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.

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.

The impact of the hepatitis B virus polymerase rtA181T mutation on replication and drug resistance is potentially affected by overlapping changes in surface gene.

UNLABELLED: The emergence of drug-resistant hepatitis B virus (HBV) is a major problem for antiviral treatment in chronic hepatitis B infection. In this study, we analyzed the evolution of drug-resistant mutations and characterized the effects of the rtA181T and rtI233V mutations on viral replication and drug resistance. We performed a clonal analysis of the HBV polymerase gene from serum samples during viral breakthrough treated with antiviral agents. A series of mutant clones containing rtA181T and/or rtI233V mutations were constructed and determined the effect of these mutations on the replication ability and drug resistance. An in vitro study revealed that the effect of the rtA181T mutation on viral replication and drug resistance is dependent on the mutations in the overlapping surface gene. Compared to the rtA181T surface missense mutation (rtA181T/sW172S), the introduction of rtA181T surface nonsense mutation (rtA181T/sW172*) resulted in decreased viral replication and increased drug resistance. Complementation assay revealed that the truncated PreS1 is responsible for reduced replication of rtA181T/sW172* mutant. Moreover, the rtA181T/sW172* mutant exhibited a defect in viral particle secretion. The rtI233V mutation that emerged during adefovir therapy reduced viral replication and conferred resistance to adefovir. Our data suggest that the impact of the rtA181T mutation on replication and drug resistance differs based on the mutation status of the corresponding surface gene. The rtI233V mutation also affects replication ability and drug resistance. This observation suggests the need for genotypic analysis of overlapping surface genes to manage antiviral drug resistance if clinical isolates harbor the rtA181T mutation. IMPORTANCE: The emergence of drug-resistant HBV that are no longer susceptible to nucleos(t)ide analogues is a major problem for antiviral treatment in chronic hepatitis B infection. Among drug-resistant mutations, the single rtA181T mutation is known to confer cross-resistance to antiviral drugs. This mutation causes intermediate or reduced susceptibility to tenofovir. Moreover, the clinical occurrence of the rtA181T mutation during antiviral therapy is also high. Our study revealed that the effect of the rtA181T mutation on viral replication and drug resistance is dependent on the mutations in the overlapping surface gene. This observation suggests the need for genotypic analysis of overlapping surface genes to manage antiviral drug resistance if clinical isolates harbor the rtA181T mutation. We believe that our study will not only extend the understanding of the drug resistance mechanism, but it will also ultimately provide new treatment options for patients with multidrug resistant HBV.

Hepatitis B virus inhibits liver regeneration via epigenetic regulation of urokinase-type plasminogen activator.

UNLABELLED: Liver regeneration after liver damage caused by toxins and pathogens is critical for liver homeostasis. Retardation of liver proliferation was reported in hepatitis B virus (HBV) X protein (HBx)-transgenic mice. However, the underlying mechanism of the HBx-mediated disturbance of liver regeneration is unknown. We investigated the molecular mechanism of the inhibition of liver regeneration using liver cell lines and a mouse model. The mouse model of acute HBV infection was established by hydrodynamic injection of viral DNA. Liver regeneration after partial hepatectomy was significantly inhibited in the HBV DNA-treated mice. Mechanism studies have revealed that the expression of urokinase-type plasminogen activator (uPA), which regulates the activation of hepatocyte growth factor (HGF), was significantly decreased in the liver tissues of HBV or HBx-expressing mice. The down-regulation of uPA was further confirmed using liver cell lines transiently or stably transfected with HBx and the HBV genome. HBx suppressed uPA expression through the epigenetic regulation of the uPA promoter in mouse liver tissues and human liver cell lines. Expression of HBx strongly induced hypermethylation of the uPA promoter by recruiting DNA methyltransferase (DNMT) 3A2. CONCLUSION: Taken together, these results suggest that infection of HBV impairs liver regeneration through the epigenetic dysregulation of liver regeneration signals by HBx.

Pre-S mutations of hepatitis B virus affect genome replication and expression of surface antigens.

BACKGROUNDS AND AIMS: In chronic hepatitis B virus (HBV) infection, quantitative HBV surface antigen (qHBsAg) is useful for monitoring viral replication and treatment responses. We aimed to determine whether pre-S mutations have any effect on circulating qHBsAg. METHODS: Plasmids expressing 1­8 amino acid deletion in pre-S1 ("pre-S1Δ1-8") and 3-25 amino acid deletion in pre-S2 ("pre-S2Δ3-25") were constructed. At 72 h posttransfection into Huh7 cells, qHBsAg were measured using electrochemiluminescence immunoassay analyzer. To mimic milieus of quasispecies, we co-transfected either pre-S1Δ1-8 or pre-S2Δ3-25 with wild type (WT). RESULTS: Pre-S mutations affected transcription and replication ability of HBV because of altered overlapping polymerase. Compared with WT, extracellular qHBsAg in pre-S1Δ1-8 and pre-S2Δ3-25 were on average 3.87-fold higher and 0.92-fold lower, respectively, whereas intracellular qHBsAg in pre-S1Δ1-8 and pre-S2Δ3-25 were 0.57-fold lower and 1.60-fold higher, respectively. Immunofluorescence staining of cellular HBsAg showed that pre-S1Δ1-8 had less staining and that pre-S2Δ3-25 had denser staining. As ratios of either pre-S1Δ1-8 or pre-S2Δ3-25:WT increased from 0:10 to 10:0 gradually, relative extracellular qHBsAg increased from 1.0 to 3.85 in pre-S1Δ1-8 co-transfection, whereas those decreased from 1.0 to 0.88 in pre-S2Δ3-25 co-transfection. CONCLUSION: Pre-S mutations exhibit different phenotypes of genome replication and HBsAg expression according to their locations. Thus, qHBsAg level for diagnosis and prognostification in chronic HBV infection should be used more cautiously, considering emergences of pre-S deletion mutants.

