Both middle and large envelope proteins can mediate neutralization of hepatitis B virus infectivity by anti-preS2 antibodies: escape by naturally occurring preS2 deletions.

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins containing preS1/preS2/S, preS2/S, and S domain alone, respectively. S and preS1 domains mediate sequential virion attachment to heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP), respectively, which can be blocked by anti-S and anti-preS1 antibodies. How anti-preS2 antibodies neutralize HBV infectivity remains enigmatic. The late stage of chronic HBV infection often selects for mutated preS2 translation initiation codon to prevent M protein expression, or in-frame preS2 deletions to shorten both L and M proteins. When introduced to infectious clone of genotype C or D, both M-minus mutations and most 5' preS2 deletions sustained virion production. Such mutant progeny viral particles were infectious in NTCP-reconstituted HepG2 cells. Neutralization experiments were performed on the genotype D clone. Although remaining susceptible to anti-preS1 and anti-S neutralizing antibodies, M-minus mutants were only partially neutralized by two anti-preS2 antibodies tested while preS2 deletion mutants were resistant. By infection experiments using viral particles with lost versus increased M protein expression, or a neutralization escaping preS2 deletion only present on L or M protein, we found that both full-length L and M proteins contributed to virus neutralization by the two anti-preS2 antibodies. Thus, immune escape could be a driving force for the selection of M-minus mutations, and especially preS2 deletions. The fact that both L and M proteins could mediate neutralization by anti-preS2 antibodies may shed light on the underlying molecular mechanism.IMPORTANCEThe large (L), middle (M), and small (S) envelope proteins of hepatitis B virus (HBV) contain preS1/preS2/S, preS2/S, and S domain alone, respectively. The discovery of heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP) as the low- and high-affinity HBV receptors could explain neutralizing potential of anti-S and anti-preS1 antibodies, respectively, but how anti-preS2 neutralizing antibodies work remains enigmatic. In this study, we found two M-minus mutants in the context of genotype D partially escaped two anti-preS2 neutralizing antibodies in NTCP-reconstituted HepG2 cells, while several naturally occurring preS2 deletion mutants escaped both antibodies. By point mutations to eliminate or enhance M protein expression, and by introducing preS2 deletion selectively to L or M protein, we found binding of anti-preS2 antibodies to both L and M proteins contributed to neutralization of wild-type HBV infectivity. Our finding may shed light on the possible mechanism(s) whereby anti-preS2 antibodies neutralize HBV infectivity.

Chimeric antigen receptors of HBV envelope proteins inhibit hepatitis B surface antigen secretion.

OBJECTIVES: Chronic hepatitis B (CHB) caused by HBV infection greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) plays critical roles in the pathogenesis of CHB. HBsAg loss is the key indicator for cure of CHB, but is rarely achieved by current approved anti-HBV drugs. Therefore, novel anti-HBV strategies are urgently needed to achieve sustained HBsAg loss. DESIGN: We developed multiple chimeric antigen receptors (CARs) based on single-chain variable fragments (scFvs, namely MA18/7-scFv and G12-scFv), respectively, targeting HBV large and small envelope proteins. Their impacts on HBsAg secretion and HBV infection, and the underlying mechanisms, were extensively investigated using various cell culture models and HBV mouse models. RESULTS: After secretory signal peptide mediated translocation into endoplasmic reticulum (ER) and secretory pathway, MA18/7-scFv and CARs blocked HBV infection and virion secretion. G12-scFv preferentially inhibited virion secretion, while both its CAR formats and crystallisable fragment (Fc)-attached versions blocked HBsAg secretion. G12-scFv and G12-CAR arrested HBV envelope proteins mainly in ER and potently inhibited HBV budding. Furthermore, G12-scFv-Fc and G12-CAR-Fc strongly suppressed serum HBsAg up to 130-fold in HBV mouse models. The inhibitory effect lasted for at least 8 weeks when delivered by an adeno-associated virus vector. CONCLUSION: CARs possess direct antiviral activity, besides the well-known application in T-cell therapy. Fc attached G12-scFv and G12-CARs could provide a novel approach for reducing circulating HBsAg.

