Increased Hepatitis B Virus Replication Research Articles

The hepatic GABAergic system promotes liver macrophage M2 polarization and mediates HBV replication in mice

Macrophages display functional phenotypic plasticity. Hepatitis B virus (HBV) infection induces polarizations of liver macrophages either to M1-like pro-inflammatory phenotype or to M2-like anti-inflammatory phenotype. Gamma-aminobutyric acid (GABA) signaling exists in various non-neuronal cells including hepatocytes and some immune cells. Here we report that macrophages express functional GABAergic signaling components and activation of type A GABA receptors (GABAARs) promotes M2-polarization thus advancing HBV replication. Notably, intraperitoneal injection of GABA or the GABAAR agonist muscimol increased HBV replication in HBV-carrier mice that were generated by hydrodynamical injection of adeno-associated virus/HBV1.2 plasmids (pAAV/HBV1.2). The GABA-augmented HBV replication in HBV-carrier mice was significantly reduced by the GABAAR inhibitor picrotoxin although picrotoxin had no significant effect on serum HBsAg levels in control HBV-carrier mice. Depletion of liver macrophages by liposomal clodronate treatment also significantly reduced the GABA-augmented HBV replication. Yet adoptive transfer of liver macrophages isolated from GABA-treated donor HBV-carrier mice into the liposomal clodronate-pretreated recipient HBV-carrier mice restored HBV replication. Moreover, GABA or muscimol treatment increased the expression of “M2” cytokines in macrophages, but had no direct effect on HBV replication in the HepG2.2.15 cells, HBV1.3-transfected Huh7, HepG2, or HepaRG cells, or HBV-infected Huh7-NTCP cells. Taken together, these results suggest that increasing GABA signaling in the liver promotes HBV replication in HBV-carrier mice by suppressing the immunity of liver macrophages, but not by increasing the susceptibility of hepatocytes to HBV infection. Our study shows that a previously unknown GABAergic system in liver macrophage has an essential role in HBV replication.

Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity.

Of 350 million people worldwide are chronically infected with hepatitis B virus (HBV) and are at risk of developing cirrhosis and hepatocellular carcinoma (HCC) later in life. HBV is the most diverse DNA virus, and its genome is composed of four open reading frames: Presurface antigen/surface antigen gene (preS/S), precore/core gene (preC/C), polymerase gene (P), and the X gene (X). HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate. The virus has 10 genotypes (A to J) with different geographical distributions. There are various HBV mutations in the HBV genome, including preC/C mutations, preS/S mutations, P gene mutations, and X gene mutations. The core promoter region plays a vital part in the replication, morphogenesis and pathogenesis of the virus. The precore region also plays a crucial role in viral replication. Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity. preC/C mutations are associated with liver disease progression. Precore mutations stop hepatitis B e antigen (HBeAg) production and basal core promoter mutations downregulate HBeAg production. Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC. The emergence of antiviral-resistant mutations is the main reason for treatment failure. This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity. Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.

CUL4B facilitates HBV replication by promoting HBx stabilization.

Objective:Hepatitis B virus (HBV) infection is a major public health problem worldwide. However, the regulatory mechanisms underlying HBV replication remain unclear. Cullin 4B-RING ubiquitin E3 ligase (CRL4B) is involved in regulating diverse physiological and pathophysiological processes. In our study, we aimed to explain the role of CUL4B in HBV infection.Methods:Cul4b transgenic mice or conditional knockout mice, as well as liver cell lines with CUL4B overexpression or knockdown, were used to assess the role of CUL4B in HBV replication. Immunoprecipitation assays and immunofluorescence staining were performed to study the interaction between CUL4B and HBx. Cycloheximide chase assays and in vivo ubiquitination assays were performed to evaluate the half-life and the ubiquitination status of HBx.Results:The hydrodynamics-based hepatitis B model in Cul4b transgenic or conditional knockout mice indicated that CUL4B promoted HBV replication (P < 0.05). Moreover, the overexpression or knockdown system in human liver cell lines validated that CUL4B increased HBV replication in an HBx-dependent manner. Importantly, immunoprecipitation assays and immunofluorescence staining showed an interaction between CUL4B and HBx. Furthermore, CUL4B upregulated HBx protein levels by inhibiting HBx ubiquitination and proteasomal degradation (P < 0.05). Finally, a positive correlation between CUL4B expression and HBV pgRNA level was observed in liver tissues from HBV-positive patients and HBV transgenic mice.Conclusions:CUL4B enhances HBV replication by interacting with HBx and disrupting its ubiquitin-dependent proteasomal degradation. CUL4B may therefore be a potential target for anti-HBV therapy.