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.

Identification and characterization of clevudine-resistant mutants of hepatitis B virus isolated from chronic hepatitis B patients.

Clevudine (CLV) is a nucleoside analog with potent antiviral activity against chronic hepatitis B virus (HBV) infection. Viral resistance to CLV in patients receiving CLV therapy has not been reported. The aim of this study was to characterize CLV-resistant HBV in patients with viral breakthrough (BT) during long-term CLV therapy. The gene encoding HBV reverse transcriptase (RT) was analyzed from chronic hepatitis B patients with viral BT during CLV therapy. Sera collected from the patients at baseline and at the time of viral BT were studied. To characterize the mutations of HBV isolated from the patients, we subjected the HBV mutants to in vitro drug susceptibility assays. Several conserved mutations were identified in the RT domain during viral BT, with M204I being the most common. In vitro phenotypic analysis showed that the mutation M204I was predominantly associated with CLV resistance, whereas L229V was a compensatory mutation for the impaired replication of the M204I mutant. A quadruple mutant (L129M, V173L, M204I, and H337N) was identified that conferred greater replicative ability and strong resistance to both CLV and lamivudine. All of the CLV-resistant clones were lamivudine resistant. They were susceptible to adefovir, entecavir, and tenofovir, except for one mutant clone. In conclusion, the mutation M204I in HBV RT plays a major role in CLV resistance and leads to viral BT during long-term CLV treatment. Several conserved mutations may have a compensatory role in replication. Drug susceptibility assays reveal that adefovir and tenofovir are the most effective compounds against CLV-resistant mutants. These data may provide additional therapeutic options for CLV-resistant patients.

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.

Association between polymorphism of tumor necrosis factor-alpha promoter and response to lamivudine treatment in patients with chronic hepatitis B.

BACKGROUND: TNF-alpha promoter polymorphism is known to play an important role in the immunopathogenesis of infection of hepatitis B virus. AIMS: We investigated whether polymorphisms of TNF-alpha promoter at position -308 or -238 had associations with the response to lamivudine treatment. METHODS: A total of 89 healthy subjects (control group) and 225 patients with chronic hepatitis B treated with lamivudine were included in this study. Polymorphisms of TNF-alpha promoter at position -308 and -238 were analyzed by polymerase chain reaction. Recruited patients were classified according to the outcome of lamivudine treatment into the responder (103 patients) or non-responder (122 patients) group. RESULTS: The numbers of A allelic polymorphism of TNF-alpha promoter at position -238 were four (2.2%) in the control, five (2.4%) in the responder and 19 (7.8%) in the non-responder group. The A allele was noted significantly more frequently in the responder than non-responder group (P = 0.012). At position -308, a significant difference was observed between the control group (14; 7.9%) and total chronic hepatitis B patients (15; 3.3%) (P = 0.015). CONCLUSIONS: Our study demonstrated that the non-response to lamivudine treatment in patients with chronic hepatitis B might be related to the A allelic polymorphism of TNF-alpha promoter at position -238.

Meta-analysis of gene expression profiles in long-term non-progressors infected with HIV-1.

BACKGROUND: In the absence of antiretroviral treatments (ARTs), a small group of individuals infected with HIV, including long-term non-progressors (LTNPs) who maintain high levels of CD4+ T cells for more than 7-10 years in the absence of ART and in particular a subgroup of LTNPs, elite controllers (ECs), who have low levels of viremia, remain clinically and/or immunologically stable for years. However, the mechanism of stable disease progression in LTNPs and ECs needs to be elucidated to help those infected with HIV-1 remain healthy. In this study, to identify the characteristics of gene expression profiles and biomarkers in LTNPs, we performed a meta-analysis using multiple gene expression profiles among LTNPs, individuals infected with HIV-1 without ART, individuals infected with HIV-1 with ART, and healthy controls. METHODS: The gene expression profiles obtained from the Gene Expression Omnibus (GEO) microarray data repositories were classified into three groups: LTNPs versus healthy controls (first group, 3 studies), LTNPs versus patients infected with HIV-1 without ART (second group, 3 studies), and LTNPs versus patients infected with HIV-1 with ART (third group, 3 studies). In addition, we considered a fourth group, patients infected with HIV-1 without ART versus healthy controls (3 studies), to exclude genes associated with HIV-1 infection in the three groups. For each group, we performed a meta-analysis using the RankProd method to identify and compare the differentially expressed genes (DEGs) in the three groups. RESULTS: We identified the 14 common DEGs in the three groups when comparing them with each other. Most belonged to immune responses, antigen processing and presentation, the interferon-gamma-mediated signaling pathway, and T cell co-stimulation. Of these DEGs, PHLDA1 was up-regulated and ACTB and ACTG1 were down-regulated in all three groups. However, the rest of the up- or down-regulated genes were discordant in the three groups. Additionally, ACTB and ACTG1 are known to inhibit viral assembly and production, and THBS1 is known to inhibit HIV-1 infection. CONCLUSIONS: These results suggest that significant genes identified in a meta-analysis provide clues to the cause of delayed disease progression and give a deeper understanding of HIV pathogenesis in LTNPs.