Influential factors on disease activity in Crohn's disease and their Roc curve evaluation: a cross-sectional study.

OBJECTIVE: This study aims to investigate the factors influencing disease activity in patients with Crohn's disease (CD) and provide insights and references for the management and prevention of CD. METHODS: We recruited CD patients who met the inclusion and exclusion criteria and were treated at the First Affiliated Hospital of Soochow University from November 2022 to June 2023. Generalized linear mixed models were used to analyze the factors affecting disease activity in CD patients. Receiver operating characteristic (ROC) curve analysis was employed to assess the predictive value of these factors for disease activity. RESULTS: A total of 268 CD participants aged 18 to 65 were included in the study, with over 68% of them in remission or experiencing mild disease activity, indicating relatively good disease control. The results of the generalized linear mixed models showed that older age, absence of diabetes, high levels of physical activity, and a low dietary inflammatory index (DII) were protective factors for lower disease activity in CD patients (p < 0.05). ROC curve analysis demonstrated that physical activity level, age, and DII all had ROC areas greater than 0.6 in predicting disease activity in CD patients (p < 0.05). CONCLUSION: The factors influencing the disease activity of CD patients are numerous and should be given attention. CD patients who are younger, have low levels of physical activity, high DII, and have diabetes are at a higher risk of increased disease activity. By reducing or avoiding the mentioned risk factors and leveraging protective factors, it is possible to mitigate the disease activity of CD to some extent.

5' preS1 Mutations To Prevent Large Envelope Protein Expression from Hepatitis B Virus Genotype A or Genotype D Markedly Increase Polymerase-Envelope Fusion Protein.

Hepatitis B virus (HBV) large (L) envelope protein is translated from 2.4-kb RNA. It contains preS1, preS2, and S domains and is detected in Western blotting as p39 and gp42. The 3.5-kb pregenomic RNA produces core and polymerase (P) proteins. We generated L-minus mutants of a genotype A clone and a genotype D clone from 1.1-mer or 1.3-mer construct, with the former overproducing pregenomic RNA. Surprisingly, mutating a preS1 ATG codon(s) or introducing a nonsense mutation soon afterwards switched secreted p39/gp42 to a p41/p44 doublet, with its amount further increased by a nonsense mutation in the core gene. A further-downstream preS1 nonsense mutation prevented p41/p44 production. Tunicamycin treatment confirmed p44 as the glycosylated form of p41. In this regard, splicing of 3.5-kb RNA to generate a junction at nucleotides (nt) 2447 to 2902 for genotype D enables translation of p43, with the N-terminal 47 residues of P protein fused to the C-terminal 371 residues of L protein. Indeed p41/p44 were detectable by an antibody against the N terminus of P protein and eliminated by a nonsense mutation at the 5' P gene or a point mutation to prevent that splicing. Therefore, lost L (and core) protein expression from the 1.1-mer or 1.3-mer construct markedly increased p41/p44 (p43), the P-L fusion protein. Cotransfection with an expression construct for L/M proteins reversed high extracellular p41/p44 associated with L-minus mutants, suggesting that L protein retains p43 in wild-type HBV to promote its intracellular degradation. Considering that p43 lacks N-terminal preS1 sequence critical for receptor binding, its physiological significance during natural infection and therapeutic potential warrant further investigation. IMPORTANCE The large (L) envelope protein of hepatitis B virus (HBV) is translated from 2.4-kb RNA and detected in Western blotting as p39 and gp42. Polymerase (P) protein is expressed at a low level from 3.5-kb RNA. The major spliced form of 3.5-kb RNA will produce a fusion protein between the first 47 residues of P protein and a short irrelevant sequence, although also at a low level. Another spliced form has the same P protein sequence fused to L protein missing its first 18 residues. We found that some point mutations to eliminate L and core protein expression from overlength HBV DNA constructs converted p39/gp42 to p41/gp44, which turned out to be the P-L fusion protein. Thus, the P-L fusion protein can be expressed at extremely high level when L protein expression is prevented. The underlying mechanism and functional significance of this variant form of L protein warrant further investigation.