When viruses collide: hepatitis B virus reactivation after hepatitis C treatment.

Treatment for hepatitis C virus (HCV) with direct-acting antivirals (DAAs) in hepatitis B virus (HBV) coinfection can result in HBV reactivation. In this issue of the JCI, Cheng and colleagues explored the role of interferon signaling in the complex interaction between HBV and HCV using cell lines, mouse models, and samples from people with coinfection. Notably, HCV enhanced interferon signaling, as measured by interferon-stimulated gene (ISG) expression, and decreased HBV transcription and replication. Blockade of interferon signaling reversed the effects on HBV replication. Further, pharmacologic inhibition of HCV replication in vitro and in coinfected humanized mice also reduced interferon signaling and, correspondingly, increased HBV replication. Intriguingly, baseline serum levels of the ISG CXCL10 predicted HBV reactivation in a cohort of coinfected people taking DAAs. Determining how interferon signaling silences HBV transcription and whether serum CXCL10 predicts HBV reactivation in a clinical setting are questions that warrant further investigation.

Hepatitis B Virus HBx Protein Mediates the Degradation of Host Restriction Factors through the Cullin 4 DDB1 E3 Ubiquitin Ligase Complex.

The hepatitis B virus (HBV) regulatory HBx protein is required for infection, and its binding to cellular damaged DNA binding protein 1 (DDB1) is critical for this function. DDB1 is an adaptor protein for the cullin 4A Really Interesting New Gene (RING) E3 ubiquitin ligase (CRL4) complex and functions by binding cellular DDB1 cullin associated factor (DCAF) receptor proteins that recruit substrates for ubiquitination and degradation. We compared the proteins found in the CRL4 complex immunoprecipitated from uninfected versus HBV-infected hepatocytes from human liver chimeric mice for insight into mechanisms by which HBV and the cell interact within the CRL4 complex. Consistent with its role as a viral DCAF, HBx was found in the HBV CRL4 complexes. In tissue culture transfection experiments, we showed that HBx expression led to decreased levels of known restriction factor structural maintenance of chromosomes protein 6 (SMC6) and putative restriction factors stromal interaction molecule 1 (STIM1, zinc finger E-box binding homeobox 2 (ZEB2), and proteasome activator subunit 4 (PSME4). Moreover, silencing of these proteins led to increased HBV replication in the HepG2-sodium taurocholate cotransporting polypeptide (NTCP) infection model. We also identified cellular DCAF receptors in CRL4 complexes from humanized mice. Increasing amounts of HBx did not reveal competitive DCAF binding to cullin4 (CUL4)-DDB1 in plasmid-transfected cells. Our results suggest a model in which HBx benefits virus replication by directly or indirectly degrading multiple cellular restriction factors.

Role of alcohol in pathogenesis of hepatitis B virus infection.

Hepatitis B virus (HBV) and alcohol abuse often contribute to the development of end-stage liver disease. Alcohol abuse not only causes rapid progression of liver disease in HBV infected patients but also allows HBV to persist chronically. Importantly, the mechanism by which alcohol promotes the progression of HBV-associated liver disease are not completely understood. Potential mechanisms include a suppressed immune response, oxidative stress, endoplasmic reticulum and Golgi apparatus stresses, and increased HBV replication. Certainly, more research is necessary to gain a better understanding of these mechanisms such that treatment(s) to prevent rapid liver disease progression in alcohol-abusing HBV patients could be developed. In this review, we discuss the aforementioned factors for the higher risk of liver diseases in alcohol-induced HBV pathogenies and suggest the areas for future studies in this field.

