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1.
Nature ; 531(7594): 386-9, 2016 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-26983541

RESUMO

Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates. Here we show that HBx achieves this by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the 'structural maintenance of chromosomes' (Smc) complex Smc5/6 for degradation. Blocking this event inhibits the stimulatory effect of HBx both on extrachromosomal reporter genes and on hepatitis B virus transcription. Conversely, silencing the Smc5/6 complex enhances extrachromosomal reporter gene transcription in the absence of HBx, restores replication of an HBx-deficient hepatitis B virus, and rescues wild-type hepatitis B virus in a DDB1-knockdown background. The Smc5/6 complex associates with extrachromosomal reporters and the hepatitis B virus genome, suggesting a direct mechanism of transcriptional inhibition. These results uncover a novel role for the Smc5/6 complex as a restriction factor selectively blocking extrachromosomal DNA transcription. By destroying this complex, HBx relieves the inhibition to allow productive hepatitis B virus gene expression.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/fisiologia , Especificidade de Hospedeiro , Transativadores/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona , DNA Viral/genética , DNA Viral/metabolismo , Genes Reporter , Genoma Viral/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Hepatócitos/virologia , Humanos , Fígado/metabolismo , Fígado/virologia , Masculino , Camundongos , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Proteólise , Transcrição Gênica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais Reguladoras e Acessórias , Replicação Viral
2.
J Virol ; 92(16)2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29848586

RESUMO

Chronic infection with hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. HBVs (family Hepadnaviridae) have been associated with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions in genome maintenance, was identified as an antiviral restriction factor of human HBV. The virus has, however, evolved to counteract this defense mechanism by degrading the complex via its regulatory HBx protein. Whether the antiviral activity of the Smc5/6 complex against hepadnaviruses is an important and evolutionarily conserved function is unknown. In this study, we used an evolutionary and functional approach to address this question. We first performed phylogenetic and positive selection analyses of the Smc5/6 complex subunits and found that they have been conserved in primates and mammals. Yet, Smc6 showed marks of adaptive evolution, potentially reminiscent of a virus-host "arms race." We then functionally tested the HBx proteins from six divergent hepadnaviruses naturally infecting primates, rodents, and bats. We demonstrate that despite little sequence homology, these HBx proteins efficiently degraded mammalian Smc5/6 complexes, independently of the host species and of the sites under positive selection. Importantly, all HBx proteins also rescued the replication of an HBx-deficient HBV in primary human hepatocytes. These findings point to an evolutionarily conserved requirement for Smc5/6 inactivation by HBx, showing that Smc5/6 antiviral activity has been an important defense mechanism against hepadnaviruses in mammals. It will be interesting to investigate whether Smc5/6 may further be a restriction factor of other, yet-unidentified viruses that may have driven some of its adaptation.IMPORTANCE Infection with hepatitis B virus (HBV) led to 887,000 human deaths in 2015. HBV has been coevolving with mammals for millions of years. Recently, the Smc5/6 complex, which has essential housekeeping functions, was identified as a restriction factor of human HBV antagonized by the regulatory HBx protein. Here we address whether the antiviral activity of Smc5/6 is an important evolutionarily conserved function. We found that all six subunits of Smc5/6 have been conserved in primates, with only Smc6 showing signatures of an "evolutionary arms race." Using evolution-guided functional analyses that included infections of primary human hepatocytes, we demonstrated that HBx proteins from very divergent mammalian HBVs could all efficiently antagonize Smc5/6, independently of the host species and sites under positive selection. These findings show that Smc5/6 antiviral activity against HBV is an important function in mammals. They also raise the intriguing possibility that Smc5/6 may restrict other, yet-unidentified viruses.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/imunologia , Interações Hospedeiro-Patógeno , Transativadores/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Evolução Molecular , Variação Genética , Vírus da Hepatite B/fisiologia , Mamíferos , Filogenia , Proteólise , Proteínas Virais Reguladoras e Acessórias
3.
J Hepatol ; 63(5): 1093-102, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26143443

RESUMO

BACKGROUND & AIMS: Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription. METHODS: We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differentiated HepaRG cells with wild-type virus or virus deficient (HBVX-) for the expression of hepatitis B virus X protein (HBx), that is required for HBV expression. RESULTS: In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA. CONCLUSION: Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , DNA Circular/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas Metiltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Northern Blotting , Southern Blotting , Células Cultivadas , DNA Circular/metabolismo , Ensaio de Imunoadsorção Enzimática , Hepatite B/metabolismo , Hepatite B/patologia , Vírus da Hepatite B/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas Metiltransferases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica
4.
Life Sci Alliance ; 6(4)2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36746533

