pre-S Region Research Articles

Impact of deletions and mutations in Hepatitis B virus envelope proteins on serological profile and clinical evolution

The Hepatitis B virus (HBV) envelope glycoproteins are essential for viral entry into the hepatocyte and are also targets for host immune response. The study of these proteins could allow us to highlight molecular hot points influencing HBV fitness, which would subsequently modify the clinical evolution of the disease, both under anti-viral therapy or without treatment. The present short communication underlines the importance of the high variability in HBV envelope proteins, in regard with the literature and in our hands, for HBV-infected patients either on anti-HBV treatment or not. We report mutations in antigenic areas of S protein, i.e. CD8+/CD4+ T-cell epitopes and B-cell epitopes in the major hydrophilic region (MHR), such as sI126N and sG145R possibly involved in the rare coexisting Hepatitis B surface Antigen (HBsAg)/anti-HBs serological pattern. We mostly report serial mutations in preS region including preS1 deletion (aa 1–6, 31–71, 38–73, 72–104) and preS2 deletion (aa132-141) in patients with various clinical evolutions. Some of these viral envelope mutations, due to immune selection pressure, may result in a worsening of the hepatic disease.

Prevalence and Characteristics of Precore Mutation in Iran and Its Correlation with Genotypes of Hepatitis B.

IntroductionMutation of the HBV precore gene prevents the production of HBeAg, which is an important target for immune responses. Distribution of this mutation varies along with frequency of HBV genotypes in accordance with geographic and ethnic variations. The general objective of this study was to evaluate the prevalence and characteristics of precore mutation in Iran and its correlation with genotypes of hepatitis B.MethodsIn this cross-sectional study, viral DNA of 182 Iranian hepatitis B surface antigen positive patients who were admitted to Bandar Abbas Blood Transfusion Organization in 2012 and 2013 was retrieved from their serum samples. HBeAg, anti-HBe, and anti-HBc IgM diagnostic tests were performed using ELISA kits. Precore and Pre-S regions were amplified using specific primers and PCR thereafter to determine the genotypes; precore mutation, PCR, and restriction fragment length polymorphism (RFLP) methods also were applied. SPSS version 12 was used for data analysis by Mann–Whitney U test, Fisher’s exact probability test, and t-test.ResultsA total of 62 patients (34.1%) had precore mutation (A1896G), and genotype D was the predominant genotype in these patients, which was followed by an unknown genotype that was suspected for genotype B. Interestingly, the relationships between precore mutation and HBeAg (p=0.037) and genotype D (p=0.005) were significant; however, no correlation was observed between this mutation and acute or chronic hepatitis and sex of patients.ConclusionThis study found high prevalence of precore mutations in southern Iran, which was significantly associated with HBeAg and genotype D.

Sequence analysis and functional characterization of full-length hepatitis B virus genomes from Korean cirrhotic patients with or without liver cancer

This study aimed to identify and characterize mutations in the hepatitis B virus (HBV) genome associated with advanced liver diseases. The 3.2-kb HBV genome of the C2 subgenotype was amplified from sera of 18 cirrhotic Korean patients with (10) or without (8) hepatocellular carcinoma (HCC), and two clones per patient were characterized by transient transfection experiments in human hepatoma cells. While A1762T/G1764A core promoter mutations were highly prevalent in both groups, the G1896A precore mutation to abolish hepatitis B e antigen (HBeAg) expression was more common in HCC clones (55% vs. 20%). High replication capacity was mostly found in HCC clones and associated with core promoter mutations, whereas more non-HCC clones harbored a nonfunctional core gene (34% vs. 8%). Large in-frame deletions in the preS region were found in 60% of HCC clones and 38% of non-HCC clones. They removed the first 11 residues of large envelope protein or impaired small envelope protein expression, or deleted a neutralizing epitope in the preS2 domain. Additional point mutations prevented middle envelope protein expression, or caused nonsense mutations in the preS or S region to truncate large and/or small envelope protein. Consequently, many clones were unable to express or secrete hepatitis B surface antigen (HBsAg). In conclusion, mutations associated with the advanced stage of chronic HBV infection are complex and diverse. Host immune pressure most likely selected for mutations in the HBV genome to abolish or reduce HBeAg or HBsAg production, to enhance genome replication, or to escape neutralizing antibodies. Some of these mutations may contribute to liver cirrhosis or HCC development.

