The GDPAL region of the pre-S1 envelope protein is important for morphogenesis of woodchuck hepatitis virus

The pre-S envelope protein of duck hepatitis B virus (DHBV) contains a region, Asp-Asp-Pro-Leu-Leu (DDPLL), that is specifically required for virus assembly and secretion (Lenhoff and Summers, J Virol 1994;68:4565-4571). We found that amino acids 201 to 205 of the pre-S envelope protein of woodchuck hepatitis virus (WHV) form a conserved amino acid cluster, Gly-Asp-Pro-Ala-Leu (GDPAL), which resembles the DDPLL sequence of DHBV. To determine whether the GDPAL region was functionally equivalent to the DDPLL region, we deleted this region from the pre-S protein of WHV or mutated individual amino acids within the region. The mutant DNA was transfected into human hepatoma cell line Huh7, and the medium was assayed for virion production by immunoprecipitation and Southern blot analysis. We found that an in-frame deletion of this small region inhibited virion formation, suggesting that the GDPAL region of the pre-S envelope protein was required for virus assembly and/or secretion of WHV. Individual replacement of alanine 204, leucine 205, or serine 206 with other amino acid residues did not affect virus production. However, substitution of either aspartic acid 202 with valine or proline 203 with leucine dramatically inhibited WHV production. Furthermore, the GDPAL mutants were individually tested for their abilities to complement a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not required to function as part of the pre-S2 protein.     

Hepatitis B virus and hepatocellular carcinoma

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.     

Fibronectin of human liver sinusoids binds hepatitis B virus: identification by an anti-idiotypic antibody bearing the internal image of the pre-S2 domain

Anti-idiotypic antibodies (anti-Ids) have been successfully used to characterize and isolate receptors of several cell ligands. To prepare an immunological probe for identification of cellular components interacting with the hepatitis B virus (HBV), polyclonal antisera against a panel of five HBV-specific monoclonal antibodies (MAbs) were produced in syngeneic BALB/c mice. MAbs to HBV used for immunization (Ab1) recognized biologically important and potentially neutralizing epitopes, located in the pre-S1, pre-S2, or S region-encoded domains of HBV proteins. All the anti-Ids (Ab2) were specific to idiotopes of the homologous Ab1 and inhibited their interaction with the corresponding viral epitopes, suggesting that they recognized unique determinants on the paratope of each immunizing Ab1. Therefore, all five generated polyclonal anti-Ids were of the Ab2 beta type and could represent internal images of viral epitopes. Ab2 raised against the pre-S2 region-specific MAb F124 bound to the extracellular matrix fibronectin of human liver sinusoids. Immunohistochemical studies demonstrated the attachment of viral and recombinant (S, M) hepatitis B surface antigen particles with the pre-S2 region-encoded epitopes to the fibronectin of human liver sinusoids. In contrast, recombinant (S, L*) hepatitis B surface antigen particles, in which the epitope recognized by F124 MAb was not expressed, did not show any binding capacity. These findings suggest that human liver fibronectin may bind HBV in vivo by the pre-S2 region-encoded epitopes in a species-restricted manner. Furthermore, binding of the circulating virus to liver sinusoids could facilitate its subsequent uptake by hepatocytes.     

Point mutations in the S and pre-S2 genes observed in two hepatitis B virus carriers positive for antibody to hepatitis B surface antigen

Two hepatitis B virus (HBV) carriers who had antibodies to HBV surface antigen (anti-HBs) were studied. Case 1 was a 47 year old woman positive for hepatitis B e antigen (HBeAg), and case 2 was a 61 year old man positive for antibody to HBeAg (anti-HBe) and DNA-polymerase (DNA-p). Neither case had received the HBV vaccine. The nucleotide sequences of the HBV-DNA extracted from the patients' sera were determined within the pre-S2 and S genes. Seven out of nine S gene clones from case 1 and six out of nine S gene clones from case 2 had an amino acid replacement from Thr or Ile to Ser at codon 126 in the alpha-determinant of the S gene. Amino acid substitution of codon 145 of the S gene previously reported was not observed. Although two previous reports on HBV escape mutant carriers with both anti-HBs and HBeAg described some deletions in the pre-S2 gene, our cases did not show these deletions. Our analysis indicated that carriers with the HBV escape mutant did not always have pre-S2 gene deletions. We found two HBV escape mutant carriers who had amino acid substitutions at codon 126 in the S gene due to point mutation without any deletions in the pre-S2 gene.     

