Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza.

The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, toward proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degradation. Consistent with intrinsic protein instability as basis for this gain-of-function, internal structural disorder is associated with the unique cytotoxic sequences of the 1918 PB1-F2 protein. Infusing mice with recombinant DDX3 protein completely rescued them from lethal infection with the 1918 PB1-F2-producing virus. Alongside NS1 protein, 1918 PB1-F2 therefore constitutes a potent IFN antagonist causative for the severe pathogenicity of the 1918 influenza strain. Our identification of molecular determinants of pathogenesis should be useful for the future design of new antiviral strategies against influenza pandemics.

The Roles of Ubiquitination in Pathogenesis of Influenza Virus Infection.

The ubiquitin system denotes a potent post-translational modification machinery that is capable of activation or deactivation of target proteins through reversible linkage of a single ubiquitin or ubiquitin chains. Ubiquitination regulates major cellular functions such as protein degradation, trafficking and signaling pathways, innate immune response, antiviral defense, and virus replication. The RNA sensor RIG-I ubiquitination is specifically induced by influenza A virus (IAV) to activate type I IFN production. Influenza virus modulates the activity of major antiviral proteins in the host cell to complete its full life cycle. Its structural and non-structural proteins, matrix proteins and the polymerase complex can regulate host immunity and antiviral response. The polymerase PB1-F2 of mutated 1918 IAV, adapts a novel IFN antagonist function by sending the DDX3 into proteasomal degradation. Ultimately the fate of virus is determined by the outcome of interplay between viral components and host antiviral proteins and ubiquitination has a central role in the encounter of virus and its host cell.

Distinctive HBV Replication Capacity and Susceptibility to Tenofovir Induced by a Polymerase Point Mutation in Hepatoma Cell Lines and Primary Human Hepatocytes.

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.

Entecavir-resistant hepatitis B virus decreases surface antigenicity: A full genome and functional characterization.

BACKGROUND AND AIM: Since polymerase and surface genes overlap in hepatitis B virus (HBV), an antiviral-induced mutation in the polymerase gene may alter the surface antigenicity in patients with chronic hepatitis B (CHB), but this possibility has not been clearly confirmed. This study aimed to determine the drug susceptibility and surface antigenicity of the patient-derived mutants. PATIENTS AND METHODS: Full-length HBV genomes isolated from four entecavir-resistant CHB patients were cloned and sequenced. Around 10 clones of full-length HBV obtained from each patient were analysed and registered in the NCBI GenBank. Representative clones were further characterized by in vitro drug susceptibility and surface antigenicity assays. RESULTS: The rtL180M + rtM204V mutations were common among all the clones analysed. Additionally, the ETV resistance mutations rtT184A/L, rtS202G and rtM250V were found among three patients. Most of the ETV-resistant mutants had amino acid alterations within the known epitopes recognized by T- and B-cells in the HBV surface and core antigens. The in vitro drug susceptibility assay showed that all tested clones were resistant to ETV treatment. However, they were all susceptible to ADV and TDF. More importantly, the rtI169T mutation in the RT domain, led to the sF161L mutation in the overlapping S gene, which decreased in surface antigenicity. CONCLUSIONS: The ETV resistance mutations can affect the antigenicity of the HBsAg proteins due to changes in the overlapping sequence of this surface antigen. Thus, the apparent decline or disappearance of HBsAg needs to be interpreted cautiously in patients with previous or current antiviral resistance mutations.

Antiviral activity of interferon-stimulated gene 20, as a putative repressor binding to hepatitis B virus enhancer II and core promoter.

