RESUMO
BACKGROUND/AIMS: The major histocompatibility class II (MHC II) transactivator, known as CIITA, is induced by Interferon gamma (IFN-γ) and plays a well-established role in regulating the expression of class II MHC molecules in antigen-presenting cells. METHODS: Primary human hepatocytes (PHH) were isolated via therapeutic hepatectomy from two donors. The hepatocellular carcinoma (HCC) cell lines HepG2 and Huh7 were used for the mechanistic study, and HBV infection was performed in HepG2-NTCP cells. HBV DNA replication intermediates and secreted antigen levels were measured using Southern blotting and ELISA, respectively. RESULTS: We identified a non-canonical function of CIITA in the inhibition of hepatitis B virus (HBV) replication in both HCC cells and patient-derived PHH. Notably, in vivo experiments demonstrated that HBV DNA and secreted antigen levels were significantly decreased in mice injected with the CIITA construct. Mechanistically, CIITA inhibited HBV transcription and replication by suppressing the activity of HBV-specific enhancers/promoters. Indeed, CIITA exerts antiviral activity in hepatocytes through ERK1/2-mediated down-regulation of the expression of hepatocyte nuclear factor 1α (HNF1α) and HNF4α, which are essential factors for virus replication. In addition, silencing of CIITA significantly abolished the IFN-γ-mediated anti-HBV activity, suggesting that CIITA mediates the anti-HBV activity of IFN-γ to some extent. HBV X protein (HBx) counteracts the antiviral activity of CIITA via direct binding and impairing its function. CONCLUSION: Our findings reveal a novel antiviral mechanism of CIITA that involves the modulation of the ERK pathway to restrict HBV transcription. Additionally, our results suggest the possibility of a new immune avoidance mechanism involving HBx.
Assuntos
Vírus da Hepatite B , Hepatócitos , Proteínas Nucleares , Transativadores , Replicação Viral , Vírus da Hepatite B/fisiologia , Humanos , Transativadores/metabolismo , Transativadores/genética , Animais , Camundongos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Hepatócitos/metabolismo , Hepatócitos/citologia , Hepatócitos/virologia , Células Hep G2 , Hepatite B/metabolismo , Interferon gama/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , DNA Viral/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
The antiviral role of the tripartite motif-containing (TRIM) protein family , a member of the E3-ubiquitin ligase family, has recently been actively studied. Hepatitis B virus (HBV) infection is a major contributor to liver diseases; however, the host factors regulated by cytokine-inducible TRIM21 to suppress HBV remain unclear. In this study, we showed the antiviral efficacy of TRIM21 against HBV in hepatoma cell lines, primary human hepatocytes isolated from patient liver tissues, and mouse model. Using TRIM21 knock-out cells, we confirmed that the antiviral effects of interferon-gamma, which suppress HBV replication, are diminished when TRIM21 is deficient. Northern blot analysis confirmed a reduction of HBV RNA levels by TRIM21. Using Luciferase reporter assay, we also discovered that TRIM21 decreases the activity of HBV enhancers, which play a crucial role in covalently closed circular DNA transcription. The participation of the RING domain and PRY-SPRY domain in the anti-HBV effect of TRIM21 was demonstrated through experiments using deletion mutants. We identified a novel interaction between TRIM21 and hepatocyte nuclear factor 4α (HNF4α) through co-immunoprecipitation assay. More specifically, ubiquitination assay revealed that TRIM21 promotes ubiquitin-mediated proteasomal degradation of HNF4α. HNF1α transcription is down-regulated as a result of the degradation of HNF4α, an activator for the HNF1α promoter. Therefore, the reduction of key HBV enhancer activators, HNF4α and HNF1α, by TRIM21 resulted in a decline in HBV transcription, ultimately leading to the inhibition of HBV replication.IMPORTANCEDespite extensive research efforts, a definitive cure for chronic hepatitis B remains elusive, emphasizing the persistent importance of this viral infection as a substantial public health concern. Although the risks associated with hepatitis B virus (HBV) infection are well known, host factors capable of suppressing HBV are largely uncharacterized. This study elucidates that tripartite motif-containing protein 21 (TRIM21) suppresses HBV transcription and consequently inhibits HBV replication by downregulating the hepatocyte nuclear factors, which are host factors associated with the HBV enhancers. Our findings demonstrate a novel anti-HBV mechanism of TRIM21 in interferon-gamma-induced anti-HBV activity. These findings may contribute to new strategies to block HBV.
Assuntos
Vírus da Hepatite B , Fator 4 Nuclear de Hepatócito , Hepatócitos , Interferon gama , Ribonucleoproteínas , Replicação Viral , Humanos , Vírus da Hepatite B/fisiologia , Animais , Camundongos , Interferon gama/farmacologia , Interferon gama/metabolismo , Hepatócitos/virologia , Hepatócitos/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hepatite B/virologia , Hepatite B/metabolismo , Células Hep G2 , Linhagem Celular TumoralRESUMO
Hepatitis B virus (HBV) is known to cause severe liver diseases such as acute or chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis B (CHB) infection is a major health problem with nearly 300 million individuals infected worldwide. Currently, nucleos(t)ide analogs (NAs) and interferon alpha are clinically approved treatments for HBV infection. NAs are potent antiviral agents that bind to HBV polymerase and block viral reverse transcription and replication. Besifovir dipivoxil maleate (BSV) is a newly developed NA against HBV in the form of acyclic nucleotide phosphonate that is available for oral administration similar to adefovir and tenofovir. Until now, resistance to BSV treatment has not been reported. In this study, we found a CHB patient who showed viral breakthrough after long-term treatment with BSV. The isolated HBV DNA from patient's serum were cloned into the replication-competent HBV 1.2 mer and the sequence of reverse transcriptase (RT) domain of HBV polymerase were analyzed. We also examined the drug susceptibility of generated clones in vitro. Several mutations were identified in HBV RT domain. A particular mutant harboring ten RT mutations showed resistance to BSV treatment in vitro. The ten mutations include rtV23I (I), rtH55R (R), rtY124H (H), rtD134E (E), rtN139K (K), rtL180M (M), rtM204V (V), rtQ267L (L), rtL269I (I) and rtL336M (M). To further identify the responsible mutations for BSV resistance, we performed in vitro drug susceptibility assay on several artificial clones. As a result, our study revealed that rtL180M (M) and rtM204V (V) mutations, already known as lamivudine-resistant mutations, confer resistance to BSV in the CHB patient.
RESUMO
Tenofovir disoproxil fumarate (TDF) has been regarded as the most potent drug for treating patients with chronic hepatitis B (CHB). However recently, viral mutations associated with tenofovir have been reported. Here, we found a CHB patient with suboptimal response after more than 4 years of TDF treatment. Clonal analysis of hepatitis B virus (HBV) isolated from sequential sera of this patient identified the seven previously reported TDF-resistant mutations (CYELMVI). Interestingly, a threonine to alanine mutation at the 301 amino acid position of the reverse-transcriptase (RT) domain, (rtT301A), was commonly accompanied with CYELMVI at a high rate (72.7%). Since the rtT301A mutation has not been reported yet, we investigated the role of this naturally occurring mutation on the viral replication and susceptibility to tenofovir in various liver cells (hepatoma cells as well as primary human hepatocytes). A cell-based phenotypic assay revealed that the rtT301A mutation dramatically impaired the replication ability with meaningful reduction in sensitivity to tenofovir in hepatoma cell lines. However, attenuated viral replication by the rtT301A mutation was significantly restored in primary human hepatocytes (PHHs). Our findings suggest that the replication capability and drug sensitivity of HBV is different between hepatoma cell lines and PHHs. Therefore, our study emphasizes that validation studies should be performed not only in the liver cancer cell lines but also in the PHHs to understand the exact viral fitness under antiviral pressure in patients.
Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Tenofovir/farmacologia , Antivirais/farmacologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Células Cultivadas , Farmacorresistência Viral/genética , Feminino , Genes Virais , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/genética , Hepatite B Crônica/virologia , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Pessoa de Meia-Idade , Mutação Puntual , DNA Polimerase Dirigida por RNA/genética , Inibidores da Transcriptase Reversa/farmacologia , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Hepatitis B virus (HBV) infection is a major factor in the development of various liver diseases such as hepatocellular carcinoma (HCC). Among HBV encoded proteins, HBV X protein (HBx) is known to play a key role in the development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor which is critical for hepatocyte differentiation. However, the expression level as well as its regulatory mechanism in HBV infection have yet to be clarified. Here, we observed the suppression of HNF4α in cells which stably express HBV whole genome or HBx protein alone, while transient transfection of HBV replicon or HBx plasmid had no effect on the HNF4α level. Importantly, in the stable HBV- or HBx-expressing hepatocytes, the downregulated level of HNF4α was restored by inhibiting the ERK signaling pathway. Our data show that HNF4α was suppressed during long-term HBV infection in cultured HepG2-NTCP cells as well as in a mouse model following hydrodynamic injection of pAAV-HBV or in mice intravenously infected with rAAV-HBV. Importantly, HNF4α downregulation increased cell proliferation, which contributed to the formation and development of tumor in xenograft nude mice. The data presented here provide proof of the effect of HBV infection in manipulating the HNF4α regulatory pathway in HCC development.
Assuntos
Carcinoma Hepatocelular/virologia , Hepatite B/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/virologia , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Hepatite B/genética , Hepatite B/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Nus , Transplante de NeoplasiasRESUMO
BACKGROUND & AIMS: Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance. METHODS: Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis. RESULTS: Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8⯱â¯0.6⯵M, whereas the IC50 values for CYE and CYEI mutants were 14.1⯱â¯1.8 and 58.1⯱â¯0.9⯵M, respectively. The IC90 value for wild-type HBV was 30⯱â¯0.5⯵M, whereas the IC90 values for CYE and CYEI mutants were 185⯱â¯0.5 and 790⯱â¯0.2⯵M, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3-778 (IC50â¯<0.4⯵M vs. IC50â¯=â¯0.4⯵M, respectively). CONCLUSIONS: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. LAY SUMMARY: Tenofovir is the most potent nucleotide analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers >10-fold resistance to tenofovir up to 8â¯years. Herein, we identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC50) and 26.3-fold (IC90) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.