Deep sequencing analysis of quasispecies in the HBV pre-S region and its association with hepatocellular carcinoma.

The association between the evolution of hepatitis B virus (HBV) quasispecies and the development of hepatocellular carcinoma (HCC) is unknown. We used deep sequencing to examine the dynamics of HBV quasispecies and their relationship to HCC development. Thirty-two chronic hepatitis B (CHB) patients with HCC (HCC group) and 32 matched CHB patients without HCC (controls) were recruited. Fourteen patients from each group had serial sera available up to 9years before the time of the present study. Deep sequencing of the HBV pre-S regions was performed. HBV quasispecies complexity, diversity, and intrapatient prevalence of pre-S deletions/mutations were analyzed. Compared with control patients, HCC patients had a significant greater quasispecies complexity (p=0.04 at the nucleotide level), greater diversity (p=0.004 and 0.009 at the nucleotide level and the amino acid level respectively), and a trend of greater complexity at the amino acid level (p=0.065). HCC patients had a higher intrapatient prevalence of pre-S deletions and point mutations (at codons 4, 27, and 167) compared with the control patients (all p<0.05). Longitudinal observation in the sera of 14 HCC patients showed that quasispecies complexity (p=0.027 and 0.024 at the nucleotide level and the amino acid level respectively) and diversity (p=0.035 and 0.031 at the nucleotide level and the amino acid level respectively) increased as the disease progressed to HCC. Increased HBV quasispecies complexity and diversity in the pre-S region, probably reflecting enhanced virus-host interplay, was associated with disease progression from CHB to HCC.

The Envelope Gene of Hepatitis B Virus Is Implicated in Both Differential Virion Secretion and Genome Replication Capacities between Genotype B and Genotype C Isolates.

Chronic infection by hepatitis B virus (HBV) genotype C is associated with a prolonged replicative phase and an increased risk of liver cancer, compared with genotype B infection. We previously found lower replication capacity but more efficient virion secretion by genotype C than genotype B isolates. Virion secretion requires interaction between core particles and ENVELOPE proteins. In the present study, chimeric constructs between genotype B and genotype C clones were generated to identify the structural basis for differential virion secretion. In addition to dimeric constructs, we also employed 1.1mer constructs, where the cytomegalovirus (CMV) promoter drove pregenomic RNA transcription. Through transient transfection experiments in Huh7 cells, we found that exchanging the entire envelope gene or just its S region could enhance virion secretion by genotype B clones while diminishing virion secretion by genotype C. Site-directed mutagenesis established the contribution of genotype-specific divergence at codons 108 and 115 in the preS1 region, as well as codon 126 in the S region, to differential virion secretion. Surprisingly, exchanging the envelope gene or just its S region, but not the core gene or 3′ S region, could markedly increase intracellular replicative DNA for genotype C clones but diminish that for genotype B, although the underlying mechanism remains to be clarified.

A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins.

PAC and its analogs represent a new class of anti-HBV agents that directly target the preS1 region of the HBV large surface protein. These agents could contribute to the development of a potent, well-tolerated, and broadly active inhibitor of HBV infection. (Hepatology 2017;65:1104-1116).

Mutational Analysis and Interactions of HBV preS1 With Asialoglycoprotein Receptor

Background: The mutations in preS1 of a large envelop protein of HBV may have profound implications in HBV receptor binding to hepatocytes and subsequent entry of the virus into host cells. Aims: This study aimed to identify the mutations in preS1 region and the receptor binding interactions of preS1 with hepatocytes. Methods: The mutations were searched through direct sequencing of the preS1 region. Sequence analysis was done through ClustalX and Jalview. Ab initio modeling of preS1 was done through Rosetta and QUARK followed by glycosylation of best model of preS1. Finally the interactions of preS1 with ASGPR was studied using PatchDock and analysis was done using MOE and pyMol. Results: Sequence comparison revealed changes in the preS1 region. Ab initio modeling results showed that preS1 is an overall unstructured protein with the presence of three structural motifs. Docking of preS1 with asialoglycoprotein receptor showed mostly hydrophobic interactions. Conclusion: In conclusion, preS1 sequences from Pakistani isolates were found to be 90% conserved and the predicted structure of preS1 was near to native structure.