Hepatitis C virus infection suppresses hepatitis B virus replication via the RIG-I-like helicase pathway

Mechanisms of hepatitis B virus (HBV) reactivation after hepatitis C virus (HCV) elimination by direct-acting antiviral (DAA) treatment in HBV/HCV-co-infected patients remain unclear. We examined RIG-I-like helicase (RLH) pathway activation by HBV mono-infection, HCV mono-infection or HBV/HCV co-infection and interference between HBV and HCV in primary human hepatocytes. Interference between HBV and HCV and HBV reactivation after DAA treatment in humanized-liver mice were assessed. HCV infection activated RLH pathway, as evidenced by RIG-I, ISG15 and ISG56 expression induction; HBV caused only RIG-I induction in vitro. RLH activation was also found in HBV/HCV-co-infected cells, and HBV replication were suppressed in HBV/HCV-co-infected than in HBV-mono-infected cells. siRNA-mediated double knockdown of ISG15 and ISG56 increased HBV replication in HBV/HCV-co-infected cells. HCV infection activated RLH pathway and suppressed HBV replication in humanized-liver mice. Subsequent elimination of HCV by DAA administration downregulated RLH pathway and upregulated HBV replication in mice. RLH pathway was activated in livers of chronic hepatitis C patients compared to those of chronic hepatitis B or non-B, non-C patients. The RLH pathway activation was downregulated by HCV elimination. In conclusion, HCV infection activated RLH pathway and suppressed HBV replication in human hepatocytes. HCV elimination upregulated HBV replication, probably through RLH pathway downregulation.

ATM and ATR Expression Potentiates HBV Replication and Contributes to Reactivation of HBV Infection upon DNA Damage.

Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.

Ovarian HBV replication following ovulation induction in female hepatitis B carriers undergoing IVF treatment: A prospective observational study.

Hepatitis B virus (HBV) can be found in ovarian tissues. This study compared HBV DNA levels in follicular fluid collected during oocyte retrieval with paired serum samples in HBV carriers after ovarian stimulation during IVF treatment for infertility. Sixty-four HBV carrier women referred to the Assisted Reproductive Units of two Hong Kong hospitals were recruited. At oocyte retrieval, the follicular fluid aspirated from the first follicle was collected for study. In 22 women, the first follicular fluid sample from both ovaries was similarly collected and studied. These women were also tested for liver function test and HBeAg. In 28 (43.8%) women, HBV DNA was detected in follicular fluid and the level correlated with serum levels (Spearman's correlation P<.001). There was concordant detection of HBV DNA in both ovaries, and the levels were significantly correlated (Spearman's correlation P=.029). In 40% of women with FF HBV DNA, the follicular fluid:serum ratio was >1.0, suggesting stimulation of HBV replication. These women also had significantly different liver function test results. Increased HBV replication exists in 40% of women with HBV DNA detected in follicular undergoing ovarian stimulation during IVF treatment.

Peretinoin, an Acyclic Retinoid, Inhibits Hepatitis B Virus Replication by Suppressing Sphingosine Metabolic Pathway In Vitro.

Hepatocellular carcinoma (HCC) frequently develops from hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently closed circular DNA (cccDNA) were evaluated by a qPCR method in HepG2.2.15 cells. Peretinoin significantly reduced the levels of intracellular HBV-DNA, nuclear cccDNA, and HBV transcript at a concentration that did not induce cytotoxicity. Conversely, other retinoids, such as 9-cis, 13-cis retinoic acid (RA), and all-trans-retinoic acid (ATRA), had no effect or rather increased HBV replication. Mechanistically, although peretinoin increased the expression of HBV-related transcription factors, as observed for other retinoids, peretinoin enhanced the binding of histone deacetylase 1 (HDAC1) to cccDNA in the nucleus and negatively regulated HBV transcription. Moreover, peretinoin significantly inhibited the expression of SPHK1, a potential inhibitor of HDAC activity, and might be involved in hepatic inflammation, fibrosis, and HCC. SPHK1 overexpression in cells cancelled the inhibition of HBV replication induced by peretinoin. This indicates that peretinoin activates HDAC1 and thereby suppresses HBV replication by inhibiting the sphingosine metabolic pathway. Therefore, peretinoin may be a novel therapeutic agent for HBV replication and chemoprevention against HCC.