Clinical isolates of hepatitis B virus genotype C have higher in vitro transmission efficiency than genotype B isolates.

Serum samples were collected from 54 hepatitis B e antigen (HBeAg)-positive Chinese patients infected with hepatitis B virus (HBV) subgenotype B2 or C2. They were compared for transmission efficiency using same volume of samples or infectivity using same genome copy number. Adding polyethylene glycol (PEG) during inoculation did not increase infectivity of fresh samples but markedly increased infectivity following prolonged sample storage. Differentiated HepaRG cells infected without PEG produced more hepatitis B surface antigen (HBsAg) and higher HBsAg/HBeAg ratio than sodium taurocholate cotransporting polypeptide (NTCP)-reconstituted HepG2 cells infected with PEG. They better supported replication of core promoter mutant in contrast to wild-type (WT) virus by HepG2/NTCP cells. Overall, subgenotype C2 samples had higher viral load than B2 samples, and in general produced more HBeAg, HBsAg, and replicative DNA following same-volume inoculation. Precore mutant was more prevalent in subgenotype B2 and had reduced transmission efficiency. When same genome copy number of viral particles was inoculated, viral signals were not necessarily higher for three WT C2 isolates than four WT B2 isolates. Using viral particles generated from cloned HBV genome, three WT C2 isolates showed slightly reduced infectivity than three B2 isolates. In conclusion, subgenotype C2 serum samples had higher transmission efficiency than B2 isolates in association with higher viral load and lower prevalence of precore mutant, but not necessarily higher infectivity. PEG-independent infection by HBV viremic serum samples is probably attributed to a labile host factor.

Lost Small Envelope Protein Expression from Naturally Occurring PreS1 Deletion Mutants of Hepatitis B Virus Is Often Accompanied by Increased HBx and Core Protein Expression as Well as Genome Replication.

Hepatitis B virus (HBV) transcribes coterminal mRNAs of 0.7 to 3.5 kb from the 3.2-kb covalently closed circular DNA, with the 2.1-kb RNA being most abundant. The 0.7-kb RNA produces HBx protein, a transcriptional transactivator, while the 3.5-kb pregenomic RNA (pgRNA) drives core and P protein translation as well as genome replication. The large (L) and small (S) envelope proteins are translated from the 2.4-kb and 2.1-kb RNAs, respectively, with the majority of the S protein being secreted as noninfectious subviral particles and detected as hepatitis B surface antigen (HBsAg). pgRNA transcription could inhibit transcription of subgenomic RNAs. The present study characterized naturally occurring in-frame deletions in the 3' preS1 region, which not only codes for L protein but also serves as the promoter for 2.1-kb RNA. The human hepatoma cell line Huh7 was transiently transfected with subgenomic expression constructs for envelope (and HBx) proteins, dimeric constructs, or constructs mimicking covalently closed circular DNA. The results confirmed lost 2.1-kb RNA transcription and HBsAg production from many deletion mutants, accompanied by increases in other (especially 2.4-kb) RNAs, intracellular HBx and core proteins, and replicative DNA but impaired virion and L protein secretion. The highest intracellular L protein levels were achieved by mutants that had residual S protein expression or retained the matrix domain in L protein. Site-directed mutagenesis of a high replicating deletion mutant suggested that increased HBx protein expression and blocked virion secretion both contributed to the high replication phenotype. Our findings could help explain why such deletions are selected at a late stage of chronic HBV infection and how they contribute to viral pathogenesis. IMPORTANCE Expression of hepatitis B e antigen (HBeAg) and overproduction of HBsAg by wild-type HBV are implicated in the induction of immune tolerance to achieve chronic infection. How HBV survives the subsequent immune clearance phase remains incompletely understood. Our previous characterization of core promoter mutations to reduce HBeAg production revealed the ability of the 3.5-kb pgRNA to diminish transcription of coterminal RNAs of 2.4 kb, 2.1 kb, and 0.7 kb. The later stage of chronic HBV infection often selects for in-frame deletions in the preS region. Here, we found that many 3' preS1 deletions prevented transcription of the 2.1-kb RNA for HBsAg production, which was often accompanied by increases in intracellular 3.5-, 0.7-, and especially 2.4-kb RNAs, HBx and core proteins, and replicative DNA but lost virion secretion. These findings established the biological consequences of preS1 deletions, thus shedding light on why they are selected and how they contribute to hepatocarcinogenesis.