Molecular characterization of hepatitis B virus among chronic hepatitis B patients from Pointe Noire, Republic of Congo.

BackgroundChronic Hepatitis B infection is a major health problem in Republic of Congo therefore molecular analysis of HBV strains is important to detect the patients at high risk of disease progression.MethodsSerum samples were obtained from 111 chronic HBV patients in Pointe Noire. HBsAg, HBeAg and HBeAb were detected. A fragment of the preS1 region of HBV was amplified and sequenced to determine genotypes, subgenotypes and to identify mutations.ResultsOf the 111 samples analyzed, 35 patients were asymptomatic carriers (ASC), 24 with a chronic active hepatitis (CAH), 33 with liver cirrhosis (LC) and 19 have a hepatocellular carcinoma (HCC). The mean age were 45 ± 13 year, 88 (79.3 %) were male and 23 (20.7 %) female. The prevalence of HBeAg was 15.3 % and 73 % of subjects were anti-HBe positive. The mean serum level of alanine aminotransferase transaminase (ALT) and aspartate transaminase (AST) was 25.1 ± 9 IU/L and 28.6 ± 10 IU/L respectively. Eighty two samples out of 111 (73.9 %) were genotyped by the analyzing of the S region of HBV, 58 (70.7 %) cases belonged to HBV genotype E and 24 (29.3 %) were genotype A with three subgenotypes; A3 (66.7 %), A4 (20.8 %) and A6 (12.5 %). Prevalence of genotype A was relatively high in CAH (33.3 %) and HCC (31.6 %) patients in comparison with other groups. The most prevalent amino acids substitutions were R38K found in 14 (17.1 %) sequences, following by H44L in 11 (13.4 %), K13E in 8 (9.8 %), N29K in 8 (9.8 %), A35E in 8 (9.8 %), V80I in 7 (8.5 %) and in 6 (7.3 %) sequences for S90T. Different substitutions located in the hepatocyte binding site were higher among patients with LC and HCC (p < 0.05).ConclusionsThis study have shown that HBV genotype E and A were the most frequent strains circulating in Republic of Congo patients. HBV pres1 substitutions found in this study were associated with severe clinical forms of liver diseases. This data have shown the importance of implementing an effective program to fight HBV infection.

Hepatitis B virus full-length genomic mutations and quasispecies in hepatocellular carcinoma.

Hepatitis B virus (HBV) full-length genomic mutations and quasispecies characteristics in hepatocellular carcinoma (HCC) were investigated. Hepatitis B virus DNA was extracted from the tumor and non-tumor tissues of 16 HCC patients. Overlapping DNA fragments covering the entire HBV genome were amplified and sequenced. To study HBV sequence at the quasispecies level, the preS region was amplified and clonally sequenced. HBV mutation profiles, quasispecies complexity and diversity, and phylogenetic characteristics were assessed. Fourteen patients had full-length HBV amplification. Hot-spot mutations at HBx aa130-131 and pre-S deletions were detected in 13 (93%) and 6 (43%) patients, respectively. Deletions in the X/preC/C regions were more frequently detected in the tumor than in the non-tumor tissues (P = 0.031). Compared with the non-tumor tissues, the tumor tissues had a lower quasispecies complexity (P = 0.014 and 0.043, at the nucleotide and amino acid levels, respectively) and diversity (P = 0.048 and 0.022, at the nucleotide and amino acid levels, respectively). Phylogenetic analysis showed that HBV sequences derived from tumor and non-tumor tissues were separately clustered, suggesting the occurrence of compartmentalization, which was confirmed by the correlation coefficient testing on both the number and length of branches of viral populations (all P < 0.02). Hepatitis B virus mutation patterns in HCC tumor tissues and non-tumor tissues were different. HBV quasispecies within the preS region were compartmentalized, and tumor tissues had a lower genome complexity and diversity. Our study suggests HBV evolution is conditioned by the differential host cellular environment in HCC tumors.

