Hepatitis B Virus Viral Replication Research Articles

Interleukin-8 in HepG2 cells: Enhancing antiviral proteins in uninfected cells but promoting HBV replication in infected cells

In vitro studies have revealed that hepatitis B virus (HBV) infection upregulates interleukin-8 (IL-8), which enhances HBV replication. Clinically, elevated IL-8 levels in chronic HBV patients are associated with diminished therapeutic efficacy of interferon-α (IFN-α). Our study advances these findings by demonstrating that IL-8 promotes the expression of myxovirus resistance A (MxA) and protein kinase R (PKR) in HepG2 cells via the PI3K-AKT pathway. However, HBV-infected cells fail to exhibit IL-8-induced upregulation of MxA and PKR, likely due to HBV's upregulation of PP2A that inhibits the PI3K-AKT pathway. Notably, IL-8 targets the C/EBPα transcription factor, increasing HBV promoter activity and viral replication, which in turn partially suppresses the expression of MxA and PKR induced by IFN-α. Our findings uncover a mechanism by which HBV may evade immune responses, suggesting potential new strategies for immunotherapy against chronic HBV infection.

G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes.

Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

Blocking neddylation elicits antiviral effect against hepatitis B virus replication.

Hepatitis B Virus (HBV) is the most common cause of chronic liver disease worldwide. The mechanisms that regulate HBV viral replication remain poorly defined. Here, we show that blocking of the neddylation elicits antiviral effect against HBV replication, indicating that NEDD8 supports viral production. To explore role of neddylation, HBV-replicating HepG2.2.15.7 cells and HBV-infected HepG2-hNTCP-30 cells were treated with siNEDD8 and MLN4924, a potent and selective NEDD8-activating enzyme inhibitor. Cell viability, intracellular and extracellular HBV DNA, covalently closed circular DNA (cccDNA), HBsAg, HBeAg, and HBcrAg were measured to assess the consequences of the various treatments on viral replication.Our data showed that HBV infection increased NEDD8 expression in human liver cell lines. Symmetrically, NEDD8 knockdown by siRNA or MLN4924 treatments decreased HBV replication in HepG2.2.15.7 and HepG2-hNTCP-30 cells. Notably, HBsAg, and HBeAg secretions were strongly suppressed in the culture supernatants, but not the HBcrAg. These results indicate that the suppression of NEDD8 decreases HBV replication. However, cccDNA steady level confirms once again its persistence and longevity in chronic infection. The manipulation of the neddylation pathway can thus provide new tools interfering with HBV persistence as well as novel therapeutic strategies against chronic hepatitis B.

Baicalin down-regulating hepatitis B virus transcription depends on the liver-specific HNF4α-HNF1α axis

Baicalin (BA) inhibits hepatitis B virus (HBV) RNAs production and reduces levels of the related hepatocyte nuclear factors (HNFs), although the underlying mechanism is unclear. In this study, we investigated the specific pathway by which BA regulates HBV transcription through the HBV-related HNFs. Following transfection of HepG2 cells with pHBV1.2, we observed that BA inhibited the production of HBV RNAs and viral proteins in a time- and dose-dependent manner. These effects were consistent with the downregulation of HNF1α, which was abolished by HNF1α-shRNA. The shRNA of HNF4α, the upstream gene of HNF1α, also remarkedly reduced HNF1α expression and impaired the anti-HBV efficacy of BA, indicating that this function of BA depended on HNF4α/HNF1α axis. Furthermore, chromatin immunoprecipitation assay showed that BA significantly reduced HNF4α-HNF1α transactivation activity. The similar effects of BA were observed in entecavir (ETV)-resistant HBVrtM204V/rtLl80M transfected HepG2 cells. Thus, we proposed a mechanism for the anti-HBV activity of BA in an HNF4α-HNF1α-dependent manner, which impaired HNF4α and HNF1α transactivation, and effectively inhibited HBV transcription and viral replication.

Differences in HBV Replication, APOBEC3 Family Expression, and Inflammatory Cytokine Levels Between Wild-Type HBV and Pre-core (G1896A) or Basal Core Promoter (A1762T/G1764A) Mutants.