Proteomics Based Identification of Autotaxin As An Anti-Hepatitis B Virus Factor and a Promoter of Hepatoma Cell Invasion and Migration

Background/Aims: Hepatitis B virus (HBV) infection is a major cause of cirrhosis and hepatocellular carcinoma. Therefore, we aimed to obtain further information on HBV pathogenesis, and to search for novel putative molecules for anti-HBV therapy. Methods: We utilized Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to identify the secretory proteins that are differentially expressed in the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line. Immunohistochemistry (IHC) was employed to assess the clinical relevance of the observations. Small interfering (si)RNA-based silencing transfection methods were carried out to study the function of ENPP2. Results: Totally, 133 unique proteins were identified as differentially expressed in HepG2.2.15 cell line compared with HepG2 cell line. Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 precursor (ENPP2) is one of the most significantly up-regulated secretory proteins associated with HBV replication. This differential expression of ENPP2 was further validated by real-time quantitative RT-PCR, Western Blot and immunohistochemical analysis. To study the function of ENPP2, we knockdown ENPP2 expression in HepG2.2.15 cell line by RNA interference. ENPP2 silencing increased HBV replication approximately 2.3-fold by enhancing, via the type I IFN signaling pathway, HBV cccDNA (covalently closed circular DNA) translation into viral RNA. Moreover, attenuation of ENPP2 expression inhibited both the invasion and migration ability of hepatoma cells in vitro via interacting with the molecules in the tumor microenvironment. Conclusion: Our study demonstrates that ENPP2 may be a novel anti-HBV target and indicate that suppression of its expression may inhibit the invasion and migration ability of hepatoma cells.

Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium

Hepatitis B virus (HBV) is designated a “metabolovirus” due to the intimate connection between the virus and host metabolism. The nutrition state of the host plays a relevant role in the severity of HBV infection. Metabolic syndrome (MS) is prone to increasing HBV DNA loads and accelerating the progression of liver disease in patients with chronic hepatitis B (CHB). Cluster of differentiation 36 (CD36), also named fatty acid translocase, is known to facilitate long-chain fatty acid uptake and contribute to the development of MS. We recently found that CD36 overexpression enhanced HBV replication. In this study, we further explored the mechanism by which CD36 overexpression promotes HBV replication. Our data showed that CD36 overexpression increased HBV replication, and CD36 knockdown inhibited HBV replication. RNA sequencing found some of the differentially expressed genes were involved in calcium ion homeostasis. CD36 overexpression elevated the cytosolic calcium level, and CD36 knockdown decreased the cytosolic calcium level. Calcium chelator BAPTA-AM could override the HBV replication increased by CD36 overexpression, and the calcium activator thapsigargin could improve the HBV replication reduced by CD36 knockdown. We further found that CD36 overexpression activated Src kinase, which plays an important role in the regulation of the store-operated Ca2+ channel. An inhibitor of Src kinase (SU6656) significantly reduced the CD36-induced HBV replication. We identified a novel link between CD36 and HBV replication, which is associated with cytosolic calcium and the Src kinase pathway. CD36 may represent a potential therapeutic target for the treatment of CHB patients with MS.

MicroRNA-125b-5p mediates post-transcriptional regulation of hepatitis B virus replication via the LIN28B/let-7 axis

ABSTRACTMicroRNAs (miRNAs) are able to modulate hepatitis B virus (HBV) replication and play an important role in the pathogenesis of HBV infection. Recently, we have identified that serum miR-125b-5p levels correlated with HBV DNA levels and liver necroinflammation. In the present study, we addressed how miR-125b-5p regulated HBV replication at the different steps, inclduing viral transcription, assembly, and virion production and investigated the underlying mechanisms. We found that miR-125b-5p overexpression increased HBV replication without altering HBV transcription. This is the first demonstration of post-transcriptional miRNA regulation of HBV replication. In contrast, transfection of miR-125b-5p inhibitor resulted in downregulation of HBV replication in hepatoma cells. Further, miR-125b-5p inhibited the phosphorylation of retinoblastoma protein and blocked cell cycle progression at the G1/S phase in hepatoma cell lines. Our results indicate that certain miRNAs are able to arrest the cell cycle at G1 phase and may increase HBV replication. RNA sequencing revealed several liver-specific metabolic pathways regulated by miR-125b-5p, which was also found to suppress LIN28B and induce let-7 family members. Additional data demonstrated that miR-125b-5p targeted the LIN28B/let-7 axis to stimulate HBV replication at a post-transcriptional step.