Prediction of HBF-0259 interactions with hepatitis B Virus receptors and surface antigen secretory factors.

Hepatitis B virus (HBV) is an etiological agent of viral hepatitis, which may lead to cirrhosis, and hepatocellular carcinoma. Current treatment strategies have not shown promising effect to date but various complications such as, drug toxicity-resistance have been reported. Study on newly discovered compounds, with minimal side effects, as specific HBV inhibitors is a fundamental subject introducing new biologic drugs. Here, we aimed to, by prediction, estimate interactions of HBF-0259 as a non-toxic anti-HBV compound on inhibiting the HBV through either interaction with the viral entry or HBsAg secreting factors using In Silico procedure. Molecular docking was performed by Hex 8.0.0 software to predict the interaction energy (Etot) between HBF-0259 and known cellular factors involved in HBV entry and HBsAg secreting factors. Hex 8.0.0 also employed to create protein-protein complexes. These interactions were then used to analyze the binding site of HBF-0259 within the assumed receptors by MGLTools software. Finally, the amino acid sequences involved in this interaction were aligned for any conservancy. Here, we showed that HBF-0259 Etot with CypA (-545.41kcal/mol) and SCCA1 (499.68kcal/mol), involved in HBsAg secretion and HBV integration, respectively, was higher than other interactions. Furthermore, HBF-0259 predicted interaction energy was even higher than those of CypA inhibitors. In addition, we claim that preS1 and/or preS2 regions within HBsAg are not suitable targets for HBF-0259. HBF-0259 has higher interaction energy with CypA and SCCA1, even more than other known receptors, co-receptors, viral ligands, and secretory factors. HBF-0259 could be introduced as potent anti-viral compound in which CypA and or SCCA1, as previously shown, are involved.

Occult HBV infection in HCC and cirrhotic tissue of HBsAg-negative patients: a virological and clinical study.

AimTo evaluate the virological and clinical characteristics of occult HBV infection (OBI) in 68 consecutive HBsAg-negative patients with biopsy-proven cirrhosis and HCC.MethodsHBV DNA was sought and sequenced in plasma, HCC tissue and non-HCC liver tissue by PCRs using primers for HBV core, surface and x regions. OBI was identified by the presence of HBV DNA in at least two different PCRs.ResultsOBI was detected in HCC tissue of 13 (20%) patients and in non-HCC liver tissue of 3 of these 13. OBI was detected in HCC tissue of 54.5% of 11 anti-HBs- negative/anti-HBc-positive patients, in 29.4% of 17 anti-HBs/anti-HBc-positive and in 5% of 40 anti-HBs/anti-HBc-negative (p < 0.0005). The 13 patients with OBI in HCC tissue more frequently than the 55 without showed Child-B or -C cirrhosis (53.9% vs. 5.5%, p < 0.0001) and BCLC-B or -C stages (46.1% vs. 1.8%, p < 0.0001). The pre-S1, pre-S2 and S region sequences in HCC tissue showed amino acid (AA) substitutions (F19L, P24L, S59F, T131I, Q129H) and deletions (in positions 4,8, 17 and 86) in the S region, AA substitutions (T40S, P124K, L54P, G76A, N222T and I273L) in pre-S1 region and AA substitutions in pre-S2 region (P41H and P66L). In the 3 patients showing OBI also in non-HCC liver tissue the S, pre-S1 and pre-S2 sequencing displayed patterns of mutations different.ConclusionsThe study showed a significant correlation between OBI and the severity of liver damage, several patterns of mutations in the S, pre-S1 and pre-S2 regions in HCC tissue, some at their first description.