Naturally occurring 5' preS1 deletions markedly enhance replication and infectivity of HBV genotype B and genotype C.

BACKGROUND AND AIMS: Deletion of 15-nucleotide or 18-nucleotide (nt) covering preS1 ATG frequently arises during chronic infection with HBV genotypes B and C. Since the second ATG is 33nt downstream, they truncate large (L) envelope protein by 11 residues like wild-type genotype D. This study characterised their functional consequences. METHODS: HBV genomes with or without deletion were amplified from a patient with advanced liver fibrosis and assembled into replication competent 1.1mer construct. Deletion, insertion or point mutation was introduced to additional clones of different genotypes. Viral particles concentrated from transfected HepG2 cells were inoculated to sodium taurocholate cotransporting polypeptide (NTCP)-reconstituted HepG2 (HepG2/NTCP) cells or differentiated HepaRG cells, and HBV RNA, DNA, proteins were monitored. RESULTS: From transfected HepG2 cells, the 15-nt and 18-nt deletions increased HBV RNA, replicative DNA and extracellular virions. When same number of viral particles was inoculated to HepG2/NTCP cells, the deletion mutants showed higher infectivity. Conversely, HBV infectivity was diminished by putting back the 18nt into naturally occurring genotype C deletion mutants and by adding 33nt to genotype D. Infectivity of full-length genotype C clones was also enhanced by mutating the first ATG codon of the preS1 region but diminished by mutating the second in-frame ATG. Removing N-terminal 11 residues from preS1 peptide 2-59 of genotype C potentiated inhibition of HBV infection and enhanced binding to HepG2/NTCP cells. CONCLUSIONS: The 15-nt and 18-nt deletions somehow increase HBV RNA, replicative DNA and virion production. Shortened L protein is more efficient at mediating HBV infection.

Effects of resistance training on exercise ability in chronic obstructive pulmonary disease subjects: A systematic review and meta-analysis.

INTRODUCTION: The resistance exercise effect on the exercise ability in chronic obstructive pulmonary disease (COPD) subjects has drawn considerable attention. However, the relationship between resistance exercise and the exercise ability of COPD subjects is conflicting. This meta-analysis was performed to evaluate this relationship. METHODS: A systematic-literature search up to July 2020 was performed in OVID, Embase, Cochrane Library, PubMed, Google scholar for randomised control trials reported relationships between resistance exercise and the exercise ability of COPD subjects, and 13 studies were detected with 1286 subjects at the baseline. Mean differences (MD) with 95% confidence intervals (CIs) were calculated comparing the resistance exercise and the exercise ability of COPD subjects using the continuous method with a random or fixed-effect model. RESULTS: A significantly higher 6-minutes walk test was observed in subjects performing resistance training (MD, 60.41; 95% CI, 39.97-80.85, P < .001) compared with non-resistance training subjects. However, no significant difference was observed between COPD subjects performing resistance exercise compared with non-resistance training COPD subjects in constant work rate cycle endurance test (MD, 1.59; 95% CI, 0.03-3.15, P = .05), unsupported upper extremity exercise test (MD, 48.77; 95% CI, -1.20 to 98.75, P < .06) and quality of life questionnaires (MD, -0.62; 95% CI, -2.49 to 1.245, P < .51). CONCLUSIONS: The resistance exercise significantly increases the 6-minutes walk test in COPD subjects. However, resistance exercise did not significantly affect the constant work rate cycle endurance test, unsupported upper extremity exercise test and quality of life questionnaires. This relationship forces us to recommend the resistance exercise to improve the 6-minutes walk test as a simple and easy evaluation of functional exercise ability in COPD subjects.

