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1.
J Virol ; 95(19): e0044421, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287051

RESUMO

DDX17 is a member of the DEAD-box helicase family proteins involved in cellular RNA folding, splicing, and translation. It has been reported that DDX17 serves as a cofactor of host zinc finger antiviral protein (ZAP)-mediated retroviral RNA degradation and exerts direct antiviral function against Raft Valley fever virus through binding to specific stem-loop structures of viral RNA. Intriguingly, we have previously shown that ZAP inhibits hepatitis B virus (HBV) replication through promoting viral RNA decay, and the ZAP-responsive element (ZRE) of HBV pregenomic RNA (pgRNA) contains a stem-loop structure, specifically epsilon, which serves as the packaging signal for pgRNA encapsidation. In this study, we demonstrated that the endogenous DDX17 is constitutively expressed in human hepatocyte-derived cells but dispensable for ZAP-mediated HBV RNA degradation. However, DDX17 was found to inhibit HBV replication primarily by reducing the level of cytoplasmic encapsidated pgRNA in a helicase-dependent manner. Immunofluorescence assay revealed that DDX17 could gain access to cytoplasm from nucleus in the presence of HBV RNA. In addition, RNA immunoprecipitation and electrophoretic mobility shift assays demonstrated that the enzymatically active DDX17 competes with HBV polymerase to bind to pgRNA at the 5' epsilon motif. In summary, our study suggests that DDX17 serves as an intrinsic host restriction factor against HBV through interfering with pgRNA encapsidation. IMPORTANCE Hepatitis B virus (HBV) chronic infection, a long-studied but yet incurable disease, remains a major public health concern worldwide. Given that HBV replication cycle highly depends on host factors, deepening our understanding of the host-virus interaction is thus of great significance in the journey of finding a cure. In eukaryotic cells, RNA helicases of the DEAD box family are highly conserved enzymes involved in diverse processes of cellular RNA metabolism. Emerging data have shown that DDX17, a typical member of the DEAD box family, functions as an antiviral factor through interacting with viral RNA. In this study, we, for the first time, demonstrate that DDX17 inhibits HBV through blocking the formation of viral replication complex, which not only broadens the antiviral spectrum of DDX17 but also provides new insight into the molecular mechanism of DDX17-mediated virus-host interaction.


Assuntos
Capsídeo/metabolismo , RNA Helicases DEAD-box/metabolismo , Vírus da Hepatite B/fisiologia , RNA Viral/metabolismo , Replicação Viral , Linhagem Celular , Linhagem Celular Tumoral , Citoplasma/metabolismo , RNA Helicases DEAD-box/química , Produtos do Gene pol/metabolismo , Vírus da Hepatite B/genética , Humanos , Conformação de Ácido Nucleico , Domínios Proteicos , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo
2.
Nat Commun ; 12(1): 3005, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021134

RESUMO

Defective cholesterol biosynthesis in eye lens cells is often associated with cataracts; however, how genes involved in cholesterol biosynthesis are regulated in lens cells remains unclear. Here, we show that Quaking (Qki) is required for the transcriptional activation of genes involved in cholesterol biosynthesis in the eye lens. At the transcriptome level, lens-specific Qki-deficient mice present downregulation of genes associated with the cholesterol biosynthesis pathway, resulting in a significant reduction of total cholesterol level in the eye lens. Mice with Qki depletion in lens epithelium display progressive accumulation of protein aggregates, eventually leading to cataracts. Notably, these defects are attenuated by topical sterol administration. Mechanistically, we demonstrate that Qki enhances cholesterol biosynthesis by recruiting Srebp2 and Pol II in the promoter regions of cholesterol biosynthesis genes. Supporting its function as a transcription co-activator, we show that Qki directly interacts with single-stranded DNA. In conclusion, we propose that Qki-Srebp2-mediated cholesterol biosynthesis is essential for maintaining the cholesterol level that protects lens from cataract development.