Absence of mutations in the p53 tumor suppressor gene in woodchuck hepatocellular carcinomas associated with hepadnavirus infection and intake of aflatoxin B1

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4-9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4-9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated a low rate of p53 mutations in HCCs of AFB1-treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development of p53 mutations less than in humans.     

Fibrosing cholestatic hepatitis in a transplant recipient with hepatitis B virus precore mutant

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for end-stage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G-->A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.     

Role of surgical interventions in the treatment of patients with trophoblastic tumor resistant to chemotherapy

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.     

Detection of the putative E2 protein of hepatitis C virus in human liver

The question was asked whether a predicted envelope protein, considered to be processed from the polyprotein precursor encoded by the putative E2/NS1 region of the hepatitis C virus (HCV) genome, may be observed in HCV-infected humans. Two polyclonal antibodies against recombinant E2/NS1 proteins were prepared and their reactivity tested against liver extracts from HCV-infected patients by immunoblotting analysis. A band corresponding to a size of 44 kDa was detected in liver extracts from patients who were positive for the HCV-specific antibody anti-C100-3 but not in liver extracts from patients who did not have anti-C100-3 antibody. Additionally, no band was detected using preimmune sera or antisera which had been preabsorbed with recombinant E2/NS1 proteins. Deglycosylation studies demonstrated that the 44 kDa protein was a glycosylated form of a 38 kDa protein which corresponds to the predicted molecular weight of the putative E2/NS1 protein. These results suggest that the 44 kDa protein is a product of the E2/NS1 region. Frequent observation of the 44 kDa band in cases of chronic active hepatitis C suggests a correlation between the expression of this protein and the progression of hepatitis.     

Genotyping by multiplex polymerase chain reaction for detection of endemic hepatitis B virus transmission

A nested polymerase chain reaction (PCR) protocol was developed for rapid genotyping of hepatitis B virus (HBV). During the first PCR round, a universal HBV primer pair was used to amplify the entire pre-S region of the HBV genome. Within the pre-S region, many nucleotide exchanges are observed. These are partly correlated to the serological hepatitis B surface antigen subtypes. Five additional subtype-specific primers were selected from that region which, together with two universal non-group-specific primers, generated specific combinations of two to four DNA fragments of defined sizes. By this approach, 55 hepatitis B surface antigen-positive patients from a pediatric oncology unit in Germany were analyzed. Fifty-four patients who had been infected within 2 years had an identical pattern in the multiplex PCR, suggesting a common source of infection and person-to-person transmission within the unit. One child who was infected 5 years later had a different PCR pattern and, therefore, must have been infected from a different source. Furthermore, 109 serum samples taken from pregnant Cameroonian women and 25 serum samples from their babies taken 6 months after birth were analyzed. In one case, mother-to-infant transmission of the virus was demonstrated. Apart from its role in epidemiological studies on HBV, multiplex PCR may also be a useful tool for rapid genetic analysis in other fields if there is a moderate degree of sequence variation which enables the design of specific primers.     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

Proteolysis of the carboxyl-terminal GPI signal independent of GPI modification as a mechanism for selective protein secretion

Variable amounts of soluble forms of a variety of glycosyl-phosphatidylinositol (GPI)-anchored proteins occur extracellularly, but the molecular mechanisms governing their release are not entirely clear. When the GPI-anchored folate receptor (FR) type beta was expressed transiently in human 293 fibroblasts, there was a roughly equal distribution of [3H]folic acid binding protein between the cell surface and the medium after 24 h over a wide range of expression levels of FR-beta. The difference in apparent molecular masses between the soluble FR-beta and the PI-PLC-treated membrane protein indicated that the former was not released from the membrane by the action of phospholipase. Brefeldin A inhibited the release of soluble FR-beta from both the transfected 293 cells and stable recombinant CHO (CHO-FR-beta) cells while pre-existing levels of cell surface FR were unaltered suggesting the absence of a precursor-product relationship between the membrane-associated FR-beta and the soluble protein in the medium. [35S]Cysteine pulse-chase analysis was consistent with this finding. Interchanging of carboxyl-terminal peptides between FR-beta and FR-alpha revealed that the nature of the processed signal for GPI modification was responsible for the quantitative membrane anchoring of FR-alpha and the production of soluble FR-beta. When total cell lysates were analyzed by Western blot, a diffuse band of apparent 41 kDa and three additional sharp bands of apparent 35, 33, and 29.3 kDa were seen. The 41 kDa band was identified as the PI-PLC sensitive cell surface receptor. Several mutant constructs of FR-beta, in which the carboxyl-terminal signal for GPI modification was either disrupted or deleted only gave the three lower bands. The three sharp bands from the wild-type and the mutant forms of FR-beta were identified as nonglycosylated (29.3 kDa) or glycosylated polypeptides in which the carboxyl-terminal peptide was at least partially proteolyzed without GPI modification. All of the mutations in the GPI signal resulted in the recovery of [3H]folic acid binding protein in the media which, similar to the wild-type FR recovered from the media, were converted to the 29.3 kDa band by N-glycanase. The results from this study indicate that a carboxyl-terminal peptide in FR-beta is efficiently proteolyzed intracellularly by a pathway that is independent of GPI signal recognition resulting in proper protein folding and secretion. Such carboxyl-terminal sequences could represent a simple adaptation for proteins whose physiologic functions reside both at the cell surface and in extracellular fluids, allowing their selective and tissue-specific release.     

Analysis of hepatitis B virus populations in an interferon-alpha-treated patient reveals predominant mutations in the C-gene and changing e-antigenicity

It is largely unknown whether hepatitis B virus (HBV) sequence variation during chronic infection hampers HBV immune recognition or the antiviral effect of cytokines on HBV production. Here we have analyzed which region of the HBV genome changes most drastically during an interferon-alpha (IFNalpha)-stimulated immune response. In addition, we have investigated whether the mutations affect viral replication, gene expression, and immune recognition of the mutant viral proteins. The study was performed with full-length HBV genomes taken longitudinally from a patient who transiently cleared HBV and seroconverted to anti-HBe during a long-term IFNalpha treatment. We found a replacement of the predominant virus population during IFNalpha therapy The virus populations differed mainly by a cluster of nucleotide changes in the C-gene and a pre-S2 deletion. Most of the newly emerging mutations localized within core/HBe B-cell epitopes, changed HBe antigenicity toward mono- and polyclonal antibodies, and also influenced the reactivity of the anti-HBc/e antibodies of the patient. All genomes tested expressed less HBeAg than wild-type HBV, while replication and IFNalpha susceptibility were similar. These data indicate that IFNalpha therapy can lead to the emergence of HBV variants with mutations mainly affecting recognition of the core/HBe proteins by antibodies. Taken together, the type of core/HBe-specific B-cell immune response, the sequence of the corresponding epitopes, and the HBe expression level appear to contribute to the decision on viral clearance or persistence.     

Changes in ceftazidime concentration in the CSF following overdose in acute kidney failure]

The diagnosis of liver diseases induced by hepatitis B virus (HBV) is supported by the detection of HBV surface antigen (HBsAg) in serum. The present study aimed to investigate the presence of HBV deoxyribonucleic acid (DNA) in patients with liver cirrhosis using a polymerase chain reaction (PCR) based on primers derived from the pre-S1 and pre-core regions. HBsAg was detected in 10 of 48 patients (21%), total anti-hepatitis B core antigen (HBc) antibodies in 54%, anti-hepatitis B e antigen (HBeAg) in 14.6%, anti-HBc immunoglobulin M in 8%, and anti-HBs in 26%; none had detectable HBeAg. HBV DNA was detected in 73% of the cirrhotic patients. All cirrhotic patients with HBsAg also had HBV DNA; HBV DNA was detected in 64.5% of those without HBsAg. We conclude that the clearance of HBsAg does not necessarily indicate termination of viraemia in patients with liver cirrhosis and the detection of HBV DNA using a PCR based on primers from the pre-S1 and pre-core regions should be included in the diagnosis of HBV infection.     

In vivo selection of duck hepatitis B virus pre-S variants which escape from neutralization

To better understand the role of specific residues within the duck hepatitis B virus (DHBV) pre-S protein in neutralization and infectivity, we have selected and identified pre-S variants which escape neutralization. A highly neutralizing monoclonal antibody (Mab 900) which recognizes an epitope 83IPQPQWTP90 localized previously on the DHBV pre-S protein, within a region suspected to mediate the virus interaction with hepatocytes, was used as immune pressure. After only two in vivo neutralization rounds with Mab 900, five different pre-S mutant genomes were identified, which harbored point mutations affecting only proline residues located at position 90 within this epitope (83IPQPQWTP90) and/or at a distance at position 5. We have shown that a single (P5L) or double proline (P5L + P90H) substitution affect neither virus replication capacity nor in vivo infectivity. However, the P5 mutation reduces mutant recognition by Mab 900 twofold, while the substitution of both prolines 5 and 90 almost completely abolishes mutant P5L + P90H reactivity with this Mab and leads to a decrease of neutralization. Therefore we describe here an experimental system which allows rapid in vivo selection and identification of DHBV pre-S variants and provide evidence that residues within and at a distance from the neutralization epitope are important in DHBV neutralization but do not affect its replication capacity and infectivity.     