Interferon-inducible MX2 is a host restriction factor of hepatitis B virus replication.

BACKGROUND & AIMS: Non-cytolytic cure of HBV-infected hepatocytes by cytokines, including type I interferons (IFNs), is of importance for resolving acute and chronic infection. However, as IFNs stimulate hundreds of genes, those most relevant for HBV suppression remain largely unknown. Amongst them are the large myxovirus resistance (Mx) GTPases. Human MX1 (or MxA) is active against many RNA viruses, while MX2 (or MxB) was recently found to restrict HIV-1, HCV, and herpesviruses. Herein, we investigated the anti-HBV activity of MX2. METHODS: The potential anti-HBV activity of MX2 and functional variants were assessed in transfected and HBV-infected hepatoma cells and primary human hepatocytes, employing multiple assays to analyze the synthesis and decay of HBV nucleic acids. The specific roles of MX2 in IFN-α-driven inhibition of HBV transcription and replication were assessed by MX2-specific shRNA interference (RNAi). RESULTS: Both MX2 alone and IFN-α substantially inhibited HBV replication, due to significant deceleration of the synthesis and slight acceleration of the turnover of viral RNA. RNAi knockdown of MX2 significantly reduced the inhibitory effects of IFN-α. Strikingly, MX2 inhibited HBV infection by reducing covalently closed circular DNA (cccDNA), most likely by indirectly impairing the conversion of relaxed circular DNA to cccDNA rather than by destabilizing existing cccDNA. Various mutations affecting the GTPase activity and oligomerization status reduced MX2's anti-HBV activity. CONCLUSION: MX2 is an important IFN-α inducible effector that decreases HBV RNA levels but can also potently inhibit HBV infection by indirectly impairing cccDNA formation. MX2 likely has the potential for therapeutic applications aimed at curing HBV infection by eliminating cccDNA. LAY SUMMARY: This study shows that the protein MX2, which is induced by interferon-α, has important anti-hepatitis B virus (HBV) effector functions. MX2 can reduce the amount of covalently closed circular DNA, which is the form of DNA that HBV uses to maintain viral persistence within hepatocytes. MX2 also reduces HBV RNA levels by downregulating synthesis of viral RNA. MX2 likely represents a novel intrinsic HBV inhibitor that could have therapeutic potential, as well as being useful for improving our understanding of the complex biology of HBV and the antiviral mechanisms of interferon-α.

An antiviral drug-resistant mutant of hepatitis B virus with high replication capacity in association with a large in-frame deletion in the preS1 region of viral surface gene.

We amplified a full-length hepatitis B virus (HBV) genome from the serum of a chronic hepatitis B patient who experienced virological breakthrough with high HBV DNA titer following adefovir (ADV) therapy. The PCR product was cloned and sequencing of the six clones revealed an isolate of C2 subgenotype. Mutation(s) in the polymerase gene responsible for ADV resistance included rtA181T (all clones) and rtN236T (four clones). The rtA181T mutation caused the W172* nonsense mutation in the overlapping S gene. In addition, all the clones harbored another nonsense mutation in the S gene (C69*) and a 207nt in-frame deletion in the preS1 region. These clones were converted to a 1.1mer construct for transient transfection of Huh7 cells. All the clones were deficient in hepatitis B surface antigen production. Three clones had similar levels of DNA replication. Comparison with a wild-type clone of the same genotype revealed a higher intracellular level of replicative DNA for clone c4, which was reduced by putting back the deleted 207nt, but not by co-transfection with an expression construct for the three surface proteins to rescue virion production. The HBcAg expression of the c4 and c4+207nt clones was mainly in the nucleus. Co-transfection with the L/M/S proteins expression construct did not alter the distribution of core. Clone c4 showed a significantly decreased susceptibility to ADV, a mild reduction in susceptibility to lamivudine and tenofovir, but remained sensitive to entecavir. In conclusion, this is an unusual ADV-resistant HBV isolate harboring two nonsense mutations in the S gene and a large in-frame deletion in the preS1 region, but still retains a high replication phenotype, which can provide a platform for recombinant vector construction.