BackgroundChronic hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC) world-wide. HBV variants, particularly the G1896A pre-core (PC) and A1762T/G1764A basal core promoter (BCP) mutations, are established risk factors for cirrhosis and HCC, but the molecular biological basis is unclear. We hypothesized that these variants result in differential HBV replication, APOBEC3 family expression, and cytokine/chemokine expression.MethodsHepG2 cells were transfected with monomeric full-length containing wild-type, PC, or BCP HBV. Cells and supernatant were collected to analyze viral infection markers (i.e., HBsAg, HBeAg, HBV DNA, and RNA). Cellular APOBEC3 expression and activity was assessed by quantitative real-time (qRT)-PCR, immunoblot, differential DNA denaturation PCR, and sequencing. Cytokine/chemokines in the supernatant and in serum from 11 CHB carriers (4 non-cirrhotic; 7 cirrhotic and/or HCC) with predominantly wild-type, PC, or BCP variants were evaluated by Luminex.ResultsHBeAg expression was reduced in PC and BCP variants, and higher supernatant HBV DNA and HBV RNA levels were found with A1762T/G1764A vs. G1896A mutant (p < 0.05). Increased APOBEC3G protein levels in wild-type vs. mutant were not associated with HBV covalently closed circular DNA G-to-A hypermutations. Differences in cytokine/chemokine expression in culture supernatants, especially IL-13 were observed amongst the variants analyzed. Noticeable increases of numerous cytokines/chemokines, including IL-4 and IL-8, were observed in ex vivo serum collected from CHB carriers with PC mutant.ConclusionHBV sequence variation leads to differences in HBV protein production (HBeAg) and viral replication in addition to altered host innate antiviral restriction factor (APOBEC3) and cytokine/chemokine expression.

Assisted reproduction in hepatitis carrier couples.

Chronic Hepatitis B virus (HBV) infection is endemic worldwide, and the prevalence is especially high in the Asia-Pacific regions. Despite its high prevalence, the literature regarding the impact of HBV infection on subfertility and fertility treatment remains limited and conflicting. Latest studies do not suggest any detrimental effect of HBV infection on the outcome of IVF/ICSI treatment in women having chronic HBV infection. There is evidence that HBV exists in ovarian tissue including oocyte and follicular fluid, and therefore has the potential risk of transmission to the embryo, which can explain the finding of vertical transmission despite immunoprophylaxis. Most recently, we have observed the evidence of HBV viral replication in female HBV carriers undergoing IVF/ICSI treatment. This raises the question of whether antiviral medication should be administered during ovarian stimulation in IVF/ICSI treatment cycles for women with chronic HBV infection to help reduce the chance of vertical transmission.

Therapeutic management with biological anti-TNF-α agent in severe psoriasis associated with chronic hepatitis B: A case report.

Systemic therapy in patients with concurrent psoriasis and chronic hepatitis B is a challenging task for both dermatologists and gastroenterologists since there is a high risk for hepatitis B virus (HBV) reactivation and hepatic toxicity under biological therapy. The therapeutic management of a patient with psoriasis and infection with the HBV is a challenge as the classical systemic treatment (methotrexate, acitretin, cyclosporine) shows a high risk of immunosuppression and/or hepatic toxicity and the biological therapy is endangered by the possibility of HBV reactivation. We present the case of a patient with moderate-severe psoriasis and chronic hepatitis B for whom we assessed the risk-benefit relation and considered useful to initiate the anti-TNF therapy concomitantly with the antiviral therapy with entecavir. The therapeutic algorithm included initiation of anti-TNF therapy with etanercept 2×50 mg/week combined with entecavir, an antiviral treatment administered continuously since the diagnosis of the HBV hepatitis, with hepatic function and viral load monitoring. After 3 months of therapy with etanercept the patient was given a dose of etanercept of 50 mg/week combined with entecavir 0.5 mg/day which he continued until week 36 when psoriatic lesions had cleared (PASI=0.6; DLQI=0). No adverse effects were registered and there was no evidence of HBV viral replication or changes in viral markers. We wish to emphasize that the use of etanercept in a patient with psoriasis and hepatitis B is a successful therapeutic alternative which may be safely used concomitantly with entecavir, with regular monitoring of viral load and hepatic function tests.

HBV T1719G mutation reduced HBV replication through mutant Enh II and HBx protein in vitro.

