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2.
Acta Pharm Sin B ; 9(4): 769-781, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31384537

RESUMO

Bicyclol is a synthetic drug for hepatoprotection in clinic since 2004. Preliminary clinical observations suggest that bicyclol might be active against hepatitis C virus (HCV) with unknown mechanism. Here, we showed that bicyclol significantly inhibited HCV replication in vitro and in hepatitis C patients. Using bicyclol as a probe, we identified glycolipid transfer protein (GLTP) to be a novel restrictive factor for HCV replication. The GLTP preferentially bound host vesicle-associated membrane protein-associated protein-A (VAP-A) in competition with the HCV NS5A, causing an interruption of the complex formation between VAP-A and HCV NS5A. As the formation of VAP-A/NS5A complex is essential for viral RNA replication, up-regulation of GLTP by bicyclol reduced the level of VAP-A/NS5A complex and thus inhibited HCV replication. Bicyclol also exhibited an inhibition on HCV variants resistant to direct-acting antiviral agents (DAAs) with an efficacy identical to that on wild type HCV. In combination with bicyclol, DAAs inhibited HCV replication in a synergistic fashion. GLTP appears to be a newly discovered host restrictive factor for HCV replication, Up-regulation of GLTP causes spontaneous restriction of HCV replication.

3.
ACS Infect Dis ; 5(7): 1139-1149, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31060350

RESUMO

Stimulator of interferon genes (STING) is an integral ER-membrane protein that can be activated by 2'3'-cGAMP synthesized by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) upon binding of double-stranded DNA. It activates interferon (IFN) and inflammatory cytokine responses to defend against infection by microorganisms. Pharmacologic activation of STING has been demonstrated to induce an antiviral state and boost antitumor immunity. We previously reported a cell-based high-throughput-screening assay that allowed for identification of small-molecule cGAS-STING-pathway agonists. We report herein a compound, 6-bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide (BNBC), that induces a proinflammatory cytokine response in a human-STING-dependent manner. Specifically, we showed that BNBC induced type I and III IFN dominant cytokine responses in primary human fibroblasts and peripheral-blood mononuclear cells (PBMCs). BNBC also induced cytokine response in PBMC-derived myeloid dendritic cells and promoted their maturation, suggesting that STING-agonist treatment could potentially regulate the activation of CD4+ and CD8+ T lymphocytes. As anticipated, treatment of primary human fibroblast cells with BNBC induced an antiviral state that inhibited the infection of several kinds of flaviviruses. Taken together, our results indicate that BNBC is a human-STING agonist that not only induces innate antiviral immunity against a broad spectrum of viruses but may also stimulate the activation of adaptive immune responses, which is important for the treatment of chronic viral infections and tumors.

4.
Cell Cycle ; 18(10): 1095-1109, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31020898

RESUMO

We have previously found that Sirt2 enhanced the outgrowth of cellular processes and MBP expression in CG4 cells, where Sirt2 expression is suppressed by transcription factor Nkx2.2. However, the detailed mechanism of Sirt2 facilitating oligodendroglial cell differentiation remained unclear. In the present study, we observed that Sirt2 partially translocated into the nuclei when CG4 cells were induced to differentiate. Sirt2 was detected at the CpG island of PDGFRα promoter via ChIP assay during the cells differentiation process rather than during the state of growth. Sirt2 deacetylated protein(s) bound to the promoter of PDGFRα and simultaneously appeared to facilitate histone3 K27 tri-methylation, both of which are suppressive signatures on gene transcription activation. In bisulfate assay, we identified that Sirt2 significantly induced DNA methylation of PDGFRα promoter compared with the control. Consistently, Sirt2 overexpression down-regulated PDGFRα expression in CG4 cells. The knock-down of PDGFRα or Sirt2 over-expression repressed cell proliferation, but knock-down of Sirt2 promoted cell proliferation. Taken together, Sirt2 translocated into the nuclei while the cells initiated a differentiation process, facilitating CG4 cell differentiation partially through epigenetic modification and suppression of PDGFRα expression. The repression of PDGFRα expression mediated by Sirt2 appeared to facilitate a transition of cellular processes, i.e. from a proliferating progenitor state to a post-mitotic state in CG4 cells.

5.
J Virol ; 93(11)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30867306

RESUMO

In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.