HBV Pre-S deletion mutation analysis: A high resolution diagnosis method.

e15612 Background: The Pre-S region(nucleotides 2854-155) locates at the S gene(encodeing the surface protein) of HBV DNA genome and consists of the Pre-S1 and Pre-S2 domains. A meta-analysis with more than 10,000 cases and dozens reports all indicate that infection of HBV with Pre-S mutant is associated with 3.77-fold increased risk of HCC. Methods: To evaluate the performance of the Pre-S deletion mutation detection system, several HBV infected serum samples with different viral titer were collected. And viral DNA was extracted with 300 uL serum. A set of Pre-S primers, which is applicable for all HBV genotypes, was used to amplify the target region with highly fidelity PCR polymerase. Results: Results with high resolution came out via a technology called capillary electrophoresis (CE). This analytic method was accomplished by Qsep100TM machine from BiOptic Inc. This Pre-S PCR system has been successfully verified with many clinical samples, and it could be applied to samples with different HBV genotypes. Traditional method often use agarose gel system to bring out results. However, it may be not applicable when it comes to samples with low viral titer. Now, this problem can be solved with the capillary electrophoresis platform. Capillary electrophoresis not only can detect the pre-S signal at E+03 viral titer grade but also provide a system with analyzing consistency. Conclusions: To sum up, the capillary electrophoresis platform is a high resolution and sensitive method for detection of Pre-S deletion, especially for low viral titer samples.

Mutational analysis of hepatitis B virus pre-S1 (9–24) fusogenic peptide

A hollow nanoparticle known as a bio-nanocapsule (BNC) consisting of hepatitis B virus (HBV) envelope L protein and liposome (LP) can encapsulate drugs and genes and thereby deliver them in vitro and in vivo to human hepatic tissues, specifically by utilizing the HBV-derived infection machinery. Recently, we identified a low pH-dependent fusogenic domain at the N-terminal part of the pre-S1 region of the HBV L protein (amino acid residues 9 to 24; NPLGFFPDHQLDPAFG), which shows membrane destabilizing activity (i.e., membrane fusion, membrane disruption, and payload release) upon interaction with target LPs. In this study, instead of BNC and HBV, we generated LPs displaying a mutated form of the pre-S1 (9–24) peptide, and performed a membrane disruption assay using target LPs containing pyranine (fluorophore) and p-xylene-bis (N-pyridinium bromide) (DPX) as a quencher. The membrane disruption activity was found to correlate with the hydrophobicity of the whole structure, while the peptide retained a random-coil structure even under low pH condition. One large hydrophobic cluster (I) and one small hydrophobic cluster (II) residing in the peptide would be connected by the protonation of residues D16 and D20, and thereby exhibit strong membrane disruption activity in a low pH-dependent manner. Furthermore, the introduction of a positively charged residue enhanced the activity significantly, suggesting that a sole positively charged residue (H17) may be important for the interaction with target LPs by electrostatic interaction. Collectively, these results suggest that the pre-S1 (9–24) peptide may be involved in the endosomal escape of the BNC's payloads, as well as in the HBV uncoating process.

Mutual antagonism between hepatitis B viral mRNA and host microRNA let-7.

The interplay between viral and host factors plays a major role in viral pathogenesis. Hepatitis B virus (HBV) infection is a global health problem that leads to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV proteins have been studied extensively about their implication in hepatocarcinogenesis, the molecular mechanisms of oncogenesis are still largely unknown. A recent concept in gene regulation, in which competitive endogenous RNAs compete for common microRNAs (miRNAs), suggests that mRNA targets are key elements in the regulation of miRNA availability. Here, we show that HBV mRNA in the preS2 region can be targeted by host miRNA let-7 g. This leads to the sequestration of let-7 g and inhibition of let-7 g function. The expression of HBV transcripts, including the preS2 region, de-repressed let-7 g targets, which may contribute to long-term oncogenesis. HBV transcript-expressing transgenic mice, but not non-targeted transcript-expressing mice, were more prone to chemically induced hepatoocarcinogenesis. Let-7 target protein expression was upregulated in human HCC tissues derived from HBV-infected patients. On the other hand, let-7 g inhibited HBV preS2 protein expression and viral products. These results suggest that the interplay between viral intermediate transcripts during HBV replication and host miRNAs is crucial to the pathogenesis of chronic viral infection.

Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability.

Hepatitis B virus (HBV) is a driver of hepatocellular carcinoma, and two viral products, X and large surface antigen (LHBS), are viral oncoproteins. During chronic viral infection, immune-escape mutants on the preS2 region of LHBS (preS2-LHBS) are gain-of-function mutations that are linked to preneoplastic ground glass hepatocytes (GGHs) and early disease onset of hepatocellular carcinoma. Here, we show that preS2-LHBS provoked calcium release from the endoplasmic reticulum (ER) and triggered stored-operated calcium entry (SOCE). The activation of SOCE increased ER and plasma membrane (PM) connections, which was linked by ER- resident stromal interaction molecule-1 (STIM1) protein and PM-resident calcium release- activated calcium modulator 1 (Orai1). Persistent activation of SOCE induced centrosome overduplication, aberrant multipolar division, chromosome aneuploidy, anchorage-independent growth, and xenograft tumorigenesis in hepatocytes expressing preS2- LHBS. Chemical inhibitions of SOCE machinery and silencing of STIM1 significantly reduced centrosome numbers, multipolar division, and xenograft tumorigenesis induced by preS2-LHBS. These results provide the first mechanistic link between calcium homeostasis and chromosome instability in hepatocytes carrying preS2-LHBS. Therefore, persistent activation of SOCE represents a novel pathological mechanism in HBV-mediated hepatocarcinogenesis.

Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals.

Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21–47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21–47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants.

Different pre-S deletion patterns and their association with hepatitis B virus genotypes.

AIMTo investigate the associations of different types of pre-S deletions with hepatitis B virus (HBV) genotypes.METHODSThe sequences of the pre-S region, basal core promoter (BCP) mutation, and precore (PC) mutation were examined through direct DNA sequencing or clonal analysis and sequencing in 273 HBV carriers, namely 55 asymptomatic carriers, 55 carriers with chronic hepatitis (CH), 55 with liver cirrhosis (LC), 53 with liver cirrhotic hepatocellular carcinoma (LC-HCC), and 55 with noncirrhotic HCC. A total of 126 HBV carriers (46.2%) harbored pre-S deletions. The DNA sequences of pre-S deletion mutants from 43 age-matched genotype B (HBV/B)-infected carriers and 43 age-matched genotype C (HBV/C)-infected carriers were further examined, aligned, and compared.RESULTSNo significant difference was observed in the mean age distribution (P = 0.464), male sex (P = 0.805), viral load (P = 0.635), or BCP mutation (P = 0.117) between the HBV/B and HBV/C groups. However, the rate of PC mutation was significantly higher in the HBV/B-infected carriers than in the HBV/C-infected carriers (P = 0.003). Both genotypes exhibited a high rate of deletion in the C-terminal half of the pre-S1 region and N-terminus of the pre-S2 region (86.0% and 79.1% in the HBV/B group; 69.8% and 72.1% in the HBV/C group, respectively). Epitope mapping showed that deletion in several epitope sites was frequent in both genotypes, particularly pS1-BT and pS2-B2. Conversely, the rate of pS2-B1 deletion was significantly higher in the HBV/B group (72.1% vs 37.2%, P = 0.002), and the rate of pS2-T deletion was significantly higher in the HBV/C group (48.8% vs 25.6%, P = 0.044). Functional mapping showed that the rate of deletion in three functional sites (the nucleocapsid binding site, start codon of M, and site for viral secretion) located in the N-terminus of the pre-S2 region was significantly higher in the HBV/B group (P < 0.05). One type of N-terminus pre-S1 deletion mutant with deletion of the start codon of the L protein was frequently observed in the HBV/C group (20.9% vs 9.3%, P = 0.228), particularly in the LC patients (42.9% vs 12.5%). Different patterns of pre-S deletions were also found between the HBV/B and HBV/C groups according to different clinical outcomes. In CH patients, deletion in the site for polymerized human serum albumin was more frequent in the HBV/B group (88.9% vs 36.4%, P = 0.028). In the LC-HCC patients, the rate of deletion in the pre-S2 region was significantly higher in the HBV/B group than in the HBV/C group (P < 0.05).CONCLUSIONHBV/B- and HBV/C-infected carriers exhibit different patterns of pre-S deletion, which may be associated with the progression of liver diseases.