Assuntos
Colesterol/biossíntese , Cristalino/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Animais , Linhagem Celular , Produtos do Gene pol , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética
3.
Viruses ; 13(3)2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802118

RESUMO

Heightened expression of human endogenous retrovirus (HERV) sequences has been associated with a range of malignancies, including prostate cancer, suggesting that they may serve as useful diagnostic or prognostic cancer biomarkers. We analysed the expression of HERV-K (Gag and Env/Np9 regions), HERV-E 4.1 (Pol and Env regions), HERV-H (Pol) and HERV-W (Gag) sequences in prostate cancer cells lines and normal prostate epithelial cells using qRT-PCR. HERV expression was also analysed in matched malignant and benign prostate tissue samples from men with prostate cancer (n = 27, median age 65.2 years (range 47-70)) and compared to prostate cancer-free male controls (n = 11). Prostate cancer epithelial cell lines exhibited a signature of HERV RNA overexpression, with all HERVs analysed, except HERV-E Pol, showing heightened expression in at least two, but more commonly all, cell lines analysed. Analysis of primary prostate material indicated increased expression of HERV-E Pol but decreased expression of HERV-E Env in both malignant and benign regions of the prostate in men with prostate cancer as compared to those without. Expression of HERV-K Gag was significantly higher in malignant regions of the prostate in men with prostate cancer as compared to matched benign regions and prostate cancer-free men (p < 0.001 for both), with 85.2% of prostate cancers donors showing malignancy-associated upregulation of HERV-K Gag RNA. HERV-K Gag protein was detected in 12/18 (66.7%) malignant tissues using immunohistochemistry, but only 1/18 (5.6%) benign tissue sections. Heightened expression of HERV-K Gag RNA and protein appears to be a sensitive and specific biomarker of prostate malignancy in this cohort of men with prostate carcinoma, supporting its potential utility as a non-invasive, adjunct clinical biomarker.


Assuntos
Retrovirus Endógenos/genética , Produtos do Gene env/genética , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Neoplasias da Próstata/genética , Idoso , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Retrovirus Endógenos/isolamento & purificação , Regulação Neoplásica da Expressão Gênica/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/metabolismo , Produtos do Gene pol/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Próstata/metabolismo , Neoplasias da Próstata/diagnóstico
4.
J Microbiol Biotechnol ; 31(1): 16-24, 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33144545

RESUMO

Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.


Assuntos
Carcinoma Hepatocelular/virologia , DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Hepatite B/enzimologia , Neoplasias Hepáticas/virologia , Fator 1 de Elongação de Peptídeos/metabolismo , Apoptose/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/farmacologia , Produtos do Gene pol , Células Hep G2 , Vírus da Hepatite B/genética , Humanos , Fator 1 de Elongação de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , RNA Interferente Pequeno
5.
Emerg Microbes Infect ; 9(1): 2381-2393, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33124952

RESUMO

Background and Aims: The drug resistance of hepatitis B virus (HBV) originates from mutations within HBV reverse transcriptase (RT) region during the prolonged antiviral therapy. So far, the characteristics of how these mutations distribute and evolve in the process of therapy have not been clarified yet. Thus we aimed to investigate these characteristics and discuss their contributing factors. Methods: HBV RT region was direct-sequenced in 285 treatment-naive and 214 post-treatment patients. Mutational frequency and Shannon entropy were calculated to identify the specific mutations differing between genotypes or treatment status. A typical putative resistance mutation rtL229V was further studied using in-vitro susceptibility assays and molecular modeling. Results: The classical resistance mutations were rarely detected among treatment-naive individuals, while the putative resistance mutations were observed at 8 AA sites. rtV191I and rtA181T/V were the only resistance mutations identified as genotype-specific mutation. Selective pressure of drug usage not only contributed to the classical resistance mutations, but also induced the changes at a putative resistance mutation site rt229. rtL229V was the major substitution at the site of rt229. It contributed to the most potent suppression of viral replication and reduced the in-vitro drug susceptibility to entecavir (ETV) when coexisting with rtM204V, consistent with the hypothesis based on the molecular modeling and clinical data analysis. Conclusions: The analysis of mutations in RT region under the different circumstances of genotypes and therapy status might pave the way for a better understanding of resistance evolution, thus providing the basis for a rational administration of antiviral therapy.


Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Produtos do Gene pol/genética , Vírus da Hepatite B/enzimologia , Hepatite B Crônica/tratamento farmacológico , Mutação , Adulto , Estudos de Casos e Controles , Linhagem Celular , Feminino , Produtos do Gene pol/química , Genótipo , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Fenótipo , Análise de Sequência de DNA , Adulto Jovem
6.
Cell ; 183(1): 185-196.e14, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007262

RESUMO

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However, the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study, we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model, even in the absence of virus neutralization.


Assuntos
Imunização Passiva/métodos , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Produtos do Gene pol/imunologia , HIV-1/imunologia , Imunoglobulina G/imunologia , Macaca mulatta/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia
7.
Viruses ; 12(8)2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32752057

RESUMO

Hepatitis B virus (HBV) polymerase seems to be very hard to express and purify sufficiently, which has long hampered the generation of anti-HBV drugs based on the nature of the polymerase. To date, there has been no useful system developed for drug screening against HBV polymerase. In this study, we successfully obtained a highly purified reverse transcriptase (RT) domain of the polymerase, which has a template/primer and substrate binding activity, and established a novel high-throughput screening (HTS) system using purified RT protein for finding novel polymerase inhibitors. To examine whether the assay system provides reliable results, we tested the small scale screening using pharmacologically active compounds. As a result, the pilot screening identified already-known anti-viral polymerase agents. Then, we screened 20,000 chemical compounds and newly identified four hits. Several of these compounds inhibited not only the HBV RT substrate and/ template/primer binding activity, but also Moloney murine leukemia virus RT activity, which has an elongation activity. Finally, these candidates did show to be effective even in the cell-based assay. Our screening system provides a useful tool for searching candidate inhibitors against HBV.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Produtos do Gene pol/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Células Hep G2 , Vírus da Hepatite B/enzimologia , Ensaios de Triagem em Larga Escala , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , DNA Polimerase Dirigida por RNA , Bibliotecas de Moléculas Pequenas , Replicação Viral/efeitos dos fármacos
8.
Genes Cells ; 25(8): 523-537, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32415897

RESUMO

Although several nucleo(s)tide analogs are available for treatment of HBV infection, long-term treatment with these drugs can lead to the emergence of drug-resistant viruses. Recent HIV-1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non-nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI-resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)-reverse transcriptase (RT) (TP-RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP-RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA-binding assay system. Then, we used TP-RT in cell-free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6-hydroxy-DL-DOPA and N-oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell-based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP-RT domain to treat HBV infection.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores da Transcriptase Reversa/farmacologia , Replicação Viral/efeitos dos fármacos , Proteínas de Transporte/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Ligação Proteica , RNA/metabolismo , Motivos de Ligação ao RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Bibliotecas de Moléculas Pequenas
9.
PLoS One ; 15(2): e0228192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023284

RESUMO

New methods of HIV-1 RNA quantification based on dual-target detection are increasingly used in HIV viral load monitoring, but clinical implications and impact of dual-target detection on HIV-1 infection management are not established. Aptima HIV-1 Quant Dx assay is a last generation HIV viral load method, that uses pol and LTR as simultaneous target, providing quantitative results based mainly on pol target, while LTR target is used to report the results when pol signal is absent. In our laboratory, about 6% of results of all HIV-1 viral load tests performed with this platform in one year period resulted from LTR signal. Interestingly, LTR-based viremia (sometimes exceeding 1,000 copies/mL) was observed in a small proportion (up to 1%) of patients under ART, considered for long time virologically suppressed on the basis of a single target (pol-based) assay. Male gender, >700 vs <200 CD4 cell/mL and dual therapy including NRTI plus either NNRTI, or PI/b or INSTI were independently associated with increased risk of LTR-based HIV-1 viral load detection by multivariable logistic regression. A significant linear correlation was observed between LTR-based HIV-1 RNA levels and PBMC-associated proviral DNA. Moreover, in a small group of patients with HIV-1 RNA levels >200 copies/mL, longitudinal assessments showed parallel kinetics between plasma viremia and proviral DNA. Sequencing of pol region for drug resistance assessment in patients with LTR-based viremia failed on plasma HIV-1 RNA, while it was successful on proviral DNA. The detection/quantification of HIV-1 viremia based only on LTR signal with a dual target assay in samples resulting undetectable with the more conventional target pol needs accurate evaluation; unravelling the biological basis of this phenomenon, here described for the first time, is mandatory to establish relevance and implication by both pathogenetic (i.e. infectivity of LTR-detected viruses, reservoir turnover, immune activation, etc.) and clinical standpoint.


