Hepatitis B Virus Large Surface Protein Research Articles

Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

Canocapavir Is a Novel Capsid Assembly Modulator Inducing a Conformational Change of the Linker Region of HBV Core Protein.

Canocapavir is a novel antiviral agent with characteristics of core protein allosteric modulators (CpAMs) that is currently in a phase II clinical trial for treatment of hepatitis B virus (HBV) infection. Herein, we show that Canocapavir prevented the encapsidation of HBV pregenomic RNA and increased the accumulation of cytoplasmic empty capsids, presumably by targeting the hydrophobic pocket at the dimer-dimer interface of HBV core protein (HBc). Canocapavir treatment markedly reduced the egress of naked capsids, which could be reversed by Alix overexpression through a mechanism other than direct association of Alix with HBc. Moreover, Canocapavir interfered with the interaction between HBc and HBV large surface protein, resulting in diminished production of empty virions. Of particular note, Canocapavir induced a conformational change of capsids, with the C-terminus of HBc linker region fully exposed on the exterior of capsids. We posit that the allosteric effect may have great importance in the anti-HBV activity of Canocapavir, given the emerging virological significance of HBc linker region. In support of this notion, the mutation at HBc V124W typically recapitulated the conformational change of the empty capsid with aberrant cytoplasmic accumulation. Collectively, our results indicate Canocapavir as a mechanistically distinct type of CpAMs against HBV infection.

The Genotype (A to H) Dependent N-terminal Sequence of HBV Large Surface Protein Affects Viral Replication, Secretion and Infectivity

Genetic variability has significant impacts on biological characteristics and pathogenicity of hepatitis B virus (HBV), in which the N-terminal sequence of the presurface 1 (preS1) region of HBV large surface protein (LHBs) displays genotype (GT) dependent genetic heterogeneity. However, the influence of this heterogeneity on its biological roles is largely unknown. By analyzing 6560 full-length genome sequences of GTA-GTH downloaded from HBVdb database, the preS1 N-terminal sequences were divided into four representative types, namely C-type (representative of GTA, GTB, and GTC), H-type (GTF and GTH), E-type (GTE and GTG), and D-type (GTD), respectively. We artificially substituted the preS1 N-termini of GTC and GTD plasmids or viral strains with each sequence of the four representative types. The roles of preS1 N-terminus on HBV replication, secretion and infectivity were investigated using HepG2 or HepG2-NTCP cells. In the transfection experiments, the results showed that the extracellular HBsAg levels and HBsAg secretion coefficients in D- and E-type strains were significantly higher than those in C- and H-type strains. D-type strain produced more extracellular HBV DNA than C-type strain. We further observed that D-, H-, and E-type strains increased the levels of intracellular replicative HBV DNAs, comparing with C-type strain. In the infection experiments, the levels of extracellular HBeAg, intracellular HBV total RNA and pgRNA/preC mRNA in D- and E-type strains were markedly higher than C and H-type ones. Our data suggest that the preS1 N-termini affect HBV replication, secretion and infectivity in a genotype dependent manner. The C- and H-type strains prefer to attenuate HBsAg secretion, while the strains of D- and E-type promoted infectivity. The existence and function of the intergenotypic shift of preS1 in naturally occurring recombination requires further investigation, as the data we acquired are mostly related to recombinant preS1 region between N-terminus of preS1 from genotypes A-H and the remaining preS1 portion of GTC or GTD.

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.

The clinical significance of the detection of hepatitis B virus large surface protein for the diagnosis and treatment

Objective To explore the correlation between hepatitis B virus large surface protein(HBV-LP)and hepatitis B virus deoxyribonucleic acid(HBV-DNA),analyze the clinical significance of detecting the two index dynamically for diagnosis and treatment of hepatitis B.Methods Enzyme linked immunosorbent assay(ELISA)and fluorescent quantitation polymerase chain reaction(FQ-PCR)were used to detect the levels of HBV-LP and HBV-DNA in serum specimen of 230 hepatitis B patients.Results There was a positive correlation between the content of HBV-LP and copy numbers of HBV-DNA in serum of hepatitis B positive cases(r=0.84,P＜0.01).The positive rate was 84.78％ in HBV-LP and 84.35％ in HBV-DNA of 230 HBV positive cases.As a result there was no significant difference(P＞0.05)in this study.Therefore,the positive rate was 63.63 ％(35/55)in HBV-LP and 58.18％(32/55)in HBV-DNA of 55 HBV negative cases.There was also no significant difference(P＞0.05)in this study.However,the positive rate of HBV-LP(82.47％)was higher than HBE(52.06％)in the 194 HBV-DNA positive cases.There was significantly different(P＜0.01).Conclusion HBV-LP and HBV-DNA are the significant index of the degree of hepatitis B virus replication in hepatitis B positive cases,especially in determining the virus replication and prognosis of treatment in HBe Ag negative,providing the reliable laboratory data for the clinical diagnosis and treatment. Key words: Hepatitis B large surface protein; Hepatitis B virus; Virus replication