BACKGROUND AND AIM: Interferon-stimulated gene 20 (ISG20) is an interferon-inducible exonuclease that inhibits the replication of several RNA viruses. In patients with chronic hepatitis B, ISG20 expression is related to the interferon-α treatment response. However, the molecular mechanism of ISG20-mediated anti-hepatitis B virus (HBV) activity is unclear. METHODS: We have investigated the effect of ISG20 on antiviral activity to address that. The life cycle of HBV was analyzed by the ectopic expression of ISG20 in HepG2 and HepG2-NTCP cells. Finally, to provide physiological relevance of our study, the expression of ISG20 from chronic hepatitis B patients was examined. RESULTS: Interferon-stimulated gene 20 was mainly induced by interferon-ß and dramatically inhibited HBV replication. In addition, ISG20 decreased HBV gene expression and transcription. Although ISG20 inhibited HBV replication by reducing viral enhancer activity, the expression of transcription factors that bind the HBV enhancer was not affected. Particularly, ISG20 suppressed HBV enhancer activity by binding to the enhancer II and core promoter (EnhII/Cp) region. CONCLUSION: Our findings suggest that ISG20 exerts the anti-HBV activity by acting as a putative repressor binding to the HBV EnhII/Cp region.

Suppression of Hepatocyte Nuclear Factor 4 α by Long-term Infection of Hepatitis B Virus Contributes to Tumor Cell Proliferation.

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.

Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients.

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 µM, whereas the IC50 values for CYE and CYEI mutants were 14.1 ±â€¯1.8 and 58.1 ±â€¯0.9 µM, respectively. The IC90 value for wild-type HBV was 30 ±â€¯0.5 µM, whereas the IC90 values for CYE and CYEI mutants were 185 ±â€¯0.5 and 790 ±â€¯0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3-778 (IC50 <0.4 µM vs. IC50 = 0.4 µM, 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 8 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.

Multiple Functions of Cellular FLIP Are Essential for Replication of Hepatitis B Virus.

Hepatitis B virus (HBV) infection is a leading cause of liver diseases; however, the host factors which facilitate the replication and persistence of HBV are largely unidentified. Cellular FLICE inhibitory protein (c-FLIP) is a typical antiapoptotic protein. In many cases of liver diseases, the expression level of c-FLIP is altered, which affects the fate of hepatocytes. We previously found that c-FLIP and its cleaved form interact with HBV X protein (HBx), which is essential for HBV replication, and regulate diverse cellular signals. In this study, we investigated the role of endogenous c-FLIP in HBV replication and its underlying mechanisms. The knockdown of endogenous c-FLIP revealed that this protein regulates HBV replication through two different mechanisms. (i) c-FLIP interacts with HBx and protects it from ubiquitin-dependent degradation. The N-terminal DED1 domain of c-FLIP is required for HBx stabilization. (ii) c-FLIP regulates the expression or stability of hepatocyte nuclear factors (HNFs), which have critical roles in HBV transcription and maintenance of hepatocytes. c-FLIP regulates the stability of HNFs through physical interactions. We verified our findings in three HBV infection systems: HepG2-NTCP cells, differentiated HepaRG cells, and primary human hepatocytes. In conclusion, our results identify c-FLIP as an essential factor in HBV replication. c-FLIP regulates viral replication through its multiple effects on viral and host proteins that have critical roles in HBV replication.IMPORTANCE Although the chronic hepatitis B virus (HBV) infection still poses a major health concern, the host factors which are required for the replication of HBV are largely uncharacterized. Our studies identify cellular FLICE inhibitory protein (c-FLIP) as an essential factor in HBV replication. We found the dual roles of c-FLIP in regulation of HBV replication: c-FLIP interacts with HBx and enhances its stability and regulates the expression or stability of hepatocyte nuclear factors which are essential for transcription of HBV genome. Our findings may provide a new target for intervention in persistent HBV infection.

Suppression of interferon-mediated anti-HBV response by single CpG methylation in the 5'-UTR of TRIM22.