THU-111 - Deep Sequencing Analysis of Hepatitis B Virus Quasispecies Characteristics in the Pre-S Region Prior to Development of Hepatocellular Carcinoma

Poster Presentations: Viral hepatitis: Hepatitis A, B, D, E – clinical (except therapy): no. THU-111

Elucidation of the early infection machinery of hepatitis B virus by using bio-nanocapsule.

Currently, hepatitis B virus (HBV), upon attaching to human hepatocytes, is considered to interact first with heparan sulfate proteoglycan (HSPG) via an antigenic loop of HBV envelope S protein. Then, it is promptly transferred to the sodium taurocholate cotransporting polypeptide (NTCP) via the myristoylated N-terminal sequence of pre-S1 region (from Gly-2 to Gly-48, HBV genotype D), and it finally enters the cell by endocytosis. However, it is not clear how HSPG passes HBV to NTCP and how NTCP contributes to the cellular entry of HBV. Owing to the poor availability and the difficulty of manipulations, including fluorophore encapsulation, it has been nearly impossible to perform biochemical and cytochemical analyses using a substantial amount of HBV. A bio-nanocapsule (BNC), which is a hollow nanoparticle consisting of HBV envelope L protein, was efficiently synthesized in Saccharomyces cerevisiae. Since BNC could encapsulate payloads (drugs, genes, proteins) and specifically enter human hepatic cells utilizing HBV-derived infection machinery, it could be used as a model of HBV infection to elucidate the early infection machinery. Recently, it was demonstrated that the N-terminal sequence of pre-S1 region (from Asn-9 to Gly-24) possesses low pH-dependent fusogenic activity, which might play a crucial role in the endosomal escape of BNC payloads and in the uncoating process of HBV. In this minireview, we describe a model in which each domain of the HBV L protein contributes to attachment onto human hepatic cells through HSPG, initiation of endocytosis, interaction with NTCP in endosomes, and consequent provocation of membrane fusion followed by endosomal escape.

Evolutionary Changes of Hepatitis B Virus Pre-S Mutations Prior to Development of Hepatocellular Carcinoma.

Background and AimsDeletions/mutations in the hepatitis B virus (HBV) pre-S region have been associated with hepatocellular carcinoma (HCC). We aimed to study the evolutionary changes of pre-S mutations prior to HCC development.MethodsWe studied the HBV pre-S sequences at 1 to 10 years preceding diagnosis of HCC in 74 patients with HBV-related HCC (HCC group). 148 chronic hepatitis B patients matched for sex and age in 2:1 ratio, who had been followed up for at least 3 years without HCC (HCC-free group) were recruited as controls. 56 and 47 patients of HCC and HCC-free groups respectively had serially stored sera for longitudinally examination at 1–3 years, 4–6 years, 7–9 years and ≥10 years prior to the recruitment of the study.ResultsCompared to the HCC-free group, higher frequencies of pre-S deletions and point mutations (at 11 codons) were observed in the HCC group (p<0.05). Multiple logistic regression analysis showed that pre-S deletions, point mutations at codon 51 and 167 were independent factors associated with HCC. Longitudinal observation showed that pre-S deletions and most of the 11 HCC-associated pre-S point mutations existed at least 10 years before HCC development, and were more prevalent preceding HCC development in patients from HCC groups than HCC-free group. The number of HCC-associated pre-S point mutations increased over time preceding HCC development, and correlated positively with the time to HCC diagnosis (r = 0.220, p = 0.005).ConclusionsHigh prevalence and cumulative evolution of pre-S mutations preceding HCC development suggested a possible carcinogenic role of pre-S mutations and their potential application in HCC risk prediction.