It was repeatedly reported that the hepatitis B virus (HBV) T1719G mutation was very common and related to progression and malignancy of liver disease. However, its effect on viral replication efficiency remains unclear. In this study, we aimed to evaluate the function and mechanisms of the T1719G mutation on viral replication capacity. Wild-type and T1719G mutation-bearing HBV1.2× plasmids were transfected into Huh7 and HepG2 cells, respectively, and HBV total RNA, 3.5kb RNA and supernatant HBV DNA were assessed using real-time PCR, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured by time-resolved fluoroimmunoassay. In order to assess Enh II activity and the binding capacity of HNF3β to Enh II sequence, dual-luciferase assay and Chromatin immunoprecipitation (ChIP)-PCR were employed, respectively. Simultaneously, the HBx or HBx-mut (T1719G) plasmid was co-transfected to evaluate the effect of HBx on viral replication. Our results showed that the T1719G mutation impaired viral replication efficacy compared with the wild type both by reducing Enh II activity and binding capacity of HNF3β with Enh II. And such reduction caused by T1719G mutation could be rescued by HBx protein. Our results show that the T1719G mutation decreases HBV viral replication capacity possibly by mutant HBx protein and altered Enh II activity.

HBV and HIV viral load but not microbial translocation or immune activation are associated with liver fibrosis among patients in South Africa

BackgroundCo-infection with HIV negatively impacts the progression of chronic hepatitis B virus (HBV) infection, including causing rapid progression to liver fibrosis. Sub-Saharan Africa represents arguably the most important intersection of high endemicity of both chronic hepatitis B virus (HBV) infection and HIV infection.MethodsWe recruited 46 HBV/HIV-co-infected; 47 HBV-monoinfected; 39 HIV-monoinfected; and 37 HBV/HIV-uninfected patients from Tygerberg Hospital, Cape Town, South Africa. All HIV-infected patients were on antiretroviral therapy for ≥3 months. Liver stiffness measurements were assessed using the Fibroscan (Fibroscan 402, Echosens). Cell-based immunomarkers were measured by flow cytometry. Soluble serum/plasma immunomarkers were measured by Luminex technology and enzyme immunoassays. HIV (COBAS/Ampliprep TaqMan HIV-1) and HBV viral loads (in-house assay) were also performed.ResultsHBV/HIV co-infected patients showed significantly higher levels of immune activation %CD8+/HLA-DR+/CD38+ (median 30%, interquartile range: 17–53) and %CD8+/PD-1 (median 22%, interquartile range: 15–33), p ≤ 0.01 compared to all other study groups. Despite this, the HBV-mono-infected group had the highest proportion of patients with advanced liver fibrosis (≥13 kPa) as measured by Fibroscan (18%). HBV mono-infected patients showed highest expression of most cytokines including IL-17 and basic fibroblastic growth factor. There was significant positive correlation between detectable HIV and HBV viral replication and liver fibrosis but not immune activation or gut translocation.DiscussionHighly-active antiretroviral therapy, including tenofovir, is effective against both HIV and HBV. Earlier therapy in the co-infected patients may therefore have controlled viral replication leading to better fibrosis scores when compared to HBV mono-infection in this study. On-going HBV and HIV viraemia, rather than microbial translocation or immune activation, appear to be the drivers of liver fibrosis. Moderate to advanced liver fibrosis in HBV-mono-infection may well indicate poor access to screening and treatment of HBV infection.

Covering the Cover

Covering the Cover

Perinatal factors influencing mother-to-child HBV transmission

Background: Chronic hepatitis B virus (HBV) infection remains a key public health problem. In China, mother-to-child transmission is recognized as the major transmission pathway. The exact mechanism by which HBV enters the fetus through the maternal placenta is unclear, hampering the development of preventative interventions. Objective: We aimed to identify perinatal factors influencing intrauterine transmission as determined by hepatitis B surface antigen (HBsAg) positivity 12 h after birth. Study design: A total of 343 HBsAg(+) newborns with HBV(+) mothers were included in this study. The maternal marker expression pattern as well as associations of maternal HBsAg status with multiple perinatal risk factors for transmission (including sex, fetal position, mode of delivery, gestational age, number of complications, and infant HBV marker levels were analyzed. Results: The majority of HBsAg(+) newborns were also HBeAg(+). The HBeAg and anti-HBe titers were significant higher in newborns from HBsAg(+) mothers than HBsAg(-) mothers (P=0.000). The frequency of ≥ 2 complications was significant higher in HBsAg(+) mothers than HBsAg(-) mothers (p=0.023). There was no significant difference in offspring in sex, mode of delivery, in utero position, or gestational age between HBsAg(+) and HBsAg(-) mothers (all P ≥ 0.05). Conclusion: The HBsAg(+) newborns of HBsAg(+) mothers showed higher HBV viral replication rates than those of HBsAg(-) mothers. Maternal HBsAg-positivity and multiple pregnancy complications were significant risk factors for uterine mother-to-child HBV transmission. Offspring of HBV-infected mothers should be screened regularly for HBV markers to identify carriers.