6.
ACS Infect Dis ; 5(5): 759-768, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30525438

RESUMO

Hepatitis B virus (HBV) core protein is a small protein with 183 amino acid residues and assembles the pregenomic (pg) RNA and viral DNA polymerase to form nucleocapsids. During the last decades, several groups have reported HBV core protein allosteric modulators (CpAMs) with distinct chemical structures. CpAMs bind to the hydrophobic HAP pocket located at the dimer-dimer interface and induce allosteric conformational changes in the core protein subunits. While Type I CpAMs, heteroaryldihydropyrimidine (HAP) derivatives, misdirect core protein dimers to assemble noncapsid polymers, Type II CpAMs, represented by sulfamoylbenzamides, phenylpropenamides, and several other chemotypes, induce the assembly of empty capsids with global structural alterations and faster mobility in native agarose gel electrophoresis. Through high throughput screening of an Asinex small molecule library containing 19 920 compounds, we identified 8 structurally distinct CpAMs. While 7 of those compounds are typical Type II CpAMs, a novel benzamide derivative, designated as BA-53038B, induced the formation of morphologically "normal" empty capsids with slow electrophoresis mobility. Drug resistant profile analyses indicated that BA-53038B most likely bound to the HAP pocket but obviously modulated HBV capsid assembly in a distinct manner. BA-53038B and other CpAMs reported herein provide novel structure scaffolds for the development of core protein-targeted antiviral agents for the treatment of chronic hepatitis B.

7.
ACS Infect Dis ; 5(5): 659-674, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29893548

RESUMO

Hepatitis B virus (HBV) has infected one-third of world population, and 240 million people are chronic carriers, to whom a curative therapy is still not available. Similar to other viruses, persistent HBV infection relies on the virus to exploit host cell functions to support its replication and efficiently evade host innate and adaptive antiviral immunity. Understanding HBV replication and concomitant host cell interactions is thus instrumental for development of therapeutics to disrupt the virus-host interactions critical for its persistence and cure chronic hepatitis B. Although the currently available cell culture systems of HBV infection are refractory to genome-wide high throughput screening of key host cellular factors essential for and/or regulating HBV replication, classic one-gene (or pathway)-at-a-time studies in the last several decades have already revealed many aspects of HBV-host interactions. An overview of the landscape of HBV-hepatocyte interaction indicates that, in addition to more tightly suppressing viral replication by directly targeting viral proteins, disruption of key viral-host cell interactions to eliminate or inactivate the covalently closed circular (ccc) DNA, the most stable HBV replication intermediate that exists as an episomal minichromosome in the nucleus of infected hepatocyte, is essential to achieve a functional cure of chronic hepatitis B. Moreover, therapeutic targeting of integrated HBV DNA and their transcripts may also be required to induce hepatitis B virus surface antigen (HBsAg) seroclearance and prevent liver carcinogenesis.

8.
Antiviral Res ; 159: 1-12, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30201396

RESUMO

Native agarose gel electrophoresis-based particle gel assay has been commonly used for examination of hepatitis B virus (HBV) capsid assembly and pregenomic RNA encapsidation in HBV replicating cells. Interestingly, treatment of cells with several chemotypes of HBV core protein allosteric modulators (CpAMs) induced the assembly of both empty and DNA-containing capsids with faster electrophoresis mobility. In an effort to determine the physical basis of CpAM-induced capsid mobility shift, we found that the surface charge, but not the size, of capsids is the primary determinant of electrophoresis mobility. Specifically, through alanine scanning mutagenesis analysis of twenty-seven charged amino acids in core protein assembly domain and hinge region, we showed that except for K7 and E8, substitution of glutamine acid (E) or aspartic acid (D) on the surface of capsids reduced their mobility, but substitution of lysine (K) or arginine (R) on the surface of capsids increased their mobility in variable degrees. However, alanine substitution of the charged amino acids that are not exposed on the surface of capsid did not apparently alter capsid mobility. Hence, CpAM-induced electrophoresis mobility shift of capsids may reflect the global alteration of capsid structure that changes the exposure and/or ionization of charged amino acid side chains of core protein. Our findings imply that CpAM inhibition of pgRNA encapsidation is possibly due to the assembly of structurally altered nucleocapsids. Practically, capsid electrophoresis mobility shift is a diagnostic marker of compounds that target core protein assembly and predicts sensitivity of HBV strains to specific CpAMs.


