Point mutation in the S gene of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr

Genomes of hepatitis B virus (HBV) were cloned from the plasma of a blood donor who carried subviral particles of three distinct subtypes in the following proportions: adr, 25%; ayr, 63%; and adyr, 12%. HBV DNA clones were classified into two groups based on a difference at only one nucleotide in the S gene. Two clones had A as nucleotide 365 that formed part of the codon for lysine as amino acid residue 122 and produced a surface antigen of subtype adr in transfected NIH 3T3 cells. The remaining four clones had G determining the codon for arginine and produced a surface antigen of subtype ayr in transfected cells. Similarly, HBV genomes were cloned from the plasma of an individual who carried subviral particles of subtypes adr (71%) and adwr (29%). Two clones had T and A as nucleotides 476 and 479, respectively. The other seven clones had C and G as the respective nucleotides. Based on a comparison with previously reported HBV genomes of various subtypes, the mutation of nucleotide 479, forming part of the codon for lysine or arginine as amino acid residue 160, was deduced to determine the w or r subtype, respectively. When NIH 3T3 cells were transfected separately with the genome of subtype adw or adr, derived from plasma containing a surface antigen of subtype adwr, and then cocultured, they produced subviral particles of either subtype adw or adr. When cells were transfected with the genomes of subtypes adw and adr simultaneously, however, subviral particles were produced that possessed w and r determinants on the selfsame particles. These results attributed the d/y or w/r subtypic change to a point mutation in the S gene and favored coinfection of hepatocytes with an HBV genome and its mutant as the mechanism of compound subtypes.     

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.     

A molecularly cloned hepatitis B virus produced in vitro is infectious in a chimpanzee.

The infectivity of hepatitis B virus (HBV) produced in vitro by HepG2 cells transfected with HBV DNA (HepG2T14) has been assayed in a chimpanzee. Following inoculation, the chimpanzee underwent a typical course of type B hepatitis infection, characterized by elevation of serum aminotransferases and by histological identification of hepatic damage. Hepatitis B surface antigen and core-related antigen appeared in the serum at weeks 5 and 7, respectively, after infection. HBV DNA was detected in serum samples, and replicative forms of the HBV genome were identified in liver biopsies. Subtype identification of hepatitis B surface antigen and restriction enzyme analysis of HBV DNA in both the inoculum and the serum of the infected chimpanzee confirmed that the hepatitis B infection observed in this animal was caused by viral particles produced by HepG2T14 cells. These findings indicate that, although HepG2 cells do not seem to be susceptible to infection by HBV in vitro, they can produce biologically active infectious virions after transfection with cloned HBV DNA.     

Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides.

A 21-mer oligodeoxynucleotide complementary to the polyadenylation signal for human hepatitis B virus (HBV) was complexed to a soluble DNA-carrier system that is targetable to hepatocytes via asialoglycoprotein receptors present on those cells. A cell line, HepG2 (2.2.15) that possesses asialoglycoprotein receptors and is permanently transfected with hepatitis B virus (ayw subtype) was exposed to complexed antisense DNA or controls. In the presence of complexed antisense DNA, the concentration of hepatitis B surface antigen in medium was 80% lower than controls after 24 h. Furthermore, during the next 6 days, there was no significant increase in surface antigen concentration in the presence of complexed antisense DNA. The inhibition could be effectively blocked by competition with an excess of free asialoglycoprotein. Total protein synthesis remained unchanged by exposure to complexed antisense sequences under identical conditions. In addition, HBV DNA in the medium and cell layers after 24-h exposure to complexed antisense sequences was 80% lower than in controls. The data indicate that antisense oligonucleotides complexed by a soluble DNA-carrier system can be targeted to cells via asialoglycoprotein receptors resulting in specific inhibition of hepatitis B viral gene expression and replication.     

Allelic subtypic determinants of hepatitis B surface antigen (i and t) that are distinct from d/y or w/r.

A monoclonal antibody (I-18) was raised against an enneapeptide representing amino acids 125 to 133 of the product of the S gene of hepatitis B virus DNA [S(125-133) segment] with a sequence of Thr-Ile-126-Pro-Ala-Gln-Gly-Thr-Ser-Met. Another monoclonal antibody (T-7) was raised against an S(125-133) segment in which Ile-126 was replaced by Thr-126. In a panel of 16 samples of hepatitis B surface antigen (HBsAg) with known S gene sequences, I-18 reacted with 5 with Ile-126. T-7 reacted with 10 HBsAg samples with Thr-126; it did not, however, react with the remaining one of subtype ayw with Thr-126 flanked by Met-125 and Thr-127. The two allelic subtypic determinants, specified by Ile-126 and Thr-126 and distinct from d/y or w/r, were named i and t after isoleucine and threonine, which regulate them. They were expressed in a mutually exclusive fashion in 216 (83%) of 260 HBsAg samples from asymptomatic carriers. They were not detected in 36 (14%) samples; the failure to detect an i or t determinant was particularly common in HBsAg samples of subtype ayw (26 [79%] of 33). A part of the S gene sequence was determined for eight HBsAg samples without a detectable i or t determinant. They had an Ile-126 or Thr-126 residue that was flanked by Thr-127, not the Pro-127 commonly possessed by HBsAg samples displaying an i or t determinant. Expression of the i/t allele, therefore, would require Pro-127. In eight (3%) of the samples, both i and t determinants were detected; the presence of i and t on the selfsame HBsAg particles was verified by sandwiching the particles between I-18 and T-7. A point mutation from thymine to cytosine at nucleotide 377 in the S gene, contributing different second letters to codon 126 (ATT for Ile and ACT for Thr), would have been responsible for the assembly of HBsAg particles with both i and t determinants by means of phenotypic mixing.     