Assuntos
Infecções por HIV/virologia , HIV-1/genética , Provírus/genética , Viremia/virologia , Adulto , Antirretrovirais/uso terapêutico , Contagem de Linfócito CD4 , DNA Viral/sangue , Farmacorresistência Viral , Feminino , Produtos do Gene pol/genética , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Repetição Terminal Longa de HIV/genética , HIV-1/isolamento & purificação , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/virologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Provírus/isolamento & purificação , RNA Viral/sangue , Carga Viral , Viremia/patologia
10.
Microbes Infect ; 22(8): 366-370, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32035224

RESUMO

The human endogenous retroviruses (HERVs) are endogenous retroviruses that are inserted into the germ cell DNA of humans over 30 million years ago. Using real-time RT-PCR we describe HERV modulation by commensal microbes in the human gut. Infants, exclusively or predominant breast milk feeding, less than 12 weeks of age, during bacteria gut colonization, were assessed for eligibility. Our data demonstrate that the colonization with commensal microbes, in particular, Bifidobacterium spp., of the gut causes modulation of HERVs.


Assuntos
Retrovirus Endógenos/genética , Microbioma Gastrointestinal/fisiologia , Transcrição Genética , Bactérias/classificação , Bactérias/isolamento & purificação , Aleitamento Materno , Retrovirus Endógenos/classificação , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Produtos do Gene pol/sangue , Produtos do Gene pol/genética , Humanos , Lactente
11.
Sci Rep ; 10(1): 263, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937823

RESUMO

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P < 0.0001), and many provirus-positive southern animals failed to express any detectable KoRV RNA. Across both populations there was a positive association between proviral load and neoplasia (P = 0.009). Potential reasons for the differences in the nature of KoRV infection between the two populations are discussed.


Assuntos
Infecções por Retroviridae/patologia , Retroviridae/genética , Envelhecimento/genética , Animais , Austrália/epidemiologia , DNA/metabolismo , Feminino , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Masculino , Phascolarctidae , Provírus/genética , RNA Viral/sangue , Retroviridae/isolamento & purificação , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/veterinária , Infecções por Retroviridae/virologia , Carga Viral
12.
Am J Physiol Gastrointest Liver Physiol ; 318(1): G162-G173, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31604033

RESUMO

Hepatitis B virus (HBV) exploits multiple strategies to evade host immune surveillance. Programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) signaling plays a critical role in regulating T cell homeostasis. However, it remains largely unknown as to how HBV infection elevates PD-L1 expression in hepatocytes. A mouse model of HBV infection was established by hydrodynamic injection with a vector containing 1.3-fold overlength HBV genome (pHBV1.3) via the tail vein. Coculture experiments with HBV-expressing hepatoma cells and Jurkat T cells were established in vitro. We observed significant decrease in the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and increase in ß-catenin/PD-L1 expression in liver tissues from patients with chronic hepatitis B and mice subjected to pHBV1.3 hydrodynamic injection. Mechanistically, decrease in PTEN enhanced ß-catenin/c-Myc signaling and PD-L1 expression in HBV-expressing hepatoma cells, which in turn augmented PD-1 expression, lowered IL-2 secretion, and induced T cell apoptosis. However, ß-catenin disruption inhibited PTEN-mediated PD-L1 expression, which was accompanied by decreased PD-1 expression, and increased IL-2 production in T cells. Luciferase reporter assays revealed that c-Myc stimulated transcriptional activity of PD-L1. In addition, HBV X protein (HBx) and HBV polymerase (HBp) contributed to PTEN downregulation and ß-catenin/PD-L1 upregulation. Strikingly, PTEN overexpression in hepatocytes inhibited ß-catenin/PD-L1 signaling and promoted HBV clearance in vivo. Our findings suggest that HBV-triggered PTEN/ß-catenin/c-Myc signaling via HBx and HBp enhances PD-L1 expression, leading to inhibition of T cell response, and promotes HBV immune evasion.NEW & NOTEWORTHY This study demonstrates that during HBV infection, HBV can increase PD-L1 expression via PTEN/ß-catenin/c-Myc signaling pathway, which in turn inhibits T cell response and ultimately promotes HBV immune evasion. Targeting this signaling pathway is a potential strategy for immunotherapy of chronic hepatitis B.