MicroRNA-137 promotes hepatitis B virus gene expression and replication via targeting the protein inhibitor of activated STAT 2.

Hepatitis B virus (HBV) infection is one of the most important infectious diseases in China. In this study, we investigated the functional role of miR-137 in HBV infection to further elucidate the mechanism underlying the associated pathology. Viral replication was determined after transfection of HEK293 cells with the replication-competent vector pHBV1.3 and miR137 mimics or inhibitors. Expression of HBV genes was determined by quantitative real-time PCR (qRT-PCR). Expression of miR-137 and protein inhibitor of activated STAT 2 (PIAS2) was determined by qRT-PCR and Western blotting. Activity of the PIAS2 3'-UTR was determined by dual-luciferase reporter assays. Transfection of HEK293 cells with pHBV1.3 increased the expression of miR-137. Co-transfection with miR-137 mimic upregulated HBV gene expression and viral replication. MiR-137 targeted the PIAS2 3'-UTR, and suppressed PIAS2 mRNA and protein expressions. SiRNA-mediated PIAS2 knockdown suppressed HBV gene expression and viral replication. PIAS2 expression rescued the promotion effect of miR-137 on HBV expression and viral replication. MiR-137 expression was significantly upregulated following HBV infection. Furthermore, miR-137 promoted the expression of HBV genes and viral replication by targeting the expression of PIAS2. Our findings might provide a new insight into the diagnosis and treatment of HBV infection.

Clonorchis sinensis Co-infection Could Affect the Disease State and Treatment Response of HBV Patients.

BackgroundClonorchis sinensis (C. sinensis) is considered to be an important parasitic zoonosis because it infects approximately 35 million people, while approximately 15 million were distributed in China. Hepatitis B virus (HBV) infection is a major public health issue. Two types of pathogens have the potential to cause human liver disease and eventually hepatocellular carcinoma. Concurrent infection with HBV and C. sinensis is often observed in some areas where C. sinensis is endemic. However, whether C. sinensis could impact HBV infection or vice versa remains unknown.Principal FindingsCo-infection with C. sinensis and HBV develops predominantly in males. Co-infected C. sinensis and HBV patients presented weaker liver function and higher HBV DNA titers. Combination treatment with antiviral and anti-C. sinensis drugs in co-infected patients could contribute to a reduction in viral load and help with liver function recovery. Excretory-secretory products (ESPs) may, in some ways, increase HBV viral replication in vitro. A mixture of ESP and HBV positive sera could induce peripheral blood mononuclear cells (PBMCs) to produce higher level of Th2 cytokines including IL-4, IL-6 and IL-10 compared to HBV alone, it seems that due to presence of ESP, the cytokine production shift towards Th2. C. sinensis/HBV co-infected patients showed higher serum IL-6 and IL-10 levels and lower serum IFN-γ levels.Conclusions/SignificancePatients with concomitant C. sinensis and HBV infection presented weaker liver function and higher HBV DNA copies. In co-infected patients, the efficacy of anti-viral treatment was better in patients who were prescribed with entecavir and praziquantel than entecavir alone. One possible reason for the weaker response to antiviral therapies in co-infected patients was the shift in cytokine production from Th1 to Th2 that may inhibit viral clearance. C. sinensis/HBV co-infection could exacerbate the imbalance of Th1/Th2 cytokine.

Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status

In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication.