Synthetic oligopeptides bearing a common or subtypic determinant of hepatitis B surface antigen.

Two determinants of hepatitis B surface Ag (HBsAg), identified by mAb raised against polypeptide components, were characterized immunochemically. One was expressed on HBsAg irrespective of the four major subtypes, i.e., adw, adr, ayw, and ayr, whereas the other was subtypic but not identical to any of d, y, w, and r determinants. The common determinant was generated by a synthetic pentadecapeptide with a sequence of Thr-Thr-Ser-Thr-Gly-Pro-Cys-Lys-Thr-Cys-Thr-Ile-Pro-Ala-Gln representing amino acids 115-129 of the S gene product, and detected invariably in 366 HBsAg samples in sera from asymptomatic carriers in Japan. The activity of the S gene product, as well as the peptide (115-129), to bind with the mAb was not affected by alkylation alone, but was completely lost after reductive alkylation. The antigenic activity was lost when the S gene product was severed between Lys122 and Thr123 by trypsin. A microconformation maintained by the -Cys121-Cys124 bond, therefore, would be required for the common determinant. The other mAb identified an epitope of HBsAg that was mimicked by a synthetic tetradecapeptide with a sequence of Thr-Cys-Thr-Ile-Pro-Ala-Gln-Gly-Thr-Ser-Met-Phe-Pro-Ser, representing amino acids 123-136 of the S gene product. Among 16 HBsAg samples with known S gene sequences, 5 with Ile126 possessed this subtypic determinant, but the remaining 11 with Thr126 did not. The 5 hepatitis B virus genomes encoding the subtypic determinant differed less than 5.6% from each other in the entire nucleotide sequence, but by 8.0% or more from any of the other 11 genomes without the capacity to encode it.     

Organization and expression of hepatitis B sequences cloned from hepatocellular carcinoma tissue DNA

We have constructed a phage lambda library of liver DNA fragments from West African patient who died of liver failure due to advanced hepatocellular carcinoma. Four hepatitis B virus (HBV) DNA-carrying recombinants have been isolated, one clone (lambda IA22) being analyzed in greatest detail. It contains approximately 3.8 kb of HBV DNA without detectable deletions or rearrangements. One site of integration lies close to the nick in free viral DNA. The restriction map of the HBV sequences is close to those published for the ay subtype. Coconvection of mouse Ltk- cells with lambda IA22 and cloned thymidine kinase gene results in the expression of gene S and the excretion of hepatitis B surface antigen (HBsAg) particles into the culture supernatant.     

A case of hepatitis B reactivation due to the hepatitis B virus escape mutant in a patient undergoing chemotherapy

A 62-year-old man had chronic hepatitis B virus (HBV) infection and was diagnosed with liver cirrhosis. At the time of diagnosis the patient’s virologic markers were positive for hepatitis B surface antigen (HBsAg), antibody to hepatitis B e antigen (anti-HBe) and antibody to hepatitis B core antigen (anti-HBc), while antibody to hepatitis B surface antigen (anti-HBs) and HBV DNA were negative. Later the patient received chemotherapy for malignancy. However, this was interrupted due to elevated liver enzymes. At the same time HBV DNA became positive. Lamivudine (LMV) therapy was administered immediately. However, the levels of serum aminotransferase and total bilirubin (TB) were still rising. Finally the patient died of fulminant hepatic failure. A sequence revealed HBV genotype C (HBsAg subtype adw) with immune escape mutations, F8L, S34L, F41S, G44V, F93C, V96G, L110I, C149Y and F161Y. The high morbidity and mortality of this complication is one of the major obstacles to completing the standard treatment for malignancy in HBV carriers. Therefore, the relative risk of antiviral prophylactic failure should be further assessed and the optimal strategy for antiviral prophylaxis in HBsAg-positive patients with oncologic and hematologic malignancies undergoing chemotherapy should be revised.     

