormones, neurotransmitters, and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions, cell signaling, and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism, namely reverse transcription. METHODS: MDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions, MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels, and qPCR was used to measure viral RNA and DNA products. RESULTS: We demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs, and through siRNA knockdown experiments, we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription. CONCLUSIONS: These studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules, raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics.
Assuntos
Arilsulfotransferase/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Macrófagos/metabolismo , Macrófagos/virologia , Transcrição Reversa , Replicação Viral , Arilsulfotransferase/genética , Diferenciação Celular , Células Cultivadas , Citosol/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Infecções por HIV/genética , Humanos , Macrófagos/citologia , Monócitos/citologiaRESUMO
Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.
Assuntos
Fatores Biológicos/genética , Fatores Biológicos/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Influenza A/crescimento & desenvolvimento , Influenza Humana/genética , Influenza Humana/virologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Biblioteca Gênica , Genoma Humano/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A/classificação , Interferência de RNA , Células Vero , Internalização do VírusRESUMO
UNLABELLED: Characterizing the cellular factors that play a role in the HIV replication cycle is fundamental to fully understanding mechanisms of viral replication and pathogenesis. Whole-genome small interfering RNA (siRNA) screens have identified positive and negative regulators of HIV replication, providing starting points for investigating new cellular factors. We report here that silencing of the deubiquitinase cylindromatosis protein (CYLD), increases HIV infection by enhancing HIV long terminal repeat (LTR)-driven transcription via the NF-κB pathway. CYLD is highly expressed in CD4(+) T lymphocytes, monocyte-derived macrophages, and dendritic cells. We found that CYLD silencing increases HIV replication in T cell lines. We confirmed the positive role of CYLD silencing in HIV infection in primary human CD4(+) T cells, in which CYLD protein was partially processed upon activation. Lastly, Jurkat T cells latently infected with HIV (JLat cells) were more responsive to phorbol 12-myristate 13-acetate (PMA) reactivation in the absence of CYLD, indicating that CYLD activity could play a role in HIV reactivation from latency. In summary, we show that CYLD acts as a potent negative regulator of HIV mRNA expression by specifically inhibiting NF-κB-driven transcription. These findings suggest a function for this protein in modulating productive viral replication as well as in viral reactivation. IMPORTANCE: HIV transcription is regulated by a number of host cell factors. Here we report that silencing of the lysine 63 deubiquitinase CYLD increases HIV transcription in an NF-κB-dependent manner. We show that CYLD is expressed in HIV target cells and that its silencing increases HIV infection in transformed T cell lines as well as primary CD4(+) T cells. Similarly, reactivation of latent provirus was facilitated in the absence of CYLD. These data suggest that CYLD, which is highly expressed in CD4(+) T cells, can control HIV transcription in productive infection as well as during reactivation from latency.
Assuntos
Infecções por HIV/genética , HIV-1/genética , NF-kappa B/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Ativação Viral/fisiologia , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Enzima Desubiquitinante CYLD , Imunofluorescência , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Células Jurkat , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , NF-kappa B/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Replicação ViralRESUMO
Transcription from the HIV-1 LTR promoter efficiently initiates but rapidly terminates because of a non-processive form of RNA polymerase II. This premature termination is overcome by assembly of an HIV-1 TAT/P-TEFb complex at the transactivation response region (TAR), a structured RNA element encoded by the first 59 nt of HIV-1 mRNA. Here we have identified a conserved DNA-binding element for the cellular transcription factor, ZASC1, in the HIV-1 core promoter immediately upstream of TAR. We show that ZASC1 interacts with TAT and P-TEFb, co-operating with TAT to regulate HIV-1 gene expression, and promoting HIV-1 transcriptional elongation. The importance of ZASC1 to HIV-1 transcription elongation was confirmed through mutagenesis of the ZASC1 binding sites in the LTR promoter, shRNAs targeting ZASC1 and expression of dominant negative ZASC1. Chromatin immunoprecipitation analysis revealed that ZASC1 recruits Tat and P-TEFb to the HIV-1 core promoter in a TAR-independent manner. Thus, we have identified ZASC1 as novel regulator of HIV-1 gene expression that functions through the DNA-dependent, RNA-independent recruitment of TAT/P-TEFb to the HIV-1 promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/metabolismo , Repetição Terminal Longa de HIV , HIV-1/metabolismo , Proteínas Nucleares/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Elongação da Transcrição Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Ligação a DNA/genética , Infecções por HIV/genética , Infecções por HIV/patologia , HIV-1/genética , Células HeLa , Humanos , Células Jurkat , Proteínas Nucleares/genética , Fator B de Elongação Transcricional Positiva/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Cellular receptors can act as molecular switches, regulating the sensitivity of microbial proteins to conformational changes that promote cellular entry. The activities of these receptor-based switches are only partially understood. In this paper, we sought to understand the mechanism that underlies the activity of the ANTXR2 anthrax toxin receptor-based switch that binds to domains 2 and 4 of the protective antigen (PA) toxin subunit. Receptor-binding restricts structural changes within the heptameric PA prepore that are required for pore conversion to an acidic endosomal compartment. The transfer cross-saturation (TCS) NMR approach was used to monitor changes in the heptameric PA-receptor contacts at different steps during prepore-to-pore conversion. These studies demonstrated that receptor contact with PA domain 2 is weakened prior to pore conversion, defining a novel intermediate in this pathway. Importantly, ANTXR2 remained bound to PA domain 4 following pore conversion, suggesting that the bound receptor might influence the structure and/or function of the newly formed pore. These studies provide new insights into the function of a receptor-based molecular switch that controls anthrax toxin entry into cells.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Receptores de Peptídeos/química , Antraz , Espectroscopia de Ressonância Magnética , Estrutura Terciária de Proteína , Receptores de Peptídeos/metabolismo , VirulênciaRESUMO
A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.