Hepatitis B virus large surface protein in serum as a candidate biomarker for evaluating hepatitis B virus infection

Aim To develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of serum hepatitis B virus large surface protein (HBV-LP), and study the clinical value of HBV-LP. Methods Serum HBV-LP levels and a panel of other HBV markers were investigated in a large population of patients with chronic HBV. The clinical value of HBV-LP was evaluated by comparing the coincidence of detection of HBV markers and the change of serum HBV-LP level during antiviral therapy. Results The ELISA was found to be sensitive and specific for the detection of HBV-LP. Serum HBV-LP level was positively correlated with HBV DNA (r = 0.743) in HBV patients. Among the five HBV markers tested, HBV-LP displayed the highest coincidence rate (94.7%) with HBV DNA. Conclusions Serum HBV-LP was strongly correlated with HBV DNA. This ELISA therefore offers a promising approach for the diagnosis and treatment monitoring of HBV patients.

Detecting the occult HBV infection: a laboratory study

To see the HBV DNA detection instance in the HBsAg negative people and to study the serological method detection strategy for detecting hepatitis B virus large surface protein (HBLP) to filtrate the occult HBV infection. The HBsAg negative serum samples were divided into HBsAb negative and positive two species according to the hepatitis B virus markers (HBVM) in daily work excepting the special HBVM modes. Total 2000 stochastic serum samples with 1000 HBsAb negative results and 1000 HBsAb positive results were collected from the copy tubes to detect HBVM with national ELISA reagent kits and put them -20 degrees C frostily. Mixed samples (8 x 30 microl) were analyzed with fluorescence quantitative PCR (FQ-PCR) and filtrated the individual positive samples. The filtrated samples were doubly tested again with American MONOLISA HBsAg ULTRA reagents. No HBV DNA positive results were found out from the 1000 HBsAb positive samples and 19 cases HBV DNA positive results were found out from the 1000 HBsAb negative samples. On these 19 samples, the HBsAg results from the American MONOLISA HBsAg ULTRA reagents were all positive and the HBLP results were all positive, too. The 19 HBV DNA quantitative results were divided into 2 cases more than 500 copies/ml, 3 cases between 400-500 copies/ ml, 3 cases between 300-400 copies/ml, 7 cases between 200-300 copies/ml and 4 cases between 100-200 copies/ml. The leaked samples tested HBsAg with national reagents are mostly from the HBsAb negative people. HBLP results may be positive on these samples and detecting HBLP marker is propitious to filtrate the occult HBV infection. This study provided a kind of serological reference for actively searching for the detecting strategy in occult HBV infection field.

Hepatitis B virus surface antigen interacts with acid alpha-glucosidase and alters glycogen metabolism

Hepatitis B virus (HBV) infection is highly correlated with hepatocellular carcinoma. Previous studies have reported that expression of hepatitis B virus pre-S2 mutant surface antigen is related to hepatoma development. An aberrant carbohydrate metabolism is a hallmark of malignant transformation. We performed yeast two-hybrid screening with HBV pre-S2-del large surface protein (pre-S2Delta) by using human liver cDNA library, and identified the acid alpha-glucosidase (acid alpha-glucosidase) as the novel cellular interacting protein of pre-S2Delta. The association of pre-S2Delta with the acid alpha-glucosidase was confirmed by confocal immunofluorescence and co-immunoprecipitation assay. Further, the acid alpha-glucosidase activity and glycogen content were analyzed in ML-1 cells expressing pre-S2Delta. The interaction between HBV large surface protein and acid alpha-glucosidase was demonstrated with co-immunoprecipitation in vitro and in vivo, and the binding was mediated through c-terminal region 889-952 amino acid of acid alpha-glucosidase. On the other hand, HBV large surface protein interacted with acid alpha-glucosidase through N-terminal region 1-157 amino acid of HBV large surface protein. Expression of HBV large surface protein enhanced acid alpha-glucosidase activity and resulted in decrease of cellular glycogen. Our result demonstrates that HBV large surface protein interacts with acid alpha-glucosidase which plays an important role in glycogen balance. Together, these data suggest a novel pathway by which HBV large surface protein affects carbohydrate metabolism.

Detecting hepatitis B virus large surface protein to filtrate the occult HBV infection.

