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H Will

Publications and source records attributed to H Will.

At least 109 records · Page 6Linked to original sources
PubMed1993

Minor envelope proteins of duck hepatitis B virus are initiated at internal pre-S AUG codons but are not essential for infectivity.

In infected liver tissue three major and several minor duck hepatitis B virus (DHBV) envelope proteins are detectable by immunoblotting. Translation initiation at the second and the most distal internal ATG codon of the Pre-S/S gene is known to lead to synthesis of two major envelope proteins (P36 and P17) whereas the origin of a further major (P28) and other minor envelope proteins is not clear. Therefore, it was investigated whether translation initiation at pre-S ATGs leads to synthesis of these other envelope proteins and, if yes, whether they are components of viral particles and essential for infectivity. Each of the five ATG codons of the pre-S region of an infectious Chinese DHBV genome (DHBV26) was mutated separated by oligonucleotide-directed mutagenesis (point mutations or deletions) and the function of the corresponding mutant viruses were tested in vitro and in vivo. Immunoblot analysis of liver cell extracts or of extracts from hepatoma cells transfected with the DHBV genomes showed expression of minor pre-S proteins of about 35, 33, and 30 kDa. These proteins were not expressed when ATG codons at nucleotide positions 825, 882, and 957, respectively, were mutated. None of the ATG mutations abolished expression of the major P28 pre-S protein. In cell culture supernatants the minor pre-S proteins P35, P33, and P30 were identified as components of viral particles. With the exception of the DHBV genome containing the mutated ATG codon 801 (translation initiation codon for the major P36 pre-S protein) all forementioned DHBV mutant genomes were infectious. These data demonstrate that minor pre-S proteins are initiated at internal AUGs of the pre-S gene and are components of viral particles but are not essential for infectivity. In contrast to previously published speculations, the results also indicate that the major pre-S protein P28 is not initiated at AUG codon 957 but probably produced by proteolysis from larger pre-S proteins.

Animals↗
PubMed1993

Hepatitis B virus C-gene variants.

The heterogeneity of hepatitis B virus (HBV) is increasingly believed to play a role in viral persistence, pathogenesis, and the type of response to antiviral therapy. One of the best studied parts of the HBV genome is the C-gene which codes for the nucleocapsid protein (HBc) and the e-antigen (HBeAg). Here we attempt to review the recent data on the sequence heterogeneity of this region and its possible implications.

Base Sequence↗
PubMed1993

Long-term response to interferon therapy in chronic hepatitis B: importance of hepatitis B virus heterogeneity.

The long-term therapeutic efficacy of alpha IFN and the influence of preC variants on the type of response were evaluated in 25 patients with chronic hepatitis B, 14 HBeAg and 11 antiHBe positive patients, treated with alpha IFN and monitored for at least four years after discontinuing therapy. In both groups of patients, serum HBV-DNA became frequently undetectable by DNA dot blot during treatment, suggesting that alpha IFN has an antiviral effect both on HBeAg and antiHBe positive chronic carriers. However, long term follow up showed that the loss of viral DNA in antiHBe carriers was only transient, because all responder patients relapsed from 1 to 48 months after IFN withdrawal. In the HBeAg positive carriers, selection for preC mutants was observed at the end of follow up in 2 patients who seroconverted to antiHBe and remained viremic. Both the frequent occurrence of reactivations in antiHBe compared to HBeAg carriers, and the association of IFN therapy with preC mutant virus selection during long term post-treatment follow up observed in this study, indicate that preC variants are more resistant to IFN therapy than preC wild type HBV. Our data suggest therefore, that IFN therapy may be less frequently able to induce a permanent remission in patients infected with preC mutants.

Alanine Transaminase↗
PubMed1993

Immunization with recombinant woodchuck hepatitis virus nucleocapsid antigen or hepatitis B virus nucleocapsid antigen protects woodchucks from woodchuck hepatitis virus infection.

Woodchucks were immunized with recombinant woodchuck hepatitis virus (WHV) nucleocapsid antigen (WHcAg) or hepatitis B virus (HBV) nucleocapsid antigen (HBcAg) and challenged with 10(6) WHV ID50. Six out of six woodchucks immunized with WHcAg and four out of six immunized with HBcAg were protected from WHV infection. Woodchucks immunized with WHcAg or HBcAg developed high serum antibody titres against WHcAg or HBcAg. Antibodies against WHc and HBc displayed little cross-reactivity (< 1%). This confirms and extends previous reports of protection against homologous challenge after immunization with HBcAg/WHcAg which are both internal viral antigens. As the dominant B-cell epitope(s) on particulate WHcAg and HBcAg appear not to be conserved it also demonstrates that antibodies against HBcAg/WHcAg are not important for this protection. Woodchucks immunized with WHcAg/HBcAg reacted with a fast serum antibody response against viral envelope proteins upon challenge with WHV, indicative of functional intrastructural/intermolecular T-cell help as one potential mechanism of protection after immunization with an internal viral antigen.

Animals↗
PubMed1993

Heterogeneity of hepatitis B virus C-gene sequences: implications for amplification and sequencing.

Occasionally direct sequencing of amplified hepatitis B virus DNA leads to weak signals on autoradiograms. Using amplified C-gene sequences we investigated whether this is due to sequence heterogeneity of virus populations and use of inappropriate primers for direct sequencing. High C-gene sequence heterogeneity (point mutations, stop codons and a one codon deletion) was observed in HBV genomes from serum of a chronic carrier who underwent interferon treatment. The type of C-gene mutations detected by direct sequencing depended on the type of primers used. Cloning and sequencing of amplified C-gene sequences demonstrated that this was due to mutations in the region complementary to the sequencing primer. These data demonstrate the existence of novel HBV C-gene mutants and imply that multiple or degenerate sequencing and amplification primers are essential for accurate evaluation of the extent of HBV C-gene heterogeneity. Based on comparative sequence analysis of all available completely or incompletely sequenced C-genes, guidelines for optimal primer design are proposed for similar studies.

Adult↗
PubMed1992

IFN enhance expression of Sp100, an autoantigen in primary biliary cirrhosis.

About 30% of patients suffering from the chronic autoimmune liver disease primary biliary cirrhosis produce autoantibodies against Sp100, a protein migrating in SDS-PAGE at a position corresponding to 100 kDa and located on discrete dot-shaped nuclear structures. The human Sp100 cDNA has recently been cloned and the deduced amino acid sequence was found to contain similarities to several transcriptional regulatory proteins; the biologic function of the Sp100 protein, however, is still unknown. In this study we present data which show that infection of HEp2 cells with influenza A virus, transformation of glial cells with SV40 DNA, and stimulation of PBL with mitogens affect the expression of the Sp100 autoantigen. These observations prompted us to investigate whether expression of the Sp100 protein is modulated by the action of IFN. Immunofluorescence staining of HEp2 and HeLa cells grown in the presence of IFN-alpha, IFN-beta, or IFN-gamma revealed an increase both in size and number of the Sp100 protein-containing nuclear dots, whereas no such effect was observed with cells treated with TNF-alpha. As measured by an immunoblot-based ELISA the amount of Sp100 protein in INF-beta-treated cells (1000 IU/ml, 18 h) was eight to nine times higher than in untreated cells. The enhanced protein expression was accompanied by an accumulation of the Sp100-specific mRNA (13-fold increase of the normal level after 10 h of INF-beta treatment of HEp2 cells). These findings characterize the Sp100 protein as a new member of IFN-modulated proteins and raise the question whether cytokine-mediated increase of Sp100 protein expression plays a role in induction of anti-Sp100 autoantibodies.

Animals↗
PubMed1992

Generation of recombinant CHO(dhfr-) cell lines by single selection for dhfr+ transformants.