Assuntos
Antígeno B7-H1/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Hepatócitos/enzimologia , Evasão da Resposta Imune , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linfócitos T/enzimologia , beta Catenina/metabolismo , Animais , Técnicas de Cocultura , Modelos Animais de Doenças , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Células Jurkat , Ativação Linfocitária , Masculino , Camundongos Endogâmicos BALB C , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/virologia , Transativadores/genética , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias
13.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31852786

RESUMO

Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-γ) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies.


Assuntos
Epitopos de Linfócito T/imunologia , Antígenos HLA/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/virologia , Linfócitos T CD8-Positivos/imunologia , Produtos do Gene pol/imunologia , Genótipo , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia , Interferon gama/imunologia , Peptídeos/imunologia , Ligação Proteica
14.
Pol J Microbiol ; 68(3): 317-322, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31880877

RESUMO

Mutations associated with the pol and the S gene can emerge as a consequence of the high replication capacity and proofreading deficiencies of hepatitis B virus during replication. The current study was constructed to evaluate primary, partial, compensatory and the escape mutations in chronic hepatitis B patients in Northern Cyprus. The samples of HBsAg positive treatment naïve 100 patients were involved in this study. HBV pol gene region was sequenced, amplified and HBV pol/S gene mutations were determined. The samples of thirty-two patients were excluded because of their low viral load (HBV < 1000 iu/ml). Among the sequenced 68 samples, there was a partial mutation (1.5%) and 36.7% displayed a resistance profile to lamivudine, adevofir, and telbivudine. Immune response escape, vaccine escape, HBIg and diagnosis escape mutations were determined in 24%, 10%, 6%, and 4% samples of the patients, respectively. Additionally, there were six different combined mutations. These data underscored that there is no concern for primary mutations in Northern Cyprus, however, we have identified a compensatory mutation (rtV173M) that may have primary mutation characteristics by combining with other mutation patterns. Additionally, HBsAg escape mutants demonstrated that detection of the S gene together with the pol gene mutations might be beneficial and important to monitor the surveillance of S variants.Mutations associated with the pol and the S gene can emerge as a consequence of the high replication capacity and proofreading deficiencies of hepatitis B virus during replication. The current study was constructed to evaluate primary, partial, compensatory and the escape mutations in chronic hepatitis B patients in Northern Cyprus. The samples of HBsAg positive treatment naïve 100 patients were involved in this study. HBV pol gene region was sequenced, amplified and HBV pol/S gene mutations were determined. The samples of thirty-two patients were excluded because of their low viral load (HBV < 1000 iu/ml). Among the sequenced 68 samples, there was a partial mutation (1.5%) and 36.7% displayed a resistance profile to lamivudine, adevofir, and telbivudine. Immune response escape, vaccine escape, HBIg and diagnosis escape mutations were determined in 24%, 10%, 6%, and 4% samples of the patients, respectively. Additionally, there were six different combined mutations. These data underscored that there is no concern for primary mutations in Northern Cyprus, however, we have identified a compensatory mutation (rtV173M) that may have primary mutation characteristics by combining with other mutation patterns. Additionally, HBsAg escape mutants demonstrated that detection of the S gene together with the pol gene mutations might be beneficial and important to monitor the surveillance of S variants.


Assuntos
Evolução Molecular , Produtos do Gene pol/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Adulto , Idoso , Antivirais/farmacologia , Chipre , Farmacorresistência Viral , Feminino , Vírus da Hepatite B/classificação , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/tratamento farmacológico , Humanos , Lamivudina/farmacologia , Masculino , Pessoa de Meia-Idade , Mutação , Adulto Jovem
15.
Cell ; 179(3): 632-643.e12, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31607510

RESUMO

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.