一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究

为了研究乙肝病毒侵染肝细胞过程中的功能蛋白 ,通过印迹免疫分析技术从人肝cDNA噬菌体表达库中筛选出一株编码乙肝表面抗原结合蛋白 (hepatitisBsurfaceantigenbindingprotein ,HBsAg BP)的cDNA克隆 .基因测序结果表明 ,该cDNA具有独立的开放阅读框架 ,编码 1个由 344个氨基酸残基构成的可溶性蛋白分子 ,属于免疫球蛋白超家族成员 .将该基因克隆到原核表达载体pTriplEx后 ,在E .coliXL1 Blue菌株中获得 4 4kD的重组蛋白 .重组蛋白经Western印迹和ELISA实验证明具有与乙肝表面抗原特异性结合的能力 .进一步经流式细胞仪实验显示 ,在纯化的重组蛋白存在的情况下 ,天然的HBsAg与肝细胞株HepG2的亲和力显著增高 .结果显示 ,该乙肝表面抗原结合蛋白可能是介导乙肝病毒对肝细胞亲和侵染的可溶性辅助受体 .     

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.     

Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.     

Stable expression and integrated hepatitis B virus genome in a human hepatoma cell line

HepG2.2.15 cell is a widely used cell model for studying HBV (hepatitis B virus) in vitro. In these cells, the HBV genome is integrated in several sites of HepG2 cellular DNA. These multiple copies may have some influence on the cellular processes. We constructed a new plasmid, pSEH-Flag-HBV, and transfected it into HepG2 cells, and then screened it with hygromycin. We then used ELISA, PCR, and RT-PCR to detect the expression of HBV in these cell lines. A cell line that stably expressed hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) was established. Using Southern blotting analysis, we found that the HBV genome was integrated as a single copy in the cellular DNA. This cell line will be a useful alternative model for HBV studies.     

Naturally occurring escape mutants of hepatitis B virus with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen.

Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.     

Presentation of an immunodominant T-cell epitope of hepatitis B surface antigen by the HLA-DPw4 molecule

Human T cells that recognize a major epitope of the hepatitis B surface antigen were studied for their ability to react with antigen when presented by mouse fibroblasts that express class II products of the human major histocompatibility gene complex after gene transfection. L cells expressing HLA-DPw4, but not those expressing HLA-DR4 or HLA-DR7, induced strong proliferative responses of antigen-specific T cells to either hepatitis B surface antigen or the synthetic peptide S1d, which bears the immunodominant T-cell epitope. These results identified a genetic restriction element of human helper T-lymphocyte responses to a major antigenic determinant of hepatitis B virus and might be important in the design of subunit vaccines to this pathogen. Peptides that induce T-cell responses that are restricted by a frequently encountered major histocompatibility complex molecule in the general population such as DPw4 would be ideal candidates as subunit vaccines.     

Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization

The complete nucleotide sequence of hepatitis B virus (HBV) DNA from Dane particles of subtype adr was determined. The 3215-bp sequence showed the presence of genes for the surface antigen (226 amino acids) and core antigen (183 amino acids), in addition to two (long and small) open reading frames (ORFs) capable of coding the 843 and 154 amino acids. These ORFs differed from those of the other adr clones so far reported [Ono et al., Nucl. Acids Res. 11 (1983) 1747–1757; Fujiyama et al., Nucl. Acids Res. 11 (1983) 4601–4610]. The gene organization of HBV DNA was found to be well conserved irrespective of subtype. The direct repeat of the undecanucleotide sequence near the 5′ ends of the short (S) and long (L) strands of HBV DNA and the two small direct repeats between both 5′ ends were found to be characteristic structures.     

Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions.

Clonal cells derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete hepatitis B surface antigen particles, nucleocapsids, and virions (M. A. Sells, M.-L. Chen, and G. Acs, Proc. Natl. Acad. Sci. USA 84:1005-1009, 1987) which elicit acute hepatitis in chimpanzees (G. Acs, M. A. Sells, R. H. Purcell, P. Price, R. Engle, M. Shapiro, and H. Popper, Proc. Natl. Acad. Sci. USA 84:4641-4644, 1987). We report here the initial characterization of the viral nucleic acids produced in this culture system. Kinetic analyses of nuclear, cytoplasmic, and extracellular HBV DNAs were performed by Southern blotting with radiolabeled HBV strand-specific probes. The results from these analyses indicate that at the stationary cellular growth phase, there is a dramatic increase in the rate at which HBV DNA accumulates. Incomplete double- and single-stranded forms of the HBV genome were detected in the nuclear and cytoplasmic fractions as well as in the extracellular medium. In addition, the nuclear DNA apparently includes multiple complete copies of the HBV genome chromosomally integrated and full-length covalently closed circular HBV DNA. Multiple HBV-specific polyadenylated RNAs with lengths of 3.5, 2.5, and 2.1 kilobases were identified by Northern (RNA) blot analysis. S1 nuclease mapping and primer extension identified a single 3' end and multiple unique initiation sites corresponding to nucleotides just 5' to the pre-S1 region, as well as upstream and within the pre-S2 and precore regions. The nucleic acid profile obtained from these analyses is essentially a facsimile of that obtained by studying liver tissue from HBV-infected individuals.