Biomedical subjects

L Notvall

Publications and source records attributed to L Notvall.

11 recordsLinked to original sources

PubMed2000

Interaction between hepatitis B virus core protein and reverse transcriptase.

Previous mutagenesis studies with hepatitis B virus (HBV) suggest that continued interactions with core are required for several steps in genomic replication. To examine core-polymerase (Pol) interactions, insect cells were coinfected with baculovirus constructs that independently expressed core and Pol. The results demonstrated several features with implications that core plays an interactive role with HBV Pol: (i) core coprecipitated with constructs expressing full-length Pol as well as the terminal protein (TP), reverse transcriptase (RT) and RNase H domains of Pol, independently; (ii) coprecipitation of core was not dependent on the presence of an epsilon stem-loop sequence; and (iii) core-Pol complexes migrated as intact capsid particles, as detected by sucrose gradient analysis. To analyze the structural and sequence requirements of core in recognition of Pol, a series of core mutants with two- to four-amino-acid insertions or carboxy-terminal deletions were assessed for Pol interaction. The results indicated that capsid formation is required but not sufficient for interaction with Pol and that the TP and RT domains of Pol have different requirements for interaction with core. To map the core binding sites on Pol, a panel of amino- and carboxy-terminal deletion mutants of the TP and RT domains of Pol were analyzed for interaction with core. At least three separate core binding sites on Pol were detected. This analysis begins to define basic requirements for core-Pol interactions, but further study is necessary to delineate the effects of these interactions on encapsidation and genome replication.

Amino Acid Sequence↗

PubMed2000

Development of a primary tamarin hepatocyte culture system for GB virus-B: a surrogate model for hepatitis C virus.

GB virus-B (GBV-B) causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Phylogenetically, GBV-B is the closest relative to hepatitis C virus (HCV), and thus GBV-B infection of tamarins represents a powerful surrogate model system for the study of HCV. In this study, the course of infection of GBV-B in tamarins was followed using a real-time 5' exonuclease (TaqMan) reverse transcription-PCR assay to determine the level of GBV-B in the serum. Peak viremia levels exceeded 10(9) genome equivalents/ml, followed by viral clearance within 14 to 16 weeks. Rechallenge of animals that had cleared infection resulted in viremia that was limited to 1 week, suggestive of a strong protective immune response. A robust tissue culture system for GBV-B was developed using primary cultures of tamarin hepatocytes. Hepatocytes obtained from a GBV-B-infected animal maintained high levels of cell-associated viral RNA and virion secretion for 42 days of culture. In vitro infection of normal hepatocytes resulted in rapid amplification of cell-associated viral RNA and secretion of up to 10(7) genome equivalents/ml of culture supernatant. In addition, infection could be monitored by immunofluorescence staining for GBV-B nonstructural NS3 protein. This model system overcomes many of the current obstacles to HCV research, including low levels of viral replication, lack of a small primate animal model, and lack of a reproducible tissue culture system.

Animals↗

PubMed1999

Mapping of the hepatitis B virus reverse transcriptase TP and RT domains by transcomplementation for nucleotide priming and by protein-protein interaction.

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction. We previously described a complementation assay for analysis of the roles of the TP and RT domains of HBV reverse transcriptase (pol) in the priming reaction. Independently expressed TP and RT domains form a complex functional for in vitro priming reactions. To map the minimal functional TP and RT domains, we prepared baculoviruses expressing amino- and carboxyl-terminal deletions of both the TP and RT domains and analyzed the proteins for the ability to participate in transcomplementation for the priming reaction. The minimal TP domain spanned amino acids 20 to 175; however, very little activity was observed without a TP domain spanning amino acids 1 to 199. The minimal RT domain spanned amino acids 300 to 775; however, little activity was observed unless the carboxyl end of the RT domain extended to amino acid 800. Thus, most of the RNase H domain was required. In previous studies, we observed a TP inhibitory domain between amino acids 199 and 344. The current analysis narrowed this domain to residues 300 to 334, which is a portion of the minimal RT domain. In addition, the ability of TP and RT deletion mutants to form stable TP-RT complexes was examined in coimmunoprecipitation assays. The minimal TP and RT domains capable of protein-protein interaction were considerably smaller than the domains required for functional interaction in the transcomplementation assays, and unlike priming activity, TP-RT interaction did not require the epsilon RNA stem-loop. These studies help to further define the complex protein-protein interactions required in HBV genome replication.

Animals↗

PubMed1999

Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein.