Assuntos
Antivirais/farmacologia , Capsídeo/metabolismo , Vírus da Hepatite B/fisiologia , RNA/metabolismo , Proteínas do Core Viral/genética , Montagem de Vírus , Regulação Alostérica , Proteínas do Capsídeo/metabolismo , Eletroforese , Ensaio de Desvio de Mobilidade Eletroforética , Células Hep G2 , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , RNA Viral/metabolismo , Replicação Viral
9.
J Virol ; 92(13)2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29669831

RESUMO

Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly.IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids.


Assuntos
Genoma Viral , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Precursores de RNA/metabolismo , RNA Viral/metabolismo , Proteínas do Core Viral/metabolismo , Montagem de Vírus , Regulação Alostérica , Capsídeo/metabolismo , Células Cultivadas , Hepatite B/genética , Hepatite B/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fosforilação , Precursores de RNA/genética , RNA Viral/genética , Proteínas do Core Viral/genética , Replicação Viral
10.
Clin Nucl Med ; 43(5): 349-351, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29517542

RESUMO

Because ovarian cancer often advances by dissemination, pulmonary metastases are rarely unaccompanied by intra-abdominal metastases. We report a case of ovarian clear cell carcinoma having multiple bilateral lung metastases and without concurrent intra-abdominal lesions, as demonstrated by FDG PET/CT scan.


Assuntos
Neoplasias Abdominais/diagnóstico por imagem , Carcinoma/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Ovarianas/patologia , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Neoplasias Abdominais/secundário , Carcinoma/diagnóstico por imagem , Feminino , Fluordesoxiglucose F18 , Humanos , Neoplasias Pulmonares/secundário , Pessoa de Meia-Idade , Neoplasias Ovarianas/diagnóstico por imagem , Compostos Radiofarmacêuticos
11.
J Virol ; 92(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29263263

RESUMO

Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes.IMPORTANCE The differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.


Assuntos
Antígenos de Diferenciação/metabolismo , Coronavirus/metabolismo , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Multimerização Proteica , Proteínas de Ligação a RNA/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Substituição de Aminoácidos , Antígenos de Diferenciação/genética , Linhagem Celular Tumoral , Coronavirus/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
12.
Antiviral Res ; 147: 37-46, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28982551

RESUMO

Stimulator of interferon genes (STING) is an endoplasmic reticulum transmembrane protein that serves as a molecular hub for activation of interferon and inflammatory cytokine response by multiple cellular DNA sensors. Not surprisingly, STING has been demonstrated to play an important role in host defense against microorganisms and pharmacologic activation of STING is considered as an attractive strategy to treat viral diseases and boost antitumor immunity. In light of this we established a HepAD38-derived reporter cell line that expresses firefly luciferase in response to the activation of cyclic GMP-AMP synthase (cGAS)-STING pathway for high throughput screening (HTS) of small molecular human STING agonists. This cell-based reporter assay required only 4 h treatment with a reference STING agonist to induce a robust luciferase signal and was demonstrated to have an excellent performance in HTS format. By screening 16,000 compounds, a dispiro diketopiperzine (DSDP) compound was identified to induce cytokine response in a manner dependent on the expression of functional human STING, but not mouse STING. Moreover, we showed that DSDP induced an interferon-dominant cytokine response in human skin fibroblasts and peripheral blood mononuclear cells, which in turn potently suppressed the replication of yellow fever virus, dengue virus and Zika virus. We have thus established a robust cell-based assay system suitable for rapid discovery and mechanistic analyses of cGAS-STING pathway agonists. Identification of DSDP as a human STING agonist enriches the pipelines of STING-targeting drug development for treatment of viral infections and cancers.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Imunidade Inata/efeitos dos fármacos , Indutores de Interferon/farmacologia , Proteínas de Membrana/agonistas , Nucleotidiltransferases/antagonistas & inibidores , Piperazinas/farmacologia , Compostos de Espiro/farmacologia , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Flavivirus/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Indutores de Interferon/química , Dose Letal Mediana , Proteínas de Membrana/genética , Camundongos , Mutação , Nucleotidiltransferases/genética , Piperazinas/química , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Compostos de Espiro/química , Fatores de Transcrição/genética , Replicação Viral/efeitos dos fármacos
13.
Int J Mol Med ; 40(6): 1792-1802, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29039494

RESUMO

The hepatitis C virus (HCV) infection is associated with various extrahepatic manifestations, which are correlated with poor outcomes, and thus increase the morbidity and mortality of chronic hepatitis C (CHC). Therefore, understanding the internal linkages between systemic manifestations and HCV infection is helpful for treatment of CHC. Yet, the mechanism by which the virus evokes the systemic diseases remains to be elucidated. In the present study, using gene set enrichment analysis (GSEA) and signaling pathway impact analysis (SPIA), a comprehensive analysis of microarray data of mRNAs was conducted in HCV-infected and -uninfected Huh7.5 cells, and signaling pathways (which are significantly activated or inhibited) and certain molecules (which are commonly important in those signaling pathways) were selected. Forty signaling pathways were selected using GSEA, and eight signaling pathways were selected with SPIA. These pathways are associated with cancer, metabolism, environmental information processing and organismal systems, which provide important information for further clarifying the intrinsic associations between syndromes of HCV infection, of which seven pathways were not previously reported, including basal transcription factors, pathogenic Escherichia coli infection, shigellosis, gastric acid secretion, dorso-ventral axis formation, amoebiasis and cholinergic synapse. Ten genes, SOS1, RAF1, IFNA2, IFNG, MTHFR, IGF1, CALM3, UBE2B, TP53 and BMP7 whose expression may be the key internal driving molecules, were selected using the online tool Anni 2.1. Furthermore, the present study demonstrated the internal linkages between systemic manifestations and HCV infection, and presented the potential molecules that are key to those linkages.


Assuntos
Hepacivirus/fisiologia , Hepacivirus/patogenicidade , Hepatite C/metabolismo , Hepatite C/fisiopatologia , Interações Hospedeiro-Patógeno/fisiologia , Transdução de Sinais/fisiologia , Algoritmos , Linhagem Celular , Biologia Computacional , Infecções por Escherichia coli , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição
14.
PLoS Pathog ; 13(9): e1006658, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28945802

RESUMO

Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.


Assuntos
DNA Circular/biossíntese , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , DNA Circular/genética , DNA Viral , DNA Polimerase Dirigida por DNA/metabolismo , Hepatócitos/virologia , Humanos , Nucleocapsídeo/efeitos dos fármacos , Nucleocapsídeo/genética , Reação em Cadeia da Polimerase em Tempo Real , Replicação Viral/fisiologia
15.
Biomed Res Int ; 2017: 1236801, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28904942

RESUMO

Use of direct-acting antivirals sometimes causes viral drug resistance, resulting in inefficiency in treated patients in real-world practice. Therefore, how to rapidly and accurately evaluate drug resistance is an urgent problem to be solved for rational use and development of antivirals in the future. Here, we aim to develop a new method by which we can evaluate easily but effectively whether a drug will still be efficient in the future treatment in infectious hepatitis C virus cell culture system. HCV-infected Huh7.5 cells were treated with drugs and the culture supernatants were replaced with fresh culture media containing the same drugs at 24 hours. The supernatants were harvested at 48 hours and incubated with naïve Huh7.5 cells. Intracellular HCV RNAs or proteins in the newly infected cells were extracted and analyzed at 48 hours or longer. Results showed that after being treated with telaprevir mutant viruses were easily detected which were resistant to telaprevir, while after being treated with sofosbuvir drug-resistant viruses did not emerge. In conclusion, the new method is simple and quick but accurate to evaluate whether a drug will be still efficient in the forthcoming therapeutic regimen and whether drug resistance will occur after long-term treatment with drugs.


Assuntos
Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Antivirais/farmacologia , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Genótipo , Hepacivirus/patogenicidade , Hepatite C/virologia , Humanos , Oligopeptídeos/farmacologia , Proteínas não Estruturais Virais/genética
16.
Artigo em Inglês | MEDLINE | ID: mdl-28717041

RESUMO

Induction of interferon and proinflammatory cytokines is a hallmark of the infection of many different viruses. However, hepatitis B virus (HBV) does not elicit a detectable cytokine response in infected hepatocytes. In order to investigate the molecular mechanism underlying the innate immune evasion, a functional cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway was reconstituted in a human hepatoma cell line supporting tetracycline-inducible HBV replication. It was demonstrated that induction of HBV replication neither activated nor inhibited this cytosolic DNA sensing pathway. However, human hepatoma cells, as well as immortalized mouse hepatocytes, express low levels of STING, which upon activation by cGAMP, the natural ligand of STING, led to induction of a proinflammatory cytokine response. Treatment of immortalized mouse hepatocytes supporting HBV replication with either cGAMP or a small molecule pharmacologic STING agonist significantly reduced viral DNA in a STING- and Janus kinase 1-dependent manner. Moreover, cGAMP treatment was able to induce inflammatory cytokine gene expression and inhibit the transcription of covalently closed circular DNA in HBV-infected human hepatoma cells expressing sodium taurocholate cotransporting polypeptide, an essential receptor for HBV infection of hepatocytes. The studies reported here and previously (F. Guo et al., Antimicrob Agents Chemother 59:1273-1281, 2015, https://doi.org/10.1128/AAC.04321-14) thus support the notion that pharmacological activation of STING in macrophages and hepatocytes induces host innate responses that can efficiently control HBV replication. Hence, despite not playing a significant role in host innate immune response to HBV infection of hepatocytes, STING is potentially a valuable target for immunotherapy of chronic hepatitis B.


Assuntos
Vírus da Hepatite B/crescimento & desenvolvimento , Hepatócitos/imunologia , Interferons/biossíntese , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Replicação Viral/genética , Animais , Antivirais/farmacologia , Linhagem Celular Tumoral , Células Hep G2 , Vírus da Hepatite B/imunologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Interferons/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Membrana/agonistas , Camundongos , Nucleotidiltransferases/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo
17.
Biosci Rep ; 37(2)2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28213360

RESUMO

Microparticles (MPs) and miRNAs have been shown to play important roles in coronary artery disease (CAD) by monitoring endothelial dysfunction. The present study aims to investigate the diagnostic value of endothelial MPs (EMPs) and miRNAs (miR-92a or miR-23a) as biomarkers in distinguishing patients with acute myocardial infarction (AMI) from those with CAD. Plasma samples from 37 patients with AMI, 42 patients with stable CAD (SCAD), and 35 healthy adults were collected for investigation in the present study. The numbers of CD31+/CD42b- MPs, CD31+/CD42b+ MPs, and CD31-/CD42b- MPs were measured by flow cytometry and the levels of miR-92a and miR-23a were analyzed using reverse transcription-quantitative PCR. Moreover, cardiac troponin I (cTnI) expression was detected by ELISA to serve as a routine diagnostic parameter. The number of CD31+/CD42b- was higher in AMI group than those in SCAD and healthy groups. Besides, the expression of miR-92a was higher in AMI group compared with two other groups. Furthermore, evidence showed that there was a positive correlation between the levels of CD31+/CD42b- MPs and miR-92a Finally, the receiver operating characteristic (ROC) curve revealed that the area value under the curve of CD31+/CD42b- MPs, miR-92a and cTnI was 0.893, 0.888, and 0.912 respectively. CD31+/CD42b- MPs and miR-92a might have great potential to provide diagnostic value for AMI and could probably regulate the endothelial dysfunction in AMI patients.


Assuntos
Micropartículas Derivadas de Células , MicroRNAs/sangue , Infarto do Miocárdio/diagnóstico , Análise de Variância , Biomarcadores/sangue , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Molécula-1 de Adesão Celular Endotelial a Plaquetas/sangue , Complexo Glicoproteico GPIb-IX de Plaquetas/análise , Prognóstico , Troponina I/sangue
18.
Expert Opin Drug Discov ; 12(1): 5-15, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27797587

RESUMO

INTRODUCTION: The current standard care of chronic hepatitis B fails to induce a durable off-drug control of hepatitis B virus (HBV) replication in the majority of treated patients. The primary reasons are its inability to eliminate the covalently closed circular (ccc) DNA, the nuclear form of HBV genome, and restoration of the dysfunctional host antiviral immune response against the virus. Accordingly, discovery and development of therapeutics to completely stop HBV replication, eliminate or functionally inactivate cccDNA as well as activate a functional antiviral immune response against HBV are the primary efforts for finding a cure for chronic hepatitis B. Area covered: Herein, the authors highlight the current efforts of HBV drug discovery and offer their opinions for the future directions of this research. Expert opinion: The authors believe that through a consecutive or overlapping three-stage antiviral and immunotherapy program to: (i) completely stop HBV replication and cccDNA amplification; (ii) reduce viral antigen load and induce HBV surface antigen (HBsAg) seroclearance through eradication or inactivation of cccDNA and RNA interference-mediated degradation of viral mRNA and (iii) activate a functional antiviral immune response against HBV through therapeutic immunization or immunotherapy, a functional cure of chronic HBV infection can be achieved in the majority of chronic HBV carriers.


Assuntos
Antivirais/farmacologia , Desenho de Drogas , Hepatite B Crônica/tratamento farmacológico , Animais , DNA Circular/genética , DNA Viral/genética , Descoberta de Drogas/métodos , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Humanos , Imunoterapia/métodos , Replicação Viral/efeitos dos fármacos
19.
Biochem Biophys Res Commun ; 477(4): 761-767, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27363341

RESUMO

Apoptotic and necrotic macrophages have long been known for their existence in atherosclerotic lesions. However, the mechanisms underlying the choice of their death pattern have not been fully elucidated. Here, we report the effects of PS-341, a potent and specific proteasome inhibitor, on the cell death of primary bone marrow-derived macrophages (BMDMs) in vitro. The results showed that PS-341 could not induce macrophage apoptosis or promote TNF-induced macrophage apoptosis, on the other hand, PS-341 could significantly inhibit macrophage necroptosis induced by TNF and pan-caspase inhibitor z-VAD treatment. Remarkably, high-dose of PS-341 showed similar inhibitory effects on macrophage necroptosis comparable to that of kinase inhibition of RIP1 through specific inhibitor Nec-1 or inhibition of RIP3 via specific genetical ablation. Furthermore, the degradation of cellular inhibitor of apoptosis proteins (cIAPs) was suppressed by PS-341, which could antagonize the activation of RIP1 kinase via post-translational mechanism. Further evidences demonstrated reduced levels of both RIP1 and RIP 3 upon PS-341 treatment, concomitantly, a more strong association of RIP1 with cIAPs and less with RIP3 was found following PS-341 treatment, these findings suggested that PS-341 may disrupt the formation of RIP1-RIP3 complex (necrosome) through stabilizing cIAPs. Collectively, our results indicated that the proteasome-mediated degradation of cIAPs could be inhibited by PS-341 and followed by limited RIP1 and RIP3 kinase activities, which were indispensable for necroptosis, thus eliciting a significant necroptosis rescue in BMDMs in vitro. Overall, our study has identified a new role of PS-341 in the cell death of BMDMs and provided a novel insight into the atherosclerotic inflammation caused by proteasome-mediated macrophage necroptosis.


Assuntos
Apoptose/efeitos dos fármacos , Bortezomib/administração & dosagem , Macrófagos/metabolismo , Macrófagos/patologia , Inibidores de Proteassoma/administração & dosagem , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo
20.
Sci Rep ; 6: 21808, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26898231

RESUMO

The cluster of differentiation 36 (CD36) is a membrane protein related to lipid metabolism. We show that HCV infection in vitro increased CD36 expression in either surface or soluble form. HCV attachment was facilitated through a direct interaction between CD36 and HCV E1 protein, causing enhanced entry and replication. The HCV co-receptor effect of CD36 was independent of that of SR-BI. CD36 monoclonal antibodies neutralized the effect of CD36 and reduced HCV replication. CD36 inhibitor sulfo-N-succinimidyl oleate (SSO), which directly bound CD36 but not SR-BI, significantly interrupted HCV entry, and therefore inhibited HCV replication. SSO's antiviral effect was seen only in HCV but not in other viruses. SSO in combination with known anti-HCV drugs showed additional inhibition against HCV. SSO was considerably safe in mice. Conclusively, CD36 interacts with HCV E1 and might be a co-receptor specific for HCV entry; thus, CD36 could be a potential drug target against HCV.


Assuntos
Antivirais/farmacologia , Antígenos CD36/genética , Hepacivirus/efeitos dos fármacos , Ácidos Oleicos/farmacologia , Receptores Virais/genética , Succinimidas/farmacologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/farmacologia , Antígenos CD36/antagonistas & inibidores , Antígenos CD36/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Regulação da Expressão Gênica , Células HEK293 , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Oligopeptídeos/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Virais/antagonistas & inibidores , Receptores Virais/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais , Testes de Toxicidade Aguda , Transgenes , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral
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