Assuntos
Gammaretrovirus/genética , Phascolarctidae/genética , RNA Interferente Pequeno/genética , Animais , Elementos de DNA Transponíveis , Gammaretrovirus/metabolismo , Gammaretrovirus/patogenicidade , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Genoma , Células Germinativas/metabolismo , Células Germinativas/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Phascolarctidae/virologia , Splicing de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismo
16.
World J Gastroenterol ; 25(33): 4985-4998, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31543688

RESUMO

BACKGROUND: Hepatitis B virus (HBV) DNA polymerase mutations usually occur to long term use of nucleos(t)ide analogues (NAs), but they can occur spontaneously in treatment-naïve chronic hepatitis B (CHB) patients. The naturally occurring HBV DNA polymerase mutations might complicate antiviral therapy with NAs, leading to the generation of drug-resistant viral mutants and disease progression. The most common substitutions are known to be YMDD-motif mutations, but their prevalence and the influence on antiviral therapy is unclear. AIM: To investigate prevalence of the naturally occurring rtM204I mutations in treatment-naïve CHB genotype C2 patients and their influence on antiviral therapy. METHODS: A total of 410 treatment-naïve CHB patients infected with HBV genotype C2 strains were enrolled in this retrospective study. Among the 410 patients, 232 were treated with NAs for at least 12 mo. Significant fibrosis was defined as fibrosis-4 index > 3.25 or aspartate aminotransferase to platelet ratio index > 1.5. Complete viral response (CVR) during NAs was defined as undetectable serum HBV DNA (< 24 IU/mL). The rtM204I variants were analyzed by a newly developed locked nucleotide probe (LNA probe) based real-time PCR (LNA-RT-PCR) method. RESULTS: The LNA-RT-PCR could discriminate rtM204I mutant-type (17 patients, 4.2%) from rtM204 wild-type (386 patients, 95.8%) in 403 of 410 patients (98.3% sensitivity). Multivariate analysis showed that naturally occurring rtM204I variants were more frequently detected in patients with significant fibrosis [odd-ratio (OR) 3.397, 95% confidence-interval (CI) 1.119-10.319, P = 0.031]. Of 232 patients receiving NAs, multivariate analysis revealed that achievement of CVR was reversely associated with naturally occurring rtM204I variants prior to NAs treatment (OR 0.014, 95%CI 0.002-0.096, P < 0.001). Almost patients receiving tenofovir achieved CVR at 12 mo of tenofovir, irrespective of pre-existence of naturally occurring rtM204I mutations (CVR rates: patients with rtM204I, 100%; patients without rtM204I, 96.6%), whereas, pre-existence of naturally-occurring rtM204I-mutations prior to NAs significantly affects CVR rates in patients receiving entecavir (at 12 mo: Patients with rtM204I, 16.7%; patients without rtM204I, 95.6%, P < 0.001). CONCLUSION: The newly developed LNA-RT-PCR method could detect naturally occurring rtM204I mutations with high-sensitivity. Theses mutations were more frequent in patients with liver fibrosis. Tenofovir is a more suitable treatment than entecavir for CHB patients carrying the naturally occurring rtM204I mutations.


Assuntos
Produtos do Gene pol/genética , Guanina/análogos & derivados , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Tenofovir/uso terapêutico , Adulto , Análise Mutacional de DNA/métodos , Sondas de DNA/genética , DNA Viral/isolamento & purificação , Farmacorresistência Viral/genética , Estudos de Viabilidade , Feminino , Guanina/farmacologia , Guanina/uso terapêutico , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Tenofovir/farmacologia
17.
Liver Int ; 39(12): 2273-2284, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31419377