The microRNA-99 family modulates hepatitis B virus replication by promoting IGF-1R/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy.

MicroRNAs are small highly conserved noncoding RNAs that are widely expressed in multicellular organisms and participate in the regulation of various cellular processes including autophagy and viral replication. Evidently, microRNAs are able to modulate host gene expression and thereby inhibit or enhance hepatitis B virus (HBV) replication. The miR-99 family members are highly expressed in the liver. Interestingly, the plasma levels of miR-99 family in the peripheral blood correspond with HBV DNA loads. Thus, we asked whether the miR-99 family regulated HBV replication and analyzed the underlying molecular mechanism. Compared with primary hepatocytes, miR-99 family expression was downregulated in hepatoma cells. Transfection of miR-99a, miR-99b, and miR-100 markedly increased HBV replication, progeny secretion, and antigen expression in hepatoma cells. However, miR-99 family had no effect on HBV transcription and HBV promoter activities, suggesting that they regulate HBV replication at posttranscriptional steps. Consistent with bioinformatic analysis and recent reports, ectopic expression of miR-99 family attenuated IGF-1R/Akt/mTOR pathway signaling and repressed insulin-stimulated activation in hepatoma cells. Moreover, the experimental data demonstrated that the miR-99 family promoted autophagy through mTOR/ULK1 signaling and thereby enhanced HBV replication. In conclusion, the miR-99 family promotes HBV replication posttranscriptionally through IGF-1R/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy.

MiR-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma.

AIMTo investigate the functional role and underlying molecular mechanism of miR-29a in hepatitis B virus (HBV) expression and replication.METHODSThe levels of miR-29a and SMARCE1 in HBV-infected HepG2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8 (CCK-8) was used to detect the viability of HepG2.2.15 cells. The relationship between miR-29a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTSmiR-29a promoted HBV replication and expression, while SMARCE1 repressed HBV replication and expression. Cell viability detection indicated that miR-29a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of miR-29a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by miR-29a overexpression.CONCLUSIONmiR-29a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, miR-29a could be a promising therapeutic target for patients with HBV infection.

Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle.

Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.

High-dose tenofovir is not effective in suppressing hepatitis B virus replication in patients with hepatocellular carcinoma progression: a preliminary result.

Backgrounds/AimsNucleos(t)ide analogues (NUCs) effectively suppress hepatitis B virus (HBV) replication, but hepatocellular carcinoma (HCC) recurrence often leads to HBV replication despite NUC therapy. The aim of this study was to determine whether high-dose tenofovir (TNF) therapy can suppresses HCC recurrence-associated HBV replication.MethodsWe performed a single-arm prospective study to assess the clinical feasibility of high-dose TNF (hdTNF). We recruited 10 patients during September 2015 and followed up for 3 months or early drop-out.ResultsAll 10 patients had HCC of advanced stages due to HCC recurrence and gradual progression. The average age of patients was 51.2±4.7 years and 9 were male. Three patients did not tolerate the increased TNF dosage and were dropped out early. The other 7 patients were relatively tolerable to the increased dosage of TNF 5 tablets per day. One patient had mild gastrointestinal symptoms and another patient complained of insomnia. Increased HBV replication and HCC progression was observed despite hdTNF for 4-8 weeks. All 7 patients showed tumor progression during the 3 month follow-up. In these patients, blood HBV DNA before hdTNF was 50-200 copies/ml; and 4-8 weeks after hdTNF, the HBV replication status was not improved with blood HBV DNA of 50-300 copies/ml. This clinical study was terminated early after these negative results were confirmed.ConclusionsThe results of this study indicated that high dose of TNF up to 5-fold the recommended dosage is not tolerated by a considerable proportion of patients and also ineffective in suppressing HCC progression-associated HBV replication.

Hepatitis B Virus (HBV) Core-Related Antigen During Nucleos(t)ide Analog Therapy Is Related to Intra-hepatic HBV Replication and Development of Hepatocellular Carcinoma.