Oncogenic circuit constituted by Ser31-HBx and Akt increases risks of chronic hepatitis and hepatocellular carcinoma

The X protein of hepatitis B virus (HBx) has been specifically implicated in the development of hepatocellular carcinoma (HCC). Clinical associations of HBx isoforms with chronic hepatitis and HCC have not been well studied. HBx has two roles in liver cells, namely pro-apoptotic and anti-apoptotic. In this report, we examined the role of Ser31-HBx in HCC and chronic hepatitis. Using the case–control study, we determined risks of chronic hepatitis and HCC conferred by hepatitis B virus (HBV) containing Ser31-HBx that was phosphorylated by Akt. Ser31-HBx isoforms conferred 3.23-fold risk of HCC in male and 3.36-fold risk in female. Ser31 isoforms were associated with 3.12-fold risk of chronic hepatitis and 3.43-fold risk of cirrhosis and also associated with higher HBV viral load and replication efficiency and lower rate of HBe loss. To determine the mechanism, we found that Ser31-HBx constituted an oncogenic circuit with Akt and cooperated with ras to transform NIH3T3 cells in contrast to non-Ser31-HBxs that did not transduce oncogenic signals. Our results give a clue to account for an underlying cause of HBx-mediated hepatocarcinogenesis. It appears that Ser31 phosphorylation of HBx by Akt plays an important role. The current study provides an example of association of HBV genome variations with risks of HCC and chronic hepatitis

Design, synthesis, and bioevaluation of paeonol derivatives as potential anti-HBV agents.

Hepatitis B virus (HBV) is a causative reagent that frequently causes progressive liver diseases, leading to the development of acute, chronic hepatitis, cirrhosis, and eventually hepatocellular carcinoma (HCC). Despite several antiviral drugs including interferon-α and nucleotide derivatives are approved for clinical treatment for HBV, critical issues remain unresolved, e.g., low-to-moderate efficacy, adverse side effects, and resistant strains. In this study, novel Paeonol-phenylsulfonyl derivatives were synthesized and their antiviral effect against HBV was evaluated. The experimental results indicated that these compounds process significant antiviral potential, including the inhibition of viral antigen expression and secretion, and the suppression of HBV viral DNA replication. Among compounds synthesized in this research, compound 2-acetyl-5-methoxyphenyl 4-methoxybenzenesulfonate (7f) had the most potent inhibitory activity with IC50 value of 0.36 μM, and high selectivity index, SI (TC50/IC50) 47.75; which exhibited an apparent inhibition effect on viral gene expression and viral propagation in cell culture model. So, we believe our compounds could serve as reservoir for antiviral drug development.

PD-1 mRNA expression is associated with clinical and viral profile and PD1 3′-untranslated region polymorphism in patients with chronic HBV infection

Programmed cell death-1 (PD-1) is involved in hepatitis B virus (HBV) infection and single-nucleotide polymorphism (SNP) rs10204525 in the 3′-untranslated region (3′ UTR) of PD1 gene was shown to be associated with the disease course of HBV infection. This study examined the associations of PD-1 mRNA expression with the clinical and viral profiles and the genotypes of rs10204525 in HBV infection. PD-1 mRNA levels in peripheral blood nuclear cells were determined by real-time quantitative reverse transcription polymerase chain reaction (PCR). PD1 rs10204525 was genotyped by bidirectional PCR amplification of specific alleles. The results showed that patients with chronic HBV infection had significantly elevated PD-1 mRNA levels than healthy controls. Patients with chronic hepatitis and hepatocellular carcinoma had significantly higher PD-1 mRNA levels than healthy controls. HBeAg (+) patients had significantly higher PD-1 mRNA levels than HBeAg (−) patients (P<0.001). PD-1 mRNA levels were sequentially increased with the elevation of HBV DNA levels. In HBV patients, but not in healthy controls, PD-1 mRNA levels were sequentially decreased from rs10204525 genotypes AA, AG to GG and the levels in genotype AA were significantly higher than in genotype GG (P=0.039). These findings suggest that increased PD-1 expression may affect the disease course of chronic HBV infection by facilitating HBV viral replication, and this may at least partially relate to PD1 3′ UTR polymorphism.

HBx affects CUL4–DDB1 function in both positive and negative manners

Hepatitis B virus (HBV) infection is a major public health problem by affecting 350 million people worldwide. The mechanisms that regulate HBV gene expression and viral replication remain poorly understood. HBx is known as the central regulator for HBV replication and is associated with the CUL4–DDB1 ubiquitin ligase through H-box motif. Here, we show that blocking the activity of DDB1 by RNA interfering inhibited viral production and gene expression of HBV, and direct association of HBx with DDB1 promoted viral activities, indicating that DDB1 function is required for viral production. On the other hand, HBx interfered with DDB1-dependent polyubiquitination of PRMT1, arginine methyltransferase 1, suggesting that HBx can also block the function of a subset of CUL4–DDB1 E3 ligases. Thus, we conclude that HBx regulates the function of DDB1 in both positive and negative manners in the context of distinct CUL4–DDB1 complexes and plays different roles in HBV replication cycle.