Increasing DNA repair capacity in bone marrow by gene transfer as a prospective tool in cancer therapy

The Rh50 glycoprotein is suspected of being involved in Rh antigen expression. We prepared Rh50 cDNA from a human bone marrow library by polymerase chain reaction and then subcloned this cDNA into various vectors. The vector containing Rh50 cDNA produced a 30-kDa nonglycosylated form of Rh50 in a rabbit reticulocyte lysate system and produced partially glycosylated Rh50 (32 kDa) when microsomes were added to the system. COS-1 cells transiently transfected with the vector containing Rh50 cDNA produced partially glycosylated Rh50 (32 kDa) recognized by a Rh50-specific antibody. Surface expression of Rh50 in K562 cells was also detected by flow cytometry using mouse monoclonal antibody (2D10) specific to Rh50. Partially glycosylated Rh50 (32 kDa) was again isolated from the lysates of K562 cells metabolically labeled with [35S]-methionine or [3H]-mannose using anti-Rh50 antisera. These systems (K562 and COS-1 cells) should prove useful for studying the transport of Rh proteins within the cell and the necessary components needed for Rh antigenicity at the cell surface.     

Dual topology of the large envelope protein of duck hepatitis B virus: determinants preventing pre-S translocation and glycosylation

The biosynthesis and topology of the large envelope protein (L protein) of hepadnaviruses was investigated using the duck hepatitis B virus (DHBV) model, which also allows the study of hepadnavirus morphogenesis in experimentally infected hepatocytes. Results from proteolysis of virus particles and from the analysis of topology and posttranslational modification of L chains synthesized in vivo or in a cell-free system both support the presence of a mixed population of L-protein molecules with their N-terminal pre-S domain located either inside or outside the virus particle. During L biosynthesis and DHBV morphogenesis, pre-S, together with the neighboring transmembrane domain (TM-I), initially remained cytoplasmically disposed and was translocated only posttranslationally. Delayed pre-S translocation into a post-endoplasmic reticulum compartment is also indicated by the absence of glycosylation at a modification-competent pre-S glycosylation site. Major features of L-protein biosynthesis and of the resulting dual topology appear to be conserved between avian and mammalian hepadnaviruses, supporting the model that pre-S domains function in part either as an internal matrix for capsid envelopment or externally as a ligand for cellular receptor binding. However, differences in the mechanisms controlling pre-S translocation were revealed by the results of mutational analyses identifying and characterizing cis-acting determinants in pre-S that delay its cotranslational translocation. Our data from DHBV demonstrate the negative influence of a cluster of positively charged amino acid residues next to TM-I, a motif that is conserved among the avian but absent from mammalian hepadnaviruses. Additional control elements, which are apparently shared between both virus groups and which may serve in chaperone binding, were mapped by deletion analysis in the central part of pre-S.     

Clinical and virological evaluation of the detection of pre-S1 and pre-S2 antigens in serum from patients with chronic hepatitis B

OBJECTIVES AND METHODS: We performed a prospective study to determine the clinical and virological significance of pre-S antigen detection in serum samples from patients with chronic hepatitis B virus infection. Four hundred thirty seven consecutive serum samples from 116 patients were tested for the presence of both pre-S1 and pre-S2 antigens by radioimmunoassay using specific monoclonal antibodies. RESULTS: The pre-S1 antigen/HBs antigen ratio, gave an estimation of the number of pre-S1 epitopes expressed on the surface of circulating viral particles, and was positively correlated with the intensity of viral replication intensity (P < 0.05). Moreover, the pre-S1 antigen/HBs antigen ratio was significantly higher in patients suffering from chronic hepatitis associated with viral replication (24% +/- 13); in anti-HBe positive patients, the pre-S1 antigen/HBs antigen ratio was higher in patients replicating a HBe antigen minus variant of the hepatitis B virus and suffering from chronic hepatitis (17% +/- 9) than in asymptomatic HBs antigen carriers (5% +/- 6) (P < 0.05). The pre-S2 antigen/HBs antigen ratio was not correlated with the level of viral replication or with the patient's clinical status. CONCLUSION: This study confirms that pre-S1 antigen detection is a reliable marker of hepatitis B virus replication which can be easily performed in chronically infected patients. This assay is especially useful in identifying anti-HBe positive carriers who replicate a minus pre-core mutant and could benefit from antiviral therapy.