N-Linked Glycosylation Is Not Essential for Sodium Taurocholate Cotransporting Polypeptide To Mediate Hepatitis B Virus Infection In Vitro.

Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a hepatitis B virus (HBV) receptor, and its overexpression in HepG2 cell lines leads to efficient secretion of hepatitis B e antigen (HBeAg) following challenge with a large dose of cell culture-derived HBV (cHBV) particles. However, NTCP-reconstituted HepG2 cells are inefficiently infected by patient serum-derived HBV (sHBV) and release very little hepatitis B surface antigen (HBsAg) following cHBV infection, unlike differentiated HepaRG cells, which are naturally susceptible to both cHBV and sHBV particles. Here, we investigated whether NTCP could explain the different behaviors of the two cell types. Endogenous NTCP protein from differentiated HepaRG cells was unglycosylated despite wild-type coding sequence. HepaRG cells stably transfected with an epitope-tagged NTCP expression construct displayed higher sHBV but not cHBV susceptibility than cells transfected with the null mutant. Tagged NTCP introduced to both HepG2 and HepaRG cells was glycosylated, with N5 and N11 being sites of N-linked glycosylation. Mutating N5, N11, or both did not alter cell surface availability of NTCP or its subcellular localization, with both the singly glycosylated and nonglycosylated forms still capable of mediating cHBV infection in HepG2 cells. In conclusion, nonglycosylated NTCP is expressed by differentiated HepaRG cells and capable of mediating cHBV infection in HepG2 cells, but it cannot explain differential susceptibility of HepaRG and HepG2/NTCP cells to cHBV versus sHBV infection and different HBsAg/HBeAg ratios following cHBV infection. The responsible host factor(s) remains to be identified.IMPORTANCE HBV can infect differentiated HepaRG cells and also HepG2 cells overexpressing NTCP, the currently accepted HBV receptor. However, HepG2/NTCP cells remain poorly susceptible to patient serum-derived HBV particles and release very little hepatitis B surface antigen following infection by cell culture-derived HBV. We found differentiated HepaRG cells expressed nonglycosylated NTCP despite a wild-type coding sequence. NTCP introduced to HepG2 cells was glycosylated at two N-linked glycosylation sites, but mutating either or both sites failed to prevent infection by cell culture-derived HBV or to confer susceptibility to serum-derived HBV. Overexpressing NTCP in HepRG cells did not increase infection by cell culture-derived HBV or distort the ratio between the two viral antigens. These findings suggest that host factors unique to HepaRG cells are required for efficient infection by serum-derived HBV, and factors other than NTCP contribute to balanced viral antigen production following infection by cell culture-derived HBV.

Identification of serum microRNAs as diagnostic biomarkers for schizophrenia.

BACKGROUND: At present, the schizophrenia diagnoses are based on the clinical symptoms and behaviors neglecting the laboratory test indicators. RESULTS: To better investigate the diagnostic potential of miRNAs for schizophrenia, we selected 14 candidate miRNAs and examined their expressions in the serums of 40 schizophrenia patients and 40 healthy controls by qRT-PCR. Ultimately three abnormally expressed microRNAs were identified, i.e., miR-34a-5p, miR-432-5p and miR-449a. Then, binary regression analysis was employed to combine 3 dysregulated miRNAs. ROC analysis revealed that the AUC of the combination of miR-432-5p + miR-449a in serums was 0.841 (95% CI: 0.791~0.887) with 90% sensitivity and 80% specificity. The AUC of the combination of miR-34a-5p + miR-432-5p + miR-449a in serums was 0.843 (95% CI: 0.791~0.887) with 90% sensitivity and 77.5% specificity. The results indicated that the combined model of miR-432-5p + miR-449a and miR-34a-5p + miR-432-5p + miR-449a have better prediction performances. CONCLUSIONS: The study concludes that the two miRNAs combinations have the potential to be used as biomarkers for schizophrenia diagnoses. The finding may be conducive to overcoming the dilemmas faced by current schizophrenia diagnosis.

Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor.

UNLABELLED: Cell culture (cc)-derived hepatitis B virus (HBV) can infect differentiated HepaRG cells, but efficient infection requires addition of polyethylene glycol (PEG) during inoculation. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor enabled ccHBV infection of NTCP reconstituted HepG2 cells, although very little hepatitis B surface antigen (HBsAg) is produced. We found infection by patient serum-derived HBV (sHBV), which required purification of viral particles through ultracentrifugation or PEG precipitation, was PEG independent and much more efficient in HepaRG cells than in HepG2/NTCP cells. In contrast to hepatitis B e antigen (HBeAg), HBsAg was not a reliable marker of productive sHBV infection at early time points. A low HBsAg/HBeAg ratio by ccHBV-infected HepG2/NTCP cells was attributable to dimethyl sulfoxide (DMSO) in culture medium, NTCP overexpression, and HBV genotype D. HepG2/NTCP cells released more viral antigens than HepG2 cells after HBV genome delivery by adeno-associated virus, and stable expression of NTCP in a ccHBV producing cell line increased viral mRNAs, proteins, replicative DNA, and covalently closed circular DNA. NTCP protein expression in HepG2/NTCP cells, despite being driven by the cytomegalovirus promoter, was markedly increased by DMSO treatment. This at least partly explains ability of DMSO to promote ccHBV infection in such cell lines. In conclusion, NTCP appeared inefficient to mediate infection by serum-derived HBV. It could promote HBV RNA transcription while inhibiting HBsAg secretion. Efficient PEG-independent sHBV infection of HepaRG cells permits comparative studies of diverse clinical HBV isolates and will help identify additional factors on virion surface promoting attachment to hepatocytes. IMPORTANCE: Currently in vitro infection with hepatitis B virus (HBV) depends on cell culture-derived HBV inoculated in the presence of polyethylene glycol. We found patient serum-derived HBV could efficiently infect differentiated HepaRG cells independent of polyethylene glycol, which represents a more physiological infection system. Serum-derived HBV has poor infectivity in HepG2 cells reconstituted with sodium taurocholate cotransporting polypeptide (NTCP), the currently accepted HBV receptor. Moreover, HepG2/NTCP cells secreted very little hepatitis B surface antigen after infection with cell culture-derived HBV, which was attributed to NTCP overexpression, genotype D virus, and dimethyl sulfoxide added to culture medium. NTCP could promote HBV RNA transcription, protein expression, and DNA replication in HepG2 cells stably transfected with HBV DNA, while dimethyl sulfoxide could increase NTCP protein level despite transcriptional control by a cytomegalovirus promoter. Therefore, this study revealed several unusual features of NTCP as an HBV receptor and established conditions for efficient serum virus infection in vitro.

Dysregulation of retinoic acid receptor diminishes hepatocyte permissiveness to hepatitis B virus infection through modulation of sodium taurocholate cotransporting polypeptide (NTCP) expression.

Sodium taurocholate cotransporting polypeptide (NTCP) is an entry receptor for hepatitis B virus (HBV) and is regarded as one of the determinants that confer HBV permissiveness to host cells. However, how host factors regulate the ability of NTCP to support HBV infection is largely unknown. We aimed to identify the host signaling that regulated NTCP expression and thereby permissiveness to HBV. Here, a cell-based chemical screening method identified that Ro41-5253 decreased host susceptibility to HBV infection. Pretreatment with Ro41-5253 inhibited the viral entry process without affecting HBV replication. Intriguingly, Ro41-5253 reduced expression of both NTCP mRNA and protein. We found that retinoic acid receptor (RAR) regulated the promoter activity of the human NTCP (hNTCP) gene and that Ro41-5253 repressed the hNTCP promoter by antagonizing RAR. RAR recruited to the hNTCP promoter region, and nucleotides -112 to -96 of the hNTCP was suggested to be critical for RAR-mediated transcriptional activation. HBV susceptibility was decreased in pharmacologically RAR-inactivated cells. CD2665 showed a stronger anti-HBV potential and disrupted the spread of HBV infection that was achieved by continuous reproduction of the whole HBV life cycle. In addition, this mechanism was significant for drug development, as antagonization of RAR blocked infection of multiple HBV genotypes and also a clinically relevant HBV mutant that was resistant to nucleoside analogs. Thus, RAR is crucial for regulating NTCP expression that determines permissiveness to HBV infection. This is the first demonstration showing host regulation of NTCP to support HBV infection.