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.

Animals↗

PubMed1997

Transcomplementation of nucleotide priming and reverse transcription between independently expressed TP and RT domains of the hepatitis B virus reverse transcriptase.

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. We recently expressed human hepatitis B virus (HBV) reverse transcriptase (pol) in insect cells by using the recombinant baculovirus system. The purified protein is active in nucleotide priming and reverse transcription reactions. In this report, we demonstrate that the tyrosine residue at amino acid number 63 within the TP (terminal protein) domain of the polymerase is the site of covalent linkage of the first nucleotide of minus-strand DNA. Analysis of pol polypeptides with mutations in the TP and RT (reverse transcriptase) domains indicated that both domains were required for in vitro nucleotide priming activity. Polymerase proteins with mutations in the TP and RT domains were not capable of complementing each other in the nucleotide priming reaction, suggesting that transcomplementation between full-length polypeptides was not possible. However, when the TP and RT domains were expressed as separate polypeptides, they formed a highly stable complex that was active in nucleotide priming and reverse transcription. The presence of an epsilon stem-loop dramatically increased the nucleotide priming activity in transcomplementation assays, even though full-length pol displayed similar activities in the absence and presence of epsilon. These data raise the possibility that in the transcomplementation assay, epsilon may play a role in the formation of a functional complex between TP and RT, rather than being required only as the template for nucleotide priming. The results indicate that using the baculovirus system, it is possible to dissect the protein-protein and protein-RNA interactions required for HBV genome replication.

Animals↗

PubMed1995

Nucleotide priming and reverse transcriptase activity of hepatitis B virus polymerase expressed in insect cells.

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. Analysis of the initial steps in this reaction as well as certain details of genome replication has been hampered by the difficulties encountered in the expression of functional hepadnavirus polymerases in heterologous systems. We have expressed human hepatitis B virus (HBV) polymerase (pol) in insect cells, using the recombinant baculovirus system. Analysis of immunoaffinity-purified pol indicated that (i) a portion of pol had initiated minus-strand DNA synthesis within infected insect cells; (ii) the pol mRNA appeared to be the template for reverse transcription; (iii) the products were small (100 to 500 nucleotides); (iv) only minus-strand DNA was synthesized; (v) the products were covalently bound to protein; and (vi) the 5' end of the minus-strand DNA mapped to DR1 by primer extension. The purified pol was also active in an in vitro polymerase assay. Analyses suggested that a different fraction of pol was active in the in vitro assays. Incubation of pol with labeled deoxyribonucleotide triphosphates resulted in the labeling of the pol polypeptide in a reaction that appeared to represent in vitro nucleotide priming. In vitro nucleotide priming was confirmed by the appearance of 32P-labeled phosphotyrosine on pol following in vitro reactions with 32P-labeled deoxyribonucleotide triphosphates. The ability to purify significant quantities of HBV pol will facilitate functional and physical analysis of this enzyme as well as the search for novel inhibitors of HBV replication.

Amino Acid Sequence↗

PubMed1993

Analysis of hepatitis C virus capsid, E1, and E2/NS1 proteins expressed in insect cells.

The baculovirus/insect cell expression system was used to express the capsid protein and glycoproteins (e1 and e2/NS1) of the hepatitis C virus (HCV). Each polypeptide domain was expressed individually using two different constructs varying at their carboxy termini in order to retain or delete hydrophobic domains that may be involved in membrane association. The capsid proteins were transported to the nucleus where they formed a single large crystal-like inclusion. The capsid proteins were phosphorylated in insect cells. The e1 and e2 polypeptides were present in both the soluble and insoluble cellular fractions. Deletion of a hydrophobic domain in the carboxy terminus of e2 resulted in the polypeptide becoming soluble but not secreted. Deletion of the carboxy terminus of e1 had no effect on solubility. Both e1 and e2 were glycosylated, with variable glycosylation of e1 giving rise to a series of polypeptides varying in apparent molecular weight. Co-infection of insect cells with viruses expressing e1 and e2 resulted in a complex that permitted the coimmunoprecipitation of e1 with antibodies to e2 and vice versa. Immunofluorescence staining of insect cells expressing e1 and e2 indicated that reactivity to e2 was more prevalent in anti-HCV positive human sera.

Animals↗

PubMed1991

Evaluation of a chimpanzee colony for antibodies to hepatitis C virus.