OBJECTIVE: Interferons (IFNs) mediate direct antiviral activity. They play a crucial role in the early host immune response against viral infections. However, IFN therapy for HBV infection is less effective than for other viral infections. DESIGN: We explored the cellular targets of HBV in response to IFNs using proteome-wide screening. RESULTS: Using LC-MS/MS, we identified proteins downregulated and upregulated by IFN treatment in HBV X protein (HBx)-stable and control cells. We found several IFN-stimulated genes downregulated by HBx, including TRIM22, which is known as an antiretroviral protein. We demonstrated that HBx suppresses the transcription of TRIM22 through a single CpG methylation in its 5'-UTR, which further reduces the IFN regulatory factor-1 binding affinity, thereby suppressing the IFN-stimulated induction of TRIM22. CONCLUSIONS: We verified our findings using a mouse model, primary human hepatocytes and human liver tissues. Our data elucidate a mechanism by which HBV evades the host innate immune system.

Cleaved c-FLIP mediates the antiviral effect of TNF-α against hepatitis B virus by dysregulating hepatocyte nuclear factors.

BACKGROUND & AIMS: Cytokines are key molecules implicated in the defense against virus infection. Tumor necrosis factor-alpha (TNF-α) is well known to block the replication of hepatitis B virus (HBV). However, the molecular mechanism and the downstream effector molecules remain largely unknown. METHODS: In this study, we investigated the antiviral effect and mechanism of p22-FLIP (FLICE-inhibitory protein) by ectopic expression in vitro and in vivo. In addition, to provide the biological relevance of our study, we examined that the p22-FLIP is involved in TNF-α-mediated suppression of HBV in primary human hepatocytes. RESULTS: We found that p22-FLIP, a newly discovered c-FLIP cleavage product, inhibited HBV replication at the transcriptional level in both hepatoma cells and primary human hepatocytes, and that c-FLIP conversion to p22-FLIP was stimulated by the TNF-α/NF-κB pathway. p22-FLIP inhibited HBV replication through the upregulation of HNF3ß but downregulation of HNF4α, thus inhibiting both HBV enhancer elements. Finally, p22-FLIP potently inhibited HBV DNA replication in a mouse model of HBV replication. CONCLUSIONS: Taken together, these findings suggest that the anti-apoptotic p22-FLIP serves a novel function of inhibiting HBV transcription, and mediates the antiviral effect of TNF-α against HBV replication.

Novel role of MHC class II transactivator in hepatitis B virus replication and viral counteraction.

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.

Gender-specific alteration of steroid metabolism and its impact on viral replication in a mouse model of hepatitis B virus infection.

Hepatitis B virus (HBV) is a sex-specific pathogen that is more severe in males than in females. Sex disparities in HBV infection have been attributed to hormonal differences between males and females. However, whether HBV infection affects the metabolic signatures of steroid hormones and how these influences viral replication remains unclear. In this study, we investigated whether HBV infection alters steroid metabolism and its effects on HBV replication. Serum samples from male and female mice obtained after the hydrodynamic injection of replication-competent HBV plasmids were subjected to quantitative steroid profiling. Serum steroid levels in mice were analyzed using an in vitro metabolism assay with the mouse liver S9 fraction. The alteration of steroids by HBV infection was observed only in male mice, particularly with significant changes in androgens, whereas no significant hormonal changes were observed in female mice. Among the altered steroids, dehydroepiandrosterone (DHEA) levels increased the most in male mice after HBV infection. An in vitro metabolism assay revealed that androgen levels were significantly reduced in HBV-infected male mice. Furthermore, the genes involved in DHEA biosynthesis were significantly upregulated in HBV-infected male mice. Interestingly, reduced dihydrotestosterone in male mice significantly inhibits viral replication by suppressing HBV promoter activity, suggesting a viral strategy to overcome the antiviral effects of steroid hormones in males. Our data demonstrated that HBV infection can cause sex-specific changes in steroid metabolism.

Antinociceptive and anti-inflammatory activity of DW-1021, the ionic complex of pelubiprofen and tramadol, in rodents.