Assuntos
Vírus da Leucose Aviária/fisiologia , Vírus do Sarcoma Aviário/fisiologia , Endocitose/fisiologia , Receptores Virais/metabolismo , Internalização do Vírus , Animais , Proteínas Aviárias/metabolismo , Linhagem Celular , Endossomos/metabolismo , Endossomos/virologia , Interações Hospedeiro-Patógeno , Humanos , Microscopia de FluorescênciaRESUMO
Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration.
Assuntos
HIV/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , beta Carioferinas/metabolismo , Regulação Viral da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , RNA Interferente Pequeno/genética , Replicação Viral , beta Carioferinas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: ZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010). METHODS: To determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies. RESULTS: Mice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection. CONCLUSIONS: These studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection.
Assuntos
Medula Óssea/virologia , Proteínas de Ligação a DNA/metabolismo , Leucemia Experimental/virologia , Vírus da Leucemia Murina de Moloney/fisiologia , Proteínas Nucleares/metabolismo , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/virologia , Replicação Viral , Animais , Proteínas de Ligação a DNA/deficiência , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiênciaRESUMO
Both treatment of disease and basic studies of complex tissues can benefit from directing viral vector infection to specific cell types. We have used a unique cell targeting method to direct viral vector transduction to cerebral cortical neurons expressing the neuregulin (NRG) receptor ErbB4; both NRG and ErbB4 have been implicated in schizophrenia, and ErbB4 expression in cerebral cortex is known to be restricted to inhibitory neurons. We find that a bridge protein composed of the avian viral receptor TVB fused to NRG, along with EnvB-pseudotyped virus, is able to direct infection selectively to ErbB4-expressing inhibitory cortical neurons in vivo. Interestingly, although ErbB4 is expressed in a broad range of cortical inhibitory cell types, NRG-dependent infection is restricted to a more selective subset of inhibitory cell types. These results demonstrate a tool that can be used for further studies of NRG and ErbB receptors in brain circuits and demonstrate the feasibility for further development of related bridge proteins to target gene expression to other specific cell types in complex tissues.
Assuntos
Receptores ErbB/genética , Vetores Genéticos , Interneurônios/metabolismo , Transdução Genética , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Sequência de Bases , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Primers do DNA/genética , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Neurregulinas/genética , Neurregulinas/metabolismo , Vírus da Raiva/genética , Ratos , Receptor ErbB-4 , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Described herein is the first-time disclosure of Linvencorvir (RG7907), a clinical compound and a hepatitis B virus (HBV) core protein allosteric modulator, for the treatment of chronic HBV infection. Built upon the core structure of hetero aryl dihydropyrimidine, RG7907 was rationally designed by combining all the drug-like features of low CYP3A4 induction, potent anti-HBV activity, high metabolic stability, low hERG liability, and favorable animal pharmacokinetic (PK) profiles. In particular, the chemistry strategy to mitigate CYP3A4 induction through introducing a large, rigid, and polar substituent at the position that has less interaction with the therapeutic biological target (HBV core proteins herein) is of general interest to the medicinal chemistry community. RG7907 demonstrated favorable animal PK, pharmacodynamics, and safety profiles with sufficient safety margins supporting its clinical development in healthy volunteers and HBV-infected patients.