RESUMO

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is the most critical factor underlying liver cirrhosis and hepatocellular carcinoma worldwide. IL-1ß and IL-18, generated by activation of the inflammasome/caspase-1 signaling pathway, play important roles in the control and clearance of HBV. However, the specific relationship between the inflammasome response and IFN-α resistance or viral persistence is yet to be established. METHODS: Blood samples of patients and supernatant fractions of HBV cell lines were collected for analysis and the effects on inflammasome activation and IL-1ß production evaluated via enzyme-linked immunosorbent assay (ELISA), western blot, quantitative RT-PCR and immunofluorescence. RESULTS: IL-1ß and IL-18 levels produced in sera of IFN-α non-responders were significantly lower than those of responders and normal donors. Additionally, expression of IL-1ß and inflammasome components was decreased in peripheral blood mononuclear cells (PBMC) of non-responders, compared with those of responders. In vitro experiments on HepG2, HepG2.2.15 and HepAD38 cell lines showed that HBV induces a significant decrease in IL-1ß production through inhibiting activation of the NF-κB signaling and inflammasome/caspase-1 pathways. And hepatitis B virus polymerase (HBV-Pol) appeared crucial for these inhibitory effects of HBV. CONCLUSION: IL-1ß production is suppressed in HBV carriers and IFN-α non-responders. HBV induces a significant decrease in IL-1ß production through inhibiting the NF-κB signaling and inflammasome pathways, for which HBV-Pol is a crucial requirement. Trial approval number: 20 173 402.


Assuntos
Produtos do Gene pol/metabolismo , Hepatite B Crônica/sangue , Interações Hospedeiro-Patógeno , Inflamassomos/metabolismo , Interleucina-1beta/sangue , Adulto , Antivirais/uso terapêutico , Estudos de Casos e Controles , Feminino , Células Hep G2 , Hepatite B Crônica/tratamento farmacológico , Hepatite C Crônica/sangue , Humanos , Interferon-alfa/uso terapêutico , Interleucina-18/sangue , Masculino , Pessoa de Meia-Idade
18.
Front Immunol ; 10: 1339, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338090

RESUMO

HERV-H endogenous retroviruses are thought to be essential to stem cell identity in humans. We embrace several decades of HERV-H research in order to relate the transcription of HERV-H loci to their genomic structure. We find that highly transcribed HERV-H loci are younger, more fragmented, and less likely to be present in other primate genomes. We also show that repeats in HERV-H LTRs are correlated to where loci are transcribed: type-I LTRs associate with stem cells while type-II repeats associate with embryonic cells. Our findings are generally in line with what is known about endogenous retrovirus biology but we find that the presence of the zinc finger motif containing region of gag is positively correlated with transcription. This leads us to suggest a possible explanation for why an unusually large proportion of HERV-H loci have been preserved in non-solo-LTR form.


Assuntos
Retrovirus Endógenos/genética , Genoma Viral/genética , Sequências Repetidas Terminais/genética , Animais , Sequência de Bases , Callithrix , Evolução Molecular , Produtos do Gene env/genética , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Genômica , Gorilla gorilla , Humanos , Macaca , Pan troglodytes , Pongo , Alinhamento de Sequência , Células-Tronco/citologia
19.
Ann Hepatol ; 18(4): 640-645, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31105017

RESUMO

INTRODUCTION AND OBJECTIVES: The hepatitis B virus (HBV) surface antigen (HBsAg) variations suggested having some effects on infection outcome. Due to some controversial issues, the aim of this study was to compare the pattern of HBsAg variation between asymptomatic carriers and HCC/cirrhosis patients. MATERIALS AND METHODS: In this cross-sectional study, 19 HCC/cirrhotic and 26 asymptomatic patients were enrolled. After viral DNA extraction, HBs gene was amplified using an in-house nested-PCR. Then, PCR products were introduced into bi-directional Sanger sequencing. The retrieved sequences were compared with references, to investigate the variation of immunologic sites, major hydrophilic region (MHR) of HBsAg as well as reverse transcriptase (RT), and also to determine genotype/subtype. RESULTS: The analysis of MHR and epitopes on HBsAg showed dozens of substitution, which occurred more prevalently in I110, P120, Y134, G159, S193, Y206, S207, I208, L213 and P214 positions. However, Y134N/F/L (P=0.04) and P120T/S (P=0.009) were significantly detected in MHR and B-cell epitope of HCC/Cirrhotic group. A number of truncation-related mutations were higher in HCC/Cirrhotic group (P>0.001), albeit only C69* stop codon was statistically significant (P=0.003). In RT, some potentially resistant substitutions such as Q215S, V191I and V214A, were revealed. Phylogenetic analysis showed that all of isolates belonged to genotype D, and the major serotype was ayw1. CONCLUSION: The higher frequency of substitutions in MHR and immune epitopes at positions such as Y134 and P120 as well as stop codons such as C69* in HCC/cirrhotic group might candidate them as predictive factors for infection outcome.