In order to establish a mammalian cell expression system with a minimum of selection steps and a stable expression of microgram amounts of recombinant protein (human tissue-type plasminogen activator mutants and chimeric proteins) per 10(6) cells per day, we investigated Chinese hamster ovary cells and the dihydrofolate reductase-deficient Chinese hamster ovary cell line CHO(dhfr-). The 1tPA expression vector pCMVtPA was cotransfected either with the SV40 enhancer sequence containing dhfr expression vector pMT2 or with the enhancerless dhfr expression vector pAdD26SV(A) into CHO(dhfr-) cells. With both dhfr expression plasmids, selection for dhfr+ transformants followed by single dilution cloning was sufficient to generate cell lines with a production level of up to 4.6 micrograms tPA/10(6) cells.day. This approach is useful if gene amplification procedures are time-consuming and impracticable because of a large number of recombinant proteins. In order to establish CHO cell lines with a tPA expression level as high as that in the case of CHO(dhfr-) cells, repeated dilution cloning is necessary.

Animals↗
PubMed1992

Inhibition of hepatitis B virus production by modified 2',3'-dideoxy-thymidine and 2',3'-dideoxy-5-methylcytidine derivatives. In vitro and in vivo studies.

The effect of analogues of both 2',3'-dideoxy-3'-fluorothymidine (FddThd) [2',3'-dideoxy-3'-fluorouridine (FddUrd), 2',3'-dideoxy-3'-fluoro-5-chlorouridine (FddClUrd), 2',3'-dideoxy-3'- fluoro-5-bromouridine (FddBrUrd) and 2',3'-dideoxy-3'-fluoro-5-bromovinyluridine (FddBVUrd)] and 2',3'-dideoxy-3'-fluorocytidine (FddCyt) [2',3'-dideoxy-3'-fluoro-5-fluorocytidine (FddFCyt), 2',3'-dideoxy-3'-fluoro-5-chlorocytidine (FddClCyt), 2',3'-dideoxy-3'-fluoro-5-methylcytidine (FddMeCyt), 2',3'-dideoxy-3'-fluoro-5-ethylcytidine (FddEtCyt), 2',3'-dideoxy-3'-chloro-5-methylcytidine (ClddMeCyt), 2',3'-dideoxy-3'-amino-5-methylcytidine (AmddMeCyt), 2',3'-dideoxy-3'-azido-5- methylcytidine (AzddMeCyt) and arabinosyl-5-methylcytosine (AraMeCyt)] were tested for their potential antiviral activity in vitro using the human hepatoblastoma cell line, Hep G2 2.2.15, which was transfected with a vector containing hepatitis B virus (HBV). It was found that FddThd, FddMeCyt, FddEtCyt, ClddMeCyt, AmddMeCyt and AraMeCyt display cytostatic activity at concentrations (CD50 values) between 0.54 (FddMeCyt) and 3.93 microM (FddEtCyt), while FddUrd, FddClUrd, FddBrUrd, FddBVUrd, FddCyt, FddFCyt, FddClCyt and AzddMeCyt do not affect cell growth at concentrations of up to 25 microM. Among the thymidine analogues tested, FddThd is the most effective antiviral agent: at a concentration of 0.03 microM a more than 90% reduction of HBV DNA synthesis was measured. On the other hand, the antiviral indexes displayed by FddClUrd, FddBrUrd and FddBVUrd are higher than tht of FddThd; FddUrd was completely inactive. The most powerful antiviral agents in the group of cytidine analogues tested in vitro were FddMeCyt (more than 90% reduction of HBVDNA synthesis at 0.10 microM) and ClddMeCyt (0.10 microM); FddClCyt, FddEtCyt, AmddMeCyt and AraMeCyt were of intermediate activity. None of the negligible antiviral activity was determined for FddUrd, FddCyt, FddFCyt and AzddMeCyt. FddThd and FddMeCyt displayed in vivo an antiviral effect in the duck/duck HBV (DHBV) animal system. Administration of 10 or 20 mg/kg (total daily dose) of FddThd and 5 or 10 mg/kg of FddMeCyt (i.m. daily) to ducks infected with DHBV for 12 days blocked virus production. Termination of treatment with FddThd of infected animals led to reappearance of the virus in the serum though at lower levels. The in vitro and the in vivo data suggest that FddThd and FddMeCyt might be promising antiviral agents for the treatment of infection caused by HBV in humans.

Animals↗
PubMed1992

Characterization of human tissue-type plasminogen activator with monoclonal antibodies: mapping of epitopes and binding sites for fibrin and lysine.

The study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding. Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D'). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA. The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

The hepatitis B virus X protein transactivation of c-fos and c-myc proto-oncogenes is mediated by multiple transcription factors.

We have constructed two expression vectors in order to study the action of the HBV 17 Kd X protein on the c-fos and c-myc promoters. The results show that the promoters contain multiple elements that respond to X protein, suggesting involvement of multiple transcription factors. The exact mechanism of the interaction remains elusive, but our data allow speculation about the factors that may be influenced.

Chloramphenicol O-Acetyltransferase↗
PubMed1992

Precore mutant hepatitis B virus infection and liver disease.

The type of hepatitis B virus ("wild-type" or precore mutant) in anti-e antigen antibody-positive carriers, viral DNA levels in the serum, and core and e antigen expression in the liver were investigated to search for a possible correlation of these factors with the severity of liver damage. Two major groups of patients were found: the patients in group A were predominantly infected with precore mutant virus and had chronic active hepatitis, expressed nuclear/cytoplasmic core and e antigens in liver biopsy specimens, and usually had high levels of viral DNA in their serum; patients in group B were infected with a mixture of wild-type and mutant viruses, had predominantly chronic persistent hepatitis, showed weaker expression of nuclear core antigen with no cytoplasmic core or e antigen, and had low viremia. A few patients were infected with viruses without precore stop-codon mutation. These data indicate a high prevalence of precore mutant viruses in anti-e carriers with chronic liver disease and suggest that monitoring of virus sequence type and DNA level may be of prognostic value for liver disease sequelae.

Adult↗
PubMed1992

Frequent and rapid emergence of mutated pre-C sequences in HBV from e-antigen positive carriers who seroconvert to anti-HBe during interferon treatment.

Hepatitis B virus (HBV) variants which cannot express e-antigen (HBeAg) are characteristic for many viremic anti-HBe positive chronic carriers who often have particularly severe and fluctuating hepatitis. Whether such variants are selected for and are less amenable to interferon treatment is under dispute. Therefore, by DNA amplification and direct sequencing we have investigated the emergence of HBV pre-C sequence variants in nine e-antigen positive chronic carriers, all of whom seroconverted to anti-HBe or lost HBeAg during interferon treatment, and in three of whom no viral DNA was detectable after interferon treatment. In most, but not all of the patients we found newly emerging pre-C sequences in a subpopulation of the viral genomes that included silent point mutations, amino acid changes, start and stop codon and frameshift mutations. The emergence of these mutations was paralleled by a drastic decrease of viremia during treatment. The observed mutations appeared most frequently during interferon treatment. Some of the mutations appeared or disappeared late after interferon treatment concomitant with anti-HBe antibody development. The appearance or lack of mutations in the pre-C region of a subpopulation of HBV of these patients was independent of successful virus elimination. These data indicate that interferon treatment is frequently associated with the simultaneous fall in titer of viral DNA by several orders of magnitude and the emergence of novel pre-C sequences, some of them preventing HBeAg expression. However, the presumably immune-mediated selection for pre-C mutant viruses and decrease in viremia under interferon treatment appears not to be prognostic for successful or unsuccessful virus elimination.

Adult↗
PubMed1992

Sensitive method for identification of minor hepatitis B mutant viruses.

There is increasing evidence for selection of hepatitis B virus (HBV) mutants during the natural course of infection, after immunization, and during interferon therapy. These mutants can prolong viral persistence, escape immune-mediated elimination, and influence the clinical sequelae of infection. Here we describe a sensitive and reliable method that allows detection of HBV mutants within a heterogeneous virus population. The method consists of amplification of HBV DNA with vector-HBV hybrid primers, efficient and enzyme-free cloning of the amplified DNA fragment, identification by hybridization with synthetic oligonucleotide probes, and sequencing of the cloned DNA fragments. We demonstrate that up to 10(5) HBV DNA fragments can be obtained in cloned form from a single amplification reaction, the ratio of HBV pre-C "wild-type" and mutant sequences of heterogeneous populations can be determined, and very minor virus subpopulations can be identified. The method can be applied to other viral or nonviral nucleic acid sequence variation studies and should help to resolve the role of mutant and wild-type viruses in pathogenesis and the forces driving their selection.

Base Sequence↗
PubMed1992

Hepatitis B virus genomes that cannot synthesize pre-S2 proteins occur frequently and as dominant virus populations in chronic carriers in Italy.

The heterogeneity of hepatitis B virus (HBV) pre-S sequences coding for envelope proteins was tested by DNA amplification and direct sequencing of viral genomes from sera of 22 unselected chronic carriers resident in Southern Italy. The sequences of the dominant viral genome populations from 15 carriers were very similar to known published "wildtype" HBV genomes and showed no deletions. In contrast, in the HBV populations of six patients, deletions in the pre-S region, mainly clustered at the amino terminal end of the pre-S2 region, were found. Four of these mutant genome populations and those from another patient cannot express pre-S2 proteins due to deletions or a mutation of the translation initiation codon. Emergence of the pre-S mutant viruses either during the natural course of infection or after interferon treatment was found in follow-up sera of one and two patients, respectively. These data indicate a high prevalence of pre-S mutant viruses which cannot express pre-S2 and normal-size pre-S1 proteins. This has important implications for the usefulness of diagnostic pre-S protein assays and possibly for interferon treatment and the efficacy of new vaccines containing pre-S proteins.

Amino Acid Sequence↗
PubMed1992

Hepatitis B virus polymerase gene: expression of the long open reading frame using the baculovirus expression system.

A recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.

Baculoviridae↗
PubMed1992

Autoantibodies to the nuclear Sp100 protein in primary biliary cirrhosis and associated diseases: epitope specificity and immunoglobulin class distribution.

Sp100, a protein with a dot-like intranuclear localization in immunofluorescence microscopy, is a major target for patient autoantibodies in primary biliary cirrhosis (PBC) and occasionally in rheumatic disorders. The human Sp100 cDNA has recently been cloned, and the deduced amino acid sequence was found to contain sequence similarities with an MHC class I domain and several transacting regulatory proteins, including HIV-1 nef proteins. In this study, recombinant Sp100 fusion proteins were used to differentiate the immunoglobulin isotypes and to map the epitopes involved in the anti-Sp100 autoimmune response. PBC patients developed IgG as well as IgM and/or IgA class anti-Sp100 autoantibodies whereas most patients with rheumatic diseases developed IgG class autoantibodies only. For epitope mapping, truncated versions of the Sp100 protein were probed for immunoreactivity in ELISA and immunoblotting. With 55 sera, 17 different reaction patterns were obtained, and at least three non-overlapping major autoantigenic domains were recognized by the majority of sera. One domain, which contains the sequence similarity with HIV nef proteins, was recognized by all anti-Sp100 sera and harbours multiple, in part discontinuous, epitopes. These data demonstrate a heterogeneous and patient-specific anti-Sp100 autoimmune response which is antigen-driven and, at least in terms of isotype composition, different in PBC and non-PBC patients.

Antigens, Nuclear↗
PubMed1992

The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.

Animals↗