Although drugs such as acetaminophen, opioids, and nonsteroidal anti-inflammatory drugs (NSAIDs), are commonly used for pain management, the side effects of these drugs such as hepatotoxicity, nephrotoxicity, nausea, and vomiting, can not be neglected. Therefore, combinations of analgesics with different mechanisms raise the possibility of developing novel analgesics. Therefore, the aim of the present study was to evaluate whether DW-1021, the ionic complex of pelubiprofen (NSAID) and tramadol (opioid), has synergic antinociceptive and anti-inflammatory effects in nociceptive as well as inflammation-induced nociceptive models compared to pelubiprofen- or tramadol-only administration. Strong synergistic antinociceptive efficacy of DW-1021 was observed in the mouse writhing test and von Frey paw withdrawal threshold test in the carrageenan-induced rats. The hot plate test in mice and the Randall-Selitto mechanical paw pressure test in carrageenan-induced rats revealed that DW-1021 had a preferable effect on relieving pain to pelubiprofen, but not as much as tramadol. In the carrageenan-induced rats, DW-1021 had a more potent effect on reducing paw inflammation (paw volume, width, and thickness) via the suppression of PGE2 production than tramadol, but less than that of pelubiprofen. Taken together, our results suggest that the administration of DW-1021, a combination of pelubiprofen and tramadol, exerted a potent effect and can be used as a potential therapeutic agent for relieving pain and inflammation.

Interaction between the Hepatitis B Virus and Cellular FLIP Variants in Viral Replication and the Innate Immune System.

During viral evolution and adaptation, many viruses have utilized host cellular factors and machinery as their partners. HBx, as a multifunctional viral protein encoded by the hepatitis B virus (HBV), promotes HBV replication and greatly contributes to the development of HBV-associated hepatocellular carcinoma (HCC). HBx interacts with several host factors in order to regulate HBV replication and evolve carcinogenesis. The cellular FADD-like IL-1ß-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) is a major factor that functions in a variety of cellular pathways and specifically in apoptosis. It has been shown that the interaction between HBx and c-FLIP determines HBV fate. In this review, we provide a comprehensive and detailed overview of the interplay between c-FLIP and HBV in various environmental circumstances. We describe strategies adapted by HBV to establish its chronic infection. We also summarize the conventional roles of c-FLIP and highlight the functional outcome of the interaction between c-FLIP and HBV or other viruses in viral replication and the innate immune system.

Dysregulation of Liver Regeneration by Hepatitis B Virus Infection: Impact on Development of Hepatocellular Carcinoma.

The liver is unique in its ability to regenerate in response to damage. The complex process of liver regeneration consists of multiple interactive pathways. About 2 billion people worldwide have been infected with hepatitis B virus (HBV), and HBV causes 686,000 deaths each year due to its complications. Long-term infection with HBV, which causes chronic inflammation, leads to serious liver-related diseases, including cirrhosis and hepatocellular carcinoma. HBV infection has been reported to interfere with the critical mechanisms required for liver regeneration. In this review, the studies on liver tissue characteristics and liver regeneration mechanisms are summarized. Moreover, the inhibitory mechanisms of HBV infection in liver regeneration are investigated. Finally, the association between interrupted liver regeneration and hepatocarcinogenesis, which are both triggered by HBV infection, is outlined. Understanding the fundamental and complex liver regeneration process is expected to provide significant therapeutic advantages for HBV-associated hepatocellular carcinoma.

Susceptibility of Drug Resistant Hepatitis B Virus Mutants to Besifovir.

Currently, interferon alpha and nucleos(t)ide analogues (NAs) are clinically available to treat hepatitis B virus (HBV) infection. Several NAs, including lamivudine (LMV), adefovir (ADV), entecavir (ETV) and tenofovir (TDF or TAF) have been approved and administered to chronic hepatitis B (CHB) patients. NAs inhibit HBV DNA synthesis by targeting the reverse transcriptase (RT) domain of HBV polymerase. Several mutations in the RT domain which lead to drug resistance against NAs have been reported, even for TDF and TAF which are highly potent with very low resistance rate. Besifovir (BFV) is a new antiviral dGMP analogue able to be used as a new NA drug for the control of CHB infection. Drug resistance to BFV is not well known due to its shorter duration of clinical use. Recently, we reported that rtL180M (M) and rtM204V (V) mutations, already resistant to LMV, are associated with BFV resistance. However, the susceptibility to BFV of previously known HBV mutants resistant to various drugs has not been studied. To investigate this, we performed in vitro drug susceptibility assays using natural and artificial mutants that are associated with resistance to LMV, ADV, ETV or TDF. As a result, LMV-resistant mutants were not susceptible to BFV and ETV-resistant clones showed partial resistance against BFV as well. However, ADV-resistant mutants were highly sensitive to BFV. In case of tenofovir-resistant mutations, the HBV mutants harboring primary mutations to tenofovir resistance were susceptible to BFV. Therefore, our study revealed that BSV may serve as an alternative drug for patients with ADV-, ETV-, TDF- or TAF-resistance.