MDM2 and p53 polymorphisms are associated with the development of hepatocellular carcinoma in patients with chronic hepatitis B virus infection.

A single-nucleotide polymorphism (SNP) in the promoter region of MDM2, SNP 309, is associated with hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus infection. The effect of p53 codon 72 polymorphism Arg72Pro on HCC risk remains inconsistent. This study evaluated the association of MDM2 and p53 polymorphisms with the presence and early onset of HCC in Korean patients with chronic hepatitis B virus (HBV) infection. In total, 583 consecutive patients with chronic HBV infection were classified according to the presence (n = 287) or absence (n = 296) of HCC. The MDM2 SNP 309 and p53 Arg72Pro were genotyped using restriction fragment length polymorphism method. The MDM2 G/G and p53 Pro/Pro genotypes were more frequent in HCC group than in non-HCC group (P < 0.001 and P = 0.004, respectively). Multivariate analysis for the presence of HCC revealed that the odds ratio (OR) for MDM2 G/G over T/T was 4.89 (P < 0.001) and that of p53 Pro/Pro over Arg/Arg was 3.03 (P = 0.006). Combined MDM2 G/G and p53 Pro/Pro had a synergistic effect on HCC risk, with an OR of 20.78 (P < 0.001). The mean age of tumor onset in patients with MDM2 G/G genotype was 50.9 years compared with 55.1 with T/T genotype (P = 0.018) and that with p53 Pro/Pro was 49.7 years compared with 52.9 with Arg/Arg (P = 0.040). Thus, MDM2 SNP 309 and p53 Arg72Pro are associated with the early development of HCC in Korean patients with chronic HBV infection.

Combination therapy of thymosin alpha-1 and lamivudine for HBeAg positive chronic hepatitis B: A prospective randomized, comparative pilot study.

BACKGROUND AND AIM: Monotherapy of lamivudine, interferon-alpha (IFN-alpha), and thymosin alpha-1 (Talpha1) is unlikely to be sufficient for the eradication of a chronic hepatitis B virus (HBV) infection. The aim of our study is to elucidate whether the combination of Talpha1 and lamivudine is superior to lamivudine monotherapy in hepatitis B e antigen (HBeAg) positive naïve patients with chronic hepatitis B. METHODS: Sixty-seven patients were assigned to two different groups in a randomized manner. The combination group (n = 34) received Talpha1 (1.6 mg subcutaneously, twice a week) and lamivudine (100 mg orally, daily) for 24 weeks, followed by continuous lamivudine therapy. The monotherapy group (n = 33) received lamivudine monotherapy continuously. RESULTS: The incidence of HBeAg seroconversion at 24 weeks was 26.5% (9/34) in the combination group and 6.1% (2/33) in the monotherapy group (P = 0.024). However, there was no statistically significant difference between 26.5% (9/34) in the combination group and 12.1% (4/33) in the monotherapy group at 52 weeks (P = 0.138). The emergence of viral breakthrough gradually increased to 35.3% (12/34) in the combination group, and to 21.2% (7/33) in the monotherapy group at 52 weeks (P = 0.201). CONCLUSIONS: The combination treatment of Talpha1 and lamivudine did not have an obvious benefit of virological and biochemical response as compared to the lamivudine monotherapy during the combination period. In addition, after the cessation of Talpha1 treatment, the combination therapy did not prevent the occurrence of viral and biochemical breakthroughs.

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.

Intracellular interleukin-32γ mediates antiviral activity of cytokines against hepatitis B virus.

Cytokines are involved in early host defense against pathogen infections. In particular, tumor necrosis factor (TNF) and interferon-gamma (IFN-γ) have critical functions in non-cytopathic elimination of hepatitis B virus (HBV) in hepatocytes. However, the molecular mechanisms and mediator molecules are largely unknown. Here we show that interleukin-32 (IL-32) is induced by TNF and IFN-γ in hepatocytes, and inhibits the replication of HBV by acting intracellularly to suppress HBV transcription and replication. The gamma isoform of IL-32 (IL-32γ) inhibits viral enhancer activities by downregulating liver-enriched transcription factors. Our data are validated in both an in vivo HBV mouse model and primary human hepatocytes. This study thus suggests that IL-32γ functions as intracellular effector in hepatocytes for suppressing HBV replication to implicate a possible mechanism of non-cytopathic viral clearance.