RESUMO

NLRP3 is a pattern recognition receptor with a well-documented role in inducing inflammasome assembly in response to cellular stress. Deregulation of its activity leads to many inflammatory disorders including gouty arthritis, Alzheimer disease, and cancer. Whereas its role in the context of cancer has been mostly explored in the immune compartment, whether NLRP3 exerts functions unrelated to immunity in cancer development remains unexplored. Here, we demonstrate that NLRP3 interacts with the ATM kinase to control the activation of the DNA damage response, independently of its inflammasome activity. NLRP3 down-regulation in both broncho- and mammary human epithelial cells significantly impairs ATM pathway activation, leading to lower p53 activation, and provides cells with the ability to resist apoptosis induced by acute genotoxic stress. Interestingly, NLRP3 expression is down-regulated in non-small cell lung cancers and breast cancers, and its expression positively correlates with patient overall survival. Our findings identify a novel non-immune function for NLRP3 in maintaining genome integrity and strengthen the concept of a functional link between innate immunity and DNA damage sensing pathways to maintain cell integrity.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Imunidade Inata , Dano ao DNA , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
5.
Clin Res Hepatol Gastroenterol ; 45(5): 101553, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33183998

RESUMO

BACKGROUND: A promising avenue for cancer treatment is exacerbating the deregulation of the DNA repair machinery that would normally protect the genome. To address the applicability of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) combined with radiotherapy for the treatment of hepatocellular carcinoma (HCC) two approaches were used: firstly, the in vitro sensitivity to the PARPi Veliparib and Talazoparib +/- radiation exposure was determined in liver cell lines and the impact of the HBV X protein (HBx) that deregulates cellular DNA damage repair via SMC5/6 degradation was investigated. Secondly, PARP expression profiles and DNA damage levels using the surrogate marker gammaH2AX were assessed in a panel of control liver vs HCC tissues. METHODS: Cell cytotoxicity was measured by clonogenic survival or relative cell growth and the DNA damage response using immunological-based techniques in Hep3B, PLC/PRF/5, HepG2- and HepaRG-derived models. Transcriptome changes due to HBx expression vs SMC6 loss were assessed by RNA sequencing in HepaRG-derived models. PARP and PARG transcripts (qPCR) and PARP1, H2AX and gammaH2AX protein levels (RPPA) were compared in control liver vs HBV-, HCV-, alcohol- and non-alcoholic steatohepatitis-associated HCC (tumor/peritumor) tissues. RESULTS: PARPi cytotoxicity was significantly enhanced when combined with X-rays (2Gy) with Talazoparib having a greater impact than Veliparib in most in vitro models. HBx expression significantly lowered survival, probably driven by SMC5/6 loss based on the transcriptome analysis and higher DNA damage levels. PARP1 and PARP2 transcript levels were significantly higher in tumor than peritumor and control tissues. The HBV/HCV/alcohol-associated tumor tissues studied had reduced H2AX but higher gammaH2AX protein levels compared to peritumor and control tissues providing evidence of increased DNA damage during liver disease progression. CONCLUSIONS: These proof-of-concept experiments support PARPi alone or combined with radiotherapy for HCC treatment, particularly for HBV-associated tumors, that warrant further investigation.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Inibidores de Poli(ADP-Ribose) Polimerases , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/radioterapia , Carcinoma Hepatocelular/virologia , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Terapia Combinada , Hepatite B/complicações , Hepatite C/complicações , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/virologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
6.
Cancers (Basel) ; 10(11)2018 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-30453567

RESUMO

Accumulating evidence supports the remarkable presence at the membrane surface of cancer cells of proteins, which are normally expressed in the intracellular compartment. Although these proteins, referred to as externalized proteins, represent a highly promising source of accessible and druggable targets for cancer therapy, the mechanisms via which they impact cancer biology remain largely unexplored. The aim of this study was to expose an externalized form of cytokeratin 8 (eK8) as a key player of colorectal tumorigenesis and characterize its mode of action. To achieve this, we generated a unique antagonist monoclonal antibody (D-A10 MAb) targeting an eight-amino-acid-long domain of eK8, which enabled us to ascertain the pro-tumoral activity of eK8 in both KRAS-mutant and wild-type colorectal cancers (CRC). We showed that this pro-tumoral activity involves a bidirectional eK8-dependent control of caspase-mediated apoptosis in vivo and of the plasminogen-induced invasion process in cellulo. Furthermore, we demonstrated that eK8 is anchored at the plasma membrane supporting this dual function. We, therefore, identified eK8 as an innovative therapeutic target in CRC and provided a unique MAb targeting eK8 that displays anti-neoplastic activities that could be useful to treat CRC, including those harboring KRAS mutations.

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