Overview of hepatitis B viral replication and genetic variability.

Chronic infection with hepatitis B virus (HBV) greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). HBV isolates worldwide can be divided into ten genotypes. Moreover, the immune clearance phase selects for mutations in different parts of the viral genome. The outcome of HBV infection is shaped by the complex interplay of the mode of transmission, host genetic factors, viral genotype and adaptive mutations, as well as environmental factors. Core promoter mutations and mutations abolishing hepatitis B e antigen (HBeAg) expression have been implicated in acute liver failure, while genotypes B, C, subgenotype A1, core promoter mutations, preS deletions, C-terminal truncation of envelope proteins, and spliced pregenomic RNA are associated with HCC development. Our efforts to treat and prevent HBV infection are hampered by the emergence of drug resistant mutants and vaccine escape mutants. This paper provides an overview of the HBV life cycle, followed by review of HBV genotypes and mutants in terms of their biological properties and clinical significance.

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.

Functional characterization of hepatitis B virus core promoter mutants revealed transcriptional interference among co-terminal viral mRNAs.

Hepatitis B virus (HBV) has a 3.2 kb circular DNA genome. It employs four promoters in conjunction with a single polyadenylation signal to generate 3.5, 2.4, 2.1 and 0.7 kb co-terminal RNAs. The 3.5 kb RNA is subdivided into the precore RNA for e-antigen expression and pregenomic RNA for genome replication. When introduced to a genotype A clone, several core promoter mutations markedly enhanced HBV genome replication, but suppressed e-antigen expression through up-regulation of pregenomic RNA at the expense of precore RNA. In this study, we found such mutations also diminished envelope proteins and hepatitis B surface antigen, products of the 2.1 and 2.4 kb subgenomic RNAs. Indeed, Northern blot analysis revealed overall increase in 3.5 kb RNA, but reduction in all subgenomic RNAs. To validate transcriptional interference, we subcloned 1.1×, 0.7× and 0.6× HBV genome, respectively, to a vector with or without a cytomegalovirus (CMV) promoter at the 5' end, so as to produce the pregenomic RNA, 2.4 kb RNA, and 2.1 kb RNA in large excess or not at all. Parallel transfection of the three pairs of constructs into a human hepatoma cell line confirmed the ability of pregenomic RNA to suppress all subgenomic transcripts and established the ability of the 2.4 and 2.1 kb RNAs to suppress the 0.7 kb RNA. Consistent with our findings, pregenomic RNA of the related duck HBV has been reported to interfere with transcription of the subgenomic RNAs. Transcriptional interference might explain why HBV produces so little 0.7 kb RNA and HBx protein despite a strong X promoter.

Interleukin-1 and tumor necrosis factor-α trigger restriction of hepatitis B virus infection via a cytidine deaminase activation-induced cytidine deaminase (AID).

Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1ß and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1ß and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1ß, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.

Impaired virion secretion by hepatitis B virus immune escape mutants and its rescue by wild-type envelope proteins or a second-site mutation.

Hepatitis B virus immune escape mutants have been associated with vaccine failure and reinfection of grafted liver despite immune prophylaxis, but their biological properties remain largely unknown. Transfection of 20 such mutants in a human hepatoma cell line identified many with severe impairment in virion secretion, which can be rescued to various extents by coexpression of wild-type envelope proteins or introduction of a novel glycosylation site. Consistent with their role in maintaining intra- or intermolecular disulfide bonds, cysteine residues within the "a" determinant are critical for virion secretion.

Primary Duck Hepatocyte Culture and Duck Hepatitis B Virus Infection Model.

Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of the hepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins.