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Biomedical subjects

C Seeger

Publications and source records attributed to C Seeger.

At least 19 recordsLinked to original sources
PubMed2001

Effect of alpha interferon on the hepatitis C virus replicon.

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.

Adaptation, Physiological↗
PubMed2001

Does a cdc2 kinase-like recognition motif on the core protein of hepadnaviruses regulate assembly and disintegration of capsids?

Hepadnaviruses are enveloped viruses, each with a DNA genome packaged in an icosahedral nucleocapsid, which is the site of viral DNA synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the absence of these proteins, nucleocapsids deliver viral DNA into the cell nucleus. Presumably, this step is identical to the delivery of viral DNA during the initiation of an infection. Unfortunately, the mechanisms triggering the disintegration of subviral core particles and delivery of viral DNA into the nucleus are not yet understood. We now report the identification of a sequence motif resembling a serine- or threonine-proline kinase recognition site in the core protein at a location that is required for the assembly of core polypeptides into capsids. Using duck hepatitis B virus, we demonstrated that mutations at this sequence motif can have profound consequences for RNA packaging, DNA replication, and core protein stability. Furthermore, we found a mutant with a conditional phenotype that depended on the cell type used for virus replication. Our results support the hypothesis predicting that this motif plays a role in assembly and disassembly of viral capsids.

Amino Acid Motifs↗
PubMed2000

Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections.

Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.

Adenoviridae↗
PubMed2000

Apoptosis and regeneration of hepatocytes during recovery from transient hepadnavirus infections.

It is well known that hepatitis B virus infections can be transient or chronic, but the basis for this dichotomy is not known. To gain insight into the mechanism responsible for the clearance of hepadnavirus infections, we have performed a molecular and histologic analysis of liver tissues obtained from transiently infected woodchucks during the critical phase of the recovery period. We found as expected that clearance from transient infections occurred subsequent to the appearance of CD4(+) and CD8(+) T cells and the production of interferon gamma and tumor necrosis factor alpha in the infected liver. These events were accompanied by a significant increase in apoptosis and regeneration of hepatocytes. Surprisingly, however, accumulation of virus-free hepatocytes was delayed for several weeks following this initial influx of lymphocytes. In addition, we observed that chronically infected animals can exhibit levels of T-cell accumulation, cytokine expression, and apoptosis that are comparable with those observed during the initial phase of transient infections. Our results are most consistent with a model for recovery predicting replacement of infected hepatocytes with regenerated cells, which by unknown mechanisms remain protected from reinfection in animals that can be cured.

Animals↗
PubMed2000

Hepatitis B virus biology.

Hepadnaviruses (hepatitis B viruses) cause transient and chronic infections of the liver. Transient infections run a course of several months, and chronic infections are often lifelong. Chronic infections can lead to liver failure with cirrhosis and hepatocellular carcinoma. The replication strategy of these viruses has been described in great detail, but virus-host interactions leading to acute and chronic disease are still poorly understood. Studies on how the virus evades the immune response to cause prolonged transient infections with high-titer viremia and lifelong infections with an ongoing inflammation of the liver are still at an early stage, and the role of the virus in liver cancer is still elusive. The state of knowledge in this very active field is therefore reviewed with an emphasis on past accomplishments as well as goals for the future.

Animals↗
PubMed1997

Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids.

Assembly of hepadnaviruses depends on the formation of a ribonucleoprotein (RNP) complex comprising the viral polymerase polypeptide and an RNA segment, epsilon, present on pregenomic RNA. This interaction, in turn, activates the reverse transcription reaction, which is primed by a tyrosine residue on the polymerase. We have shown recently that the formation of this RNP complex in an avian hepadnavirus, the duck hepatitis B virus, depends on cellular factors that include the heat shock protein 90 (Hsp90). We now report that RNP formation also requires ATP hydrolysis and the function of p23, a recently identified chaperone partner for Hsp90. Furthermore, we also provide evidence that the chaperone complex is incorporated into the viral nucleocapsids in a polymerase-dependent reaction. Based on these findings, we propose a model for hepadnavirus assembly and priming of viral DNA synthesis where a dynamic, energy-driven process, mediated by a multi-component chaperone complex consisting of Hsp90, p23 and, potentially, additional factors, maintains the reverse transcriptase in a specific conformation that is competent for RNA packaging and protein priming of viral DNA synthesis.

Adenosine Triphosphate↗
PubMed1997

Why are hepadnaviruses DNA and not RNA viruses?

Virion assembly in hepadnaviruses is a two-step process leading to (1) the packaging of viral pregenomic RNA and reverse transcriptase into nucleocapsids and (2) the assembly of nucleocapsids with envelope components, which results in the formation of mature virus particles. Characteristically, both steps are intimately coupled to viral DNA synthesis. While assembly of nucleocapsids is coupled to the protein priming of reverse transcription, virion formation is linked to genome maturation.