Detecting hepatitis B virus large surface protein (HBLP) with serological method to filtrate the occult HBV infection and study the clinical detection strategy. Two thousands HBsAg negative stochastic serum samples were collected from the copy tubes in daily work to detect hepatitis B Virus markers (HBVM) with national EHSA regent kits and put them -20 degrees C frostily. The 2000 samples were detected with HBLP and filtrated the positive samples. The HBsAg markers were doubly counterchecked with other two adding kinds of national ELISA regent kits (total 3 kinds) at the filtrated samples. The last samples were doubly tested again with American MONOLISA HBsAg ULTRA regents. HBV DNA levels were quantitative analyzed with fluorescence quantitative PCR (FQ-PCR) and taking the mean results. Fifteen HBLP positive samples were detected out from the 2000 serum samples. We educed the conform negative results from the three kinds of national regents but conform positive results from the American MONOLISA HBsAg ULTRA regents in repeating HBsAg detection at the 15 samples. The HBV DNA FQ-PCR quantitative results were all less than 500 copies/mi and divided into two cases hetween 400-500 copies/nil, three cases 300-400 copies/ml, five cases 200-300 copies/ml, four cases 100-200 copies/ml and one case was less than l00copies/ml. The false HBsAg negative results for serum samples are more generally through national regents than through importations and HBLP results mayhe are positive in these samples. Detecting HBLP marker is propitious to filtrate the occult HBV infection. This study provided a kind of serological reference for actively searching for the detecting strategy in occult HBV infection field.

Application of serum hepatitis B virus large surface protein detection in the therapy of hepatitis B by adefovir

Objective Detecting the serum hepatitis B virus large surface protein(HBV-LP) and hepatitis B virus DNA(HBV-DNA) load to study the correlation between HBV-LP and HBV-DNA and to evaluate the application value of the effect of HBV-LP on antiviral therapy.Methods HBV-DNA was detected by fluorescent quantitative PCR(FQ-PCR),and serum HBV-LP was detected by enzyme linked immunosorhent assay(ELISA).Results Both HBV-DNA and HSV-LP in the patients who were treated effectively with adefovir decreased,HBV-DNA in the patients who were treated ineffectively with adefovir had never turned negative and HBV-LP retained in high level and HBV-LP showed in high level or no change while HBV-DNA had turned negative temporally in the patients having relapsed symptoms to adefovir.Conclusion Measuring serum HBV-LP dynamically can be useful in evaluation of adefovir therapeutic effect. Key words: Hepatitis B virus; Hepatitis B vires large surface protein; Antiviral therapy

Application of serum hepatitis B virus large surface protein determination in lamivudine anti-viral therapy for patients with hepatitis B

To study the correlation between serum hepatitis B virus large surface protein (HBV-LP) and hepatitis B virus DNA (HBV-DNA) load in hepatitis B patients treated with lamivudine, and the application of serum HBV-LP measurement in evaluation of lamivudine therapeutic effect and end point judgement. HBV DNA was detected by fluorescent quantitative PCR (FQ-PCR), serum HBV-LP was detected by enzyme linked immunosorbent assay (ELISA). Both HBV-DNA and HBV-LP decreased in patients responding to lamivudine therapy, and HBV-LP turned negative 3 months later than HBV-DNA. In patients resistant to lamivudine therapy, neither HBV-DNA nor HBV-LP turned negative. In patients whose symptoms relapsed, HBV-LP didn't turn negative, while HBV-DNA was undetectable only transitorily. Measuring serum HBV-LP dynamically can be useful in evaluation of lamivudine therapeutic effect.

Endoplasmic Reticulum Stress Stimulates the Expression of Cyclooxygenase-2 through Activation of NF-κB and pp38 Mitogen-activated Protein Kinase

Expression of mutant proteins or viral infection may interfere with proper protein folding activity in the endoplasmic reticulum (ER). Several pathways that maintain cellular homeostasis were activated in response to these ER disturbances. Here we investigated which of these ER stress-activated pathways induce COX-2 and potentially oncogenesis. Tunicamycin and brefeldin A, two ER stress inducers, increased the expression of COX-2 in ML-1 or MCF-7 cells. Nuclear translocation of NF-kappaB and activation of pp38 MAPK were observed during ER stress. IkappaBalpha kinase inhibitor Bay 11-7082 or IkappaBalpha kinase dominant negative mutant significantly inhibited the induction of COX-2. pp38 MAPK inhibitor SB203580 or eIF2alpha phosphorylation inhibitor 2-aminopurine attenuated the nuclear NF-kappaB DNA binding activity and COX-2 induction. Expression of mutant hepatitis B virus (HBV) large surface proteins, inducers of ER stress, enhanced the expression of COX-2 in ML-1 and HuH-7 cells. Transgenic mice showed higher expression of COX-2 protein in liver and kidney tissue expressing mutant HBV large surface protein in vivo. Similarly, increased expression of COX-2 mRNA was observed in human hepatocellular carcinoma tissue expressing mutant HBV large surface proteins. In ML-1 cells expressing mutant HBV large surface protein, anchorage-independent growth was enhanced, and the enhancement was abolished by the addition of specific COX-2 inhibitors. Thus, ER stress due either to expression of viral surface proteins or drugs can stimulate the expression of COX-2 through the NF-kappaB and pp38 kinase pathways. Our results provide important insights into cellular carcinogenesis associated with latent endoplasmic reticulum stress.