The chimpanzee is the only species other than man that is generally susceptible to infection by hepatitis C virus (HCV). Aspects of future studies on vaccines and therapeutics for HCV may continue to depend on the chimpanzee. In an attempt to determine the HCV status of the animals in a chimpanzee colony, the recently developed enzyme immunoassay (EIA) for antibodies to HCV was used. The results of the assay indicated that only 31.3% of the animals that had previously been inoculated with a non-A, non-B hepatitis agent were currently positive in the assay. A retrospective analysis suggested that an additional 20% of the chimpanzees had been positive at some time following infection. Seroconversion to an anti-HCV antibody response using this assay did not appear to correlate with the severity of the initial disease or the development of chronic liver damage. Examination of the EIA-positive samples using the second generation recombinant immunoblot assay (RIBA) containing four HCV antigens suggested that chimpanzees responded differentially to these antigens. Examination of serum samples from 139 uninoculated animals by EIA revealed seven positive samples and ten samples with borderline values. The nature of the reactivities in most of the positive samples could not be resolved, but analysis by RIBA indicated that at least one animal in the breeder colony had been exposed to HCV. Due to the low seroconversion rate and the uncertainties surrounding many of the positive reactions, this assay cannot be used to determine the initial source or extent of spread of HCV infections in the uninoculated animals.

Animals↗

PubMed1991

Immunogenicity of hepatitis B surface antigen derived from the baculovirus expression vector system: a mouse potency study.

A standard mouse potency test was performed to evaluate the immunogenicity of recombinant hepatitis B surface antigen (HBsAg) produced in the baculovirus/insect cell expression system. Groups of NIH Swiss mice were immunized with serial four-fold amounts of either baculovirus-derived HBsAg adsorbed to aluminum sulfate or a commercially available yeast-derived recombinant HBsAg vaccine preparation. Results from these experiments showed that the effective dose of baculovirus- and yeast-derived HBsAg vaccine preparations necessary to seroconvert 50% of the animals were similar. The duration of the antibody response to HBsAg was studied in mice immunized with the highest doses of the two recombinant vaccine preparations 3 and 6 months after injection. No decrease in the anti-HBs response was observed 6 months after injection. No decrease in the anti-HBs response was observed 6 months after immunization with either of the two vaccine preparations. These results indicate that the baculovirus-derived recombinant HBsAg could serve as an alternative vaccine candidate for hepatitis B virus.

Animals↗

PubMed1990

Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity.

The hepatitis B virus core open reading frame with and without the precore domain was expressed in insect cells using a baculovirus expression system. Precore antigen was not properly processed in insect cells and was present in highly insoluble cytoplasmic aggregates. Core antigen without the precore domain formed core particles with a diameter of 28 nm that were secreted into the medium. Both core and precore antigens were phosphorylated in insect cells. The immune response in mice to both antigens yielded antibodies with a high degree of preferential reactivity for the homologous immunizing polypeptide. A kinase activity that phosphorylated core antigen was associated with highly purified core particles. The kinase activity resembled that previously demonstrated for core particles purified from the cytoplasm of infected hepatocytes and detergent-treated Dane particles. Partial resistance of the phosphate-label to phosphatase treatment suggested that some of the phosphorylated sites are in the interior of the particle. The presence of kinase activity in recombinant core particles demonstrated that this activity is not derived from another hepatitis B virus-encoded polypeptide, and the lack of a kinase consensus sequence in the core open reading frame suggests that the kinase is of cellular origin.

Animals↗

PubMed1989

Expression and characterization of hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system.

The baculovirus Autographa californica nuclear polyhedrosis virus was used as an expression vector to produce hepatitis B virus surface antigen with and without the pre-S domain. The S gene product was expressed as both fusion and nonfusion polypeptides. No difference was observed in the posttranslational modification of the fusion and nonfusion polypeptides. The S proteins were not secreted into the medium but were inserted into the endoplasmic reticulum, glycosylated, and partially extruded into the lumen of the endoplasmic reticulum as 22-nm lipoprotein particles. The oligosaccharide chains on the insect cell-derived S protein were of the N-linked high-mannose form, in contrast to the complex-type oligosaccharides detected on plasma-derived hepatitis B virus surface antigen. The pre-S-S polypeptides were inserted into the endoplasmic reticulum, glycosylated, and modified by fatty acid acylation with myristic acid. A procedure was developed to purify the S protein from cellular membranes by using detergent extraction and immunoaffinity chromatography. The purified S protein was in the form of protein-detergent micelles and was highly antigenic and immunogenic.

Animals↗