DNA, Viral↗
PubMed1997

The hepatitis B virus X protein: the quest for a role in viral replication and pathogenesis.

Although the biological importance of hepatitis B virus X protein (HBX) in the life cycle of hepatitis B virus has been well established, the cellular and molecular basis of its function remains largely undefined. Despite the association of multiple activities with HBX, none of them appear to provide a unifying hypothesis regarding the true biological function of HBX. Identification and characterization of cellular targets of HBX remain an essential goal in the elucidation of the molecular mechanisms of HBX. Using the Saccharomyces cerevisiae two-hybrid system, we have identified and characterized a novel subunit of the proteasome complex (XAPC7) that interacts specifically with HBX. We also showed that HBX binds specifically to XAPC7 in vitro. Mutagenesis studies have defined the domains of interaction to be critical for the function of HBX. Furthermore, overexpression of XAPC7 appeared to activate transcription by itself and antisense expression of XAPC7 was able to block transactivation by HBX. Therefore, the proteasome complex is possibly a functional target of HBX in cells.

Hepatitis B virus↗
PubMed1997

Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.

We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

Cell Line↗
PubMed1997

PNA-mediated purification of PCR amplifiable human genomic DNA from whole blood.

A simple and rapid procedure whereby human genomic DNA can be purified in a PCR amplifiable form from whole blood is described. In a first step, human genomic DNA is hybridized in solution to a biotinylated peptide nucleic acid (PNA), which forms a high-affinity triplex with A7 sequence motifs in the target DNA. The complex is then captured onto paramagnetic streptavidin-coated particles, which are subsequently transferred directly into the PCR. The purification method effectively removes inhibitors of the PCR from as much as 500 microL of whole blood.

Biotin↗
PubMed1996

Mutagenesis of a hepatitis B virus reverse transcriptase yields temperature-sensitive virus.

Replication of the hepadnavirus genome is catalyzed by a multifunctional reverse transcriptase (the pol protein) that exhibits DNA polymerase and DNA priming activities and has the ability to transfer RNA and DNA strands across the viral genome. A salient feature of this enzyme is the ability to prime RNA-directed DNA synthesis with protein rather than with RNA. This is reflected in its unique physical make up, which includes an amino-terminal (TP) domain that is separated by a spacer from the reverse transcriptase (RT) domain. To establish a structure function relationship for the pol protein, we examined 52 mutants for their ability to replicate viral DNA in vitro and in cultured cells. We demonstrated that the role of the TP domain is limited to the early steps of viral DNA synthesis including RNA packaging and protein priming. Both the TP and the RT domains are required for the interaction with epsilon RNA, which is the template for the protein-priming reaction and serves as the RNA packaging signal. In addition, we report the isolation of a thermosensitive variant of a hepadnavirus that will permit investigations of individual steps of the viral replication cycle under synchronized conditions.

Animals↗
PubMed1996

Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP.

Animals↗
PubMed1995

Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli.

We have characterized four genes from the 52-min region on the Escherichia coli linkage map. Three of these genes are directly involved in the metabolism of xanthosine, whereas the function of the fourth gene is unknown. One of the genes (xapA) encodes xanthosine phosphorylase. The second gene, named xapB, encodes a polypeptide that shows strong similarity to the nucleoside transport protein NupG. The genes xapA and xapB are located clockwise of a gene identified as xapR, which encodes a positive regulator belonging to the LysR family and is required for the expression of xapA and xapB. The genes xapA and xapB form an operon, and their expression was strictly dependent on the presence of both the XapR protein and the inducer xanthosine. Expression of the xapR gene is constitutive and not autoregulated, unlike the case for many other LysR family proteins. In minicells, the XapB polypeptide was found primarily in the membrane fraction, indicating that XapB is a transport protein like NupG and is involved in the transport of xanthosine.

Amino Acid Sequence↗
PubMed1994

Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase.

All known DNA polymerases require primers for the initiation of DNA synthesis. While cellular polymerases and reverse transcriptases use free hydroxyl groups of RNA or DNA, the DNA polymerases of certain animal viruses and bacteriophages depend upon hydroxyl groups of amino acid residues within proteins as primers for DNA synthesis. Recently, the reverse transcriptase of a hepadnavirus has been shown to prime RNA-directed DNA synthesis from an internal site of the polypeptide (G.H. Wang and C. Seeger, Cell 71:663-670, 1992). In this report we demonstrate that a tyrosine residue of the polymerase polypeptide is the site of a phosphodiester linkage with the first nucleotide of minus-strand DNA. This tyrosine residue is located within an amino-terminal domain of the polymerase polypeptide and is indispensable for the priming of reverse transcription. Our results demonstrate that the hepatitis B virus reverse transcriptase can initiate DNA synthesis without the requirement for tRNA as a primer.

DNA, Viral↗