Identification and Characterization of Besifovir-Resistant Hepatitis B Virus Isolated from a Chronic Hepatitis B Patient.

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.

Hepatocyte Growth Factor-Dependent Antiviral Activity of Activated cdc42-Associated Kinase 1 Against Hepatitis B Virus.

Activated cdc42-associated kinase 1 (ACK1) is a well-known non-receptor tyrosine kinase that regulates cell proliferation and growth through activation of cellular signaling pathways, including mitogen-activated protein kinase (MAPK). However, the anti-HBV activity of ACK1 has not been elucidated. This study aimed to investigate the role of ACK1 in the HBV life cycle and the mechanism underlying the anti-HBV activity of ACK1. To examine the antiviral activity of ACK1, we established HepG2-ACK1 cells stably overexpressing ACK1. The HBV life cycle, including HBeAg/HBsAg secretion, HBV DNA/transcription, and enhancer activity, was analyzed in HepG2 and HepG2-ACK1 cells with HBV replication-competent HBV 1.2mer (HBV 1.2). Finally, the anti-HBV activity of ACK1 was examined in an HBV infection system. ACK1 suppressed HBV gene expression and transcription in HepG2 and HepG2-ACK1 cells. Furthermore, ACK1 inhibited HBV replication by decreasing viral enhancer activity. ACK1 exhibited its anti-HBV activity via activation of Erk1/2, which consequently downregulated the expression of HNF4α binding to HBV enhancers. Furthermore, hepatocyte growth factor (HGF) induced ACK1 expression at an early stage. Finally, ACK1 mediated the antiviral effect of HGF in the HBV infection system. These results indicated that ACK1 induced by HGF inhibited HBV replication at the transcriptional level by activating the MAPK-HNF signaling pathway. Our findings suggest that ACK1 is a potentially novel upstream molecule of MAPK-mediated anti-HBV activity.

Direct Detection of Drug-Resistant Hepatitis B Virus in Serum Using a Dendron-Modified Microarray.

BACKGROUND/AIMS: Direct sequencing is the gold standard for the detection of drug-resistance mutations in hepatitis B virus (HBV); however, this procedure is time-consuming, labor-intensive, and difficult to adapt to high-throughput screening. In this study, we aimed to develop a dendron-modified DNA microarray for the detection of genotypic resistance mutations and evaluate its efficiency. METHODS: The specificity, sensitivity, and selectivity of dendron-modified slides for the detection of representative drug-resistance mutations were evaluated and compared to those of conventional slides. The diagnostic accuracy was validated using sera obtained from 13 patients who developed viral breakthrough during lamivudine, adefovir, or entecavir therapy and compared with the accuracy of restriction fragment mass polymorphism and direct sequencing data. RESULTS: The dendron-modified slides significantly outperformed the conventional microarray slides and were able to detect HBV DNA at a very low level (1 copy/µL). Notably, HBV mutants could be detected in the chronic hepatitis B patient sera without virus purification. The validation of our data revealed that this technique is fully compatible with sequencing data of drug-resistant HBV. CONCLUSIONS: We developed a novel diagnostic technique for the simultaneous detection of several drug-resistance mutations using a dendron-modified DNA microarray. This technique can be directly applied to sera from chronic hepatitis B patients who show resistance to several nucleos(t)ide analogues.