Hepatitis B virus replication is blocked by a 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) inhibitor of the viral ribonuclease H activity

Nucleos(t)ide analog drugs profoundly suppress Hepatitis B virus (HBV) replication but rarely cure the infection, so therapy is usually life-long. The nucleos(t)ide analogs inhibit the viral DNA polymerase and often push HBV to the brink of extinction, so it may be possible to eradicate HBV by suppressing HBV replication further. The HBV ribonuclease H (RNaseH) is a logical new drug target because it is the second of only two viral enzymes essential for viral replication. We recently developed a low throughput screening pipeline for inhibitors of the HBV RNaseH and viral replication. Here, we screened a series of twenty-three nitrogen-based polyoxygenated heterocycles including sixteen 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives for anti-HBV RNaseH activity. Nine compounds inhibited the HBV RNaseH, but activity was marginal for eight of them. Compound #1 [2-hydroxyisoquinoline-1,3(2H,4H)-dione, HID] was the best hit with an IC50 of 28.1μM and an EC50 of 4.2μM. It preferentially suppressed accumulation of the viral plus-polarity DNA strand in replication inhibition assays, indicating that replication was blocked due to suppression of HBV RNaseH activity. It had a CC50 of 75μM, yielding a therapeutic index of ∼18. The EC50 value was 7-fold lower than the IC50, possibly due to cellular retention or metabolism of the compound, or higher affinity for the full-length enzyme than the recombinant form used for screening. These data indicate that the 2-hydroxyisoquinoline-1,3(2H,4H)-diones will have different structure–activity relationships for the HBV and HIV RNaseHs. Therefore, HID compounds may provide a foundation for development of more effective RNaseH inhibitors of HBV replication.

The Influence of Hepatitis B Viral Load and Pre-S Deletion Mutations on Post-Operative Recurrence of Hepatocellular Carcinoma and the Tertiary Preventive Effects by Anti-Viral Therapy

BackgroundWhether or not hepatitis B virus (HBV) genotypes, mutations, and viral loads determine outcomes for patients with HBV-induced hepatocellular carcinoma (HCC) remains controversial.AimsTo study the influence of HBV viral factors on prognoses for patients with HBV-induced HCC after resection surgery and investigate if antiviral therapy could counteract the adverse effects of viral factors.MethodsA total of 333 HBV-related HCC patients who underwent tumor resection were enrolled retrospectively. Serum HBV DNA levels, mutations, anti-viral therapy, and other clinical variables were analyzed for their association with post-operative recurrence.ResultsAfter a median follow-up of 45.9 months, 208 patients had HCC recurrence after resection. The 5-year overall survival and recurrence-free survival rates were 55.4% and 35.3%, respectively. Multivariate analysis showed indocyanine green retention rate at 15 minutes >10%, gamma-glutamyltransferase (GGT) level >60 U/L, macroscopic and microscopic venous invasion, and the absence of anti-viral therapy were significant risk factors for recurrence. Anti-viral therapy could decrease recurrence in patients with early stage HCC, but the effect was less apparent in those with the Barcelona-Clinic Liver Cancer stage C HCC. For patients without antiviral therapy after resection, serum HBV DNA levels >106 copies/mL, GGT >60 U/L, and macroscopic and microscopic venous invasion were significant risk factors predicting recurrence. Among the 216 patients without anti-viral therapy but with complete HBV surface gene mapping data, 73 were with pre-S deletion mutants. Among patients with higher serum HBV DNA levels, those with pre-S deletion had significantly higher rates of recurrence. Moreover, multivariate analysis showed multi-nodularity, macroscopic venous invasion, cirrhosis, advanced tumor cell differentiation, and pre-S deletion were significant risk factors predictive of recurrence.ConclusionsOngoing HBV viral replication and pre-S deletion are crucial for determining post-operative tumor recurrence. Anti-viral therapy can help reduce recurrence and improve prognosis, especially for those with early stage HCC.