Although nucleos(t)ide analog (NA) therapy effectively reduces the hepatitis B virus (HBV) DNA load in the serum of patients with chronic hepatitis B, it does not completely reduce the incidence of hepatocellular carcinoma (HCC). A total of 109 patients who had chronic hepatitis B and were receiving NA therapy were analyzed. Multivariate Cox regression analysis showed that age (>60 years had a hazard ratio [HR] of 2.66), FIB-4 index (an index of >2.1 had a HR of 2.57), and the presence of HBV core-related antigen (HBcrAg; HR, 3.53) during treatment were significantly associated with the development of HCC. The amount of HBV DNA and pregenomic RNA in liver were significantly higher in 16 HBcrAg-positive patients, compared with 12 HBcrAg-negative patients, suggesting active HBV replication in HBcrAg-positive livers. Hepatic gene expression profiling showed that HBV-promoting transcriptional factors, including HNF4α, PPARα, and LRH1, were upregulated in HBcrAg-positive livers. HepAD38 cells overexpressing LRH1 increased HBV replication, characterized by higher HBV DNA and pregenomic RNA levels, during long-term exposure to entecavir. Conversely, overexpression of precore/core in HepG2 cells increased levels of these transcriptional factors. Metformin efficiently repressed HBV replication in primary human hepatocytes. Modulating HBV transcriptional factors by metformin in combination with NA therapy would potentiate anti-HBV activity and reduce the incidence of HCC in HBcrAg-positive patients.

Prognostic values of fluctuations in serum levels of alanine transaminase in inactive carrier state of HBV infection.

Background:Current guidelines introduce periodic monitoring of serum alanine transaminase (ALT) as the first-line modality in follow-up patients, with a hepatitis B virus (HBV) inactive carrier state.Objectives:This study aimed to determine the incidence rate and patterns of ALT fluctuations and prognostic values for the development of chronic HBV e antigen (HBeAg)-negative hepatitis B (CHB), HBV surface antigen (HBsAg) seroclearance, and liver-related complications.Patients and Methods:Treatment-naïve patients with a chronic HBV infection, HBeAg(-)/HBeAb(+), normal ALT levels, and HBV DNA < 2000 IU/mL, were followed-up every 6-12 months by assessing serum ALT levels. Serum HBV DNA was measured in cases of elevated ALT levels.Results:A total of 399 patients were followed-up for 8.9 years; ALT > upper limit of normal (ULN, i.e. 40 IU/L) was detected in 103 (25.8%) patients, with an annual incidence rate of 2.9%. ALT elevation was associated with; male gender, age, and higher serum ALT levels at study entry. Among the cases of ALT elevations, 16 (15.5%) patients had ALT levels > 2 × ULN. There were 38 (36.9%) patients who had ALT levels that remained > ULN over six months, and 21 (20.4%) patients experienced at least two episodes of ALT elevations. In 15 (14.6%) patients, elevated ALT levels were associated with increased HBV replication (i.e. HBV DNA > 2 000 IU/mL) and these were considered as CHB. However, elevation of ALT levels, even in the absence of HBV replication, increased the risk for the development of CHB up to 8-fold in prospective follow-ups. HBsAg seroclearance, cirrhosis, and hepatocellular carcinoma were detected in 43 (10.8%), 4 (1%), and 1 (0.25%) patients, respectively.Conclusions:Fluctuations in serum ALT levels may change the prognosis of a HBV inactive carrier state.

Keratin 8 is involved in hepatitis B virus replication

Hepatitis B virus (HBV) infection can result in fatal liver diseases, including cirrhosis or liver failure, and its replication and pathogenesis depend on the critical interplay between viral and host factors. This study investigated HBV replication-related host proteins and the effect of candidate proteins on HBV replication. Isobaric tags for relative and absolute quantitation (iTRAQ) were used to measure HBV replication-related proteins in HepG2 cells and HepG2.2.15 cells. KRT8 was up-regulated in HepG2.2.15 cells but not in HepG2 cells, and KRT8 was overexpressed in an HBV-infected patient's liver tissue. This result suggested that KRT8 is involved in HBV replication. To further clarify the relationship between KRT8 and HBV replication, KRT8 gene expression was inhibited by siRNA. The silencing of KRT8 mildly suppressed HBV replication. Moreover, overexpressed KRT8 significantly increased HBV replication, and the inhibition of HBV DNA did not suppress KRT8 expression. Thus, the host protein KRT8 is involved in the replication of HBV DNA, and it dramatically enhances HBV replication.