Assuntos
Carcinoma Hepatocelular/virologia , Portador Sadio/virologia , Farmacorresistência Viral/genética , Produtos do Gene pol/genética , Antígenos de Superfície da Hepatite B/genética , Hepatite B Crônica/virologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Adulto , Infecções Assintomáticas , DNA Viral/análise , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/genética , Feminino , Genótipo , Humanos , Evasão da Resposta Imune/genética , Masculino , Pessoa de Meia-Idade , Mutação , Adulto Jovem
20.
Mikrobiyol Bul ; 53(2): 144-155, 2019 Apr.
Artigo em Turco | MEDLINE | ID: mdl-31130119

RESUMO

Chronic hepatitis B (CHB) is an important public health problem affecting over 240 million people all around the world. The aim of the treatment in chronic hepatitis B is to prevent progression to cirrhosis and liver cancer. Interferons (standard and peginterferon) (Peg-IFN) and nucleoside/nucleotide analogues (NAs) are widely used in the treatment of CHB. The use of long-term therapy can however result in drug resistant mutations, which can lead to treatment failure. In patients with chronic hepatitis B, in addition to primary drug resistance mutations in the pol gene, compensatory mutations were reported. The genom of HBV polymerase (pol) gene overlaps with the envelope (S) gene. Nucleoside/nucleotide analogue (NA) resistance mutations in the pol gene of HBV, either from selection of primary or secondary resistance mutations, typically result in changes in HBsAg. Recent studies have conferred a new acronym for these HBV pol/S gene overlap mutants; ADAPVEMs, for antiviral drug-associated potential vaccine-escape mutants. The aim of this study was to investigate clinically and epidemiologically significant HBV pol/S gene mutations in NA treated CHB patients. In the study, a total of 100 patients who received nucleoside/nucleotide analogue therapy for one year or more were included. The levels of HBV DNA from serum samples were detected by the commercial real-time PCR assay and the mutations of pol/S genes by direct sequencing. Sixteen samples with low HBV DNA levels (> 200 IU/ml) could not be interpreted by sequencing due to insufficient amplification. Of the remaining 84 patients that could be sequenced HBV pol gene of HBV, 53 (63.09%) were males and 31 (36.91%) were women and the mean age was 47 ± 14.99 years (range: 20-67). Primary/secondary drug mutations (rtM204I/V, rtI169S, rtL180M, rtT184L, rtA194V, rtM204I/rtL91I, rtQ149K, rtQ215H/S, rtN238D) were detected in 38 (45.2%) of the patients. Because of the HBV pol/S gene overlapping, in 27 patients immun-selected amino acid substitutions (sI110L, sT127P, sS114A, sT123A), in nine patients HBIg selected escape mutants (sP120R, sT123N, sE164D, sY134F, sQ129H, sT118A, sP127K), in seven patients vaccine escape mutants (sT126I, sP120S, sG145A, s S193L) and in one patient misdiagnosis of HBsAg (sT131I) were detected. In addition, antiviral drug-associated potential vaccine-escape mutants were detected in 13 (15.4%) patients. In patients with chronic HBV, NAs including commonly used lamivudine were observed to have the potential for ADAPVEM to emerge during treatment. It was concluded that after determination of antiviral drug resistance and ADAPVEMs replanning of treatment should be done in the NA treatment of patients with CHB.


Assuntos
Antivirais , Farmacorresistência Viral , Produtos do Gene pol , Vírus da Hepatite B , Hepatite B Crônica , Mutação , Proteínas do Envelope Viral , Adulto , Idoso , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Feminino , Produtos do Gene pol/genética , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Humanos , Lamivudina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação/genética , Nucleotídeos/uso terapêutico , Proteínas do Envelope Viral/genética , Adulto Jovem
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