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C Seeger

Publications and source records attributed to C Seeger.

At least 37 records · Page 2Linked to original sources
PubMed1994

Role of RNA in enzymatic activity of the reverse transcriptase of hepatitis B viruses.

The hepadnavirus reverse transcriptase is a multifunction enzyme. In addition to its role in DNA synthesis, the polymerase is required for RNA packaging and also functions as the primer for minus-strand DNA synthesis. Previously, we demonstrated that the protein-priming activity of the polymerase requires a viral RNA segment, termed epsilon, which serves as a template for the synthesis of a short DNA oligomer that is covalently attached to the reverse transcriptase (G.-H. Wang and C. Seeger, J. Virol. 67:6507-6512, 1993). We now report that epsilon is sufficient for activation of the reverse transcriptase to prime DNA synthesis through the formation of a stable RNA-protein (RNP) complex. We also demonstrate that the binding reaction depends on sequence-specific determinants on epsilon. Moreover, our results indicate that two genetically separated domains of the reverse transcriptase are required for formation of the RNP complex. Finally, we show that the polymerase has a DNA polymerase activity in the absence of epsilon which does not depend on the protein-priming mechanism.

Animals↗
PubMed1994

Woodchuck hepatitis virus X protein is required for viral infection in vivo.

The X gene of the mammalian hepadnaviruses is believed to encode a protein of 17 kDa which has been shown to transactivate a wide range of viral and cellular promoters. The necessity for X gene expression during the viral life cycle in vivo has recently been suggested (H.-S. Chen, S. Kaneko, R. Girones, R. W. Anderson, W. E. Hornbuckle, B. C. Tennant, P. J. Cote, J. L. Gerin, R. H. Purcell, and R. H. Miller, J. Virol. 67:1218-1226, 1993). We have independently constructed two variants of woodchuck hepatitis virus (WHV) with mutations in the X coding region. Transient transfection of two different hepatoma cell lines showed that these WHV X gene mutants were competent for virus replication in vitro. To determine whether X expression was required for viral replication in vivo, we injected mutant and wild-type genomes into the livers of susceptible woodchucks. While the wild-type WHV genomes were infectious in all animals examined, the mutant genomes did not initiate a WHV infection in woodchucks. These results indicate that the X gene of the hepadnaviruses plays a major role in viral replication in vivo.

Animals↗
PubMed1993

Novel mechanism for reverse transcription in hepatitis B viruses.

Reverse transcription of all retroviruses and most retroid elements requires tRNA as a primer for DNA synthesis. However, in hepatitis B viruses the viral polymerase itself acts as a primer for reverse transcription (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992). We have now demonstrated that in order to prime DNA synthesis, the polymerase binds to an RNA hairpin, which then serves as a template for the formation of a short DNA primer that is covalently linked to protein. Following its synthesis, the nascent DNA strand apparently dissociates from its template and reanneals with complementary sequences at the 3' end of the RNA genome, where DNA synthesis continues. Since this RNA hairpin also functions as a packaging signal for viral RNA, hepadnaviruses have adopted a replication strategy that relies on the same signal for two biochemically distinct events, RNA packaging and reverse transcription. This mechanism is without precedent among all known retroid elements and among other viruses and bacteriophages that use protein as a primer for RNA or DNA synthesis. It could provide an effective target for antiviral therapy, which is required for the treatment of more than 300 million carriers of hepatitis B virus.

Base Sequence↗
PubMed1993

Differential activation of myc gene family members in hepatic carcinogenesis by closely related hepatitis B viruses.

Woodchucks infected with woodchuck hepatitis virus (WHV) and ground squirrels infected with ground squirrel hepatitis virus (GSHV) both develop hepatocellular carcinoma (HCC), but WHV-associated tumors arise more frequently and much earlier in life. These differences are preserved when the oncogenic potentials of the two viruses are examined in the same host (woodchucks). We examined RNA and genomic DNA from tumors arising from WHV- and GSHV-infected woodchucks to determine whether these viruses use the same oncogenic pathway. N-myc RNA was not expressed in normal liver but was expressed in 10 of 13 WHV-associated HCCs examined. Southern blot analysis showed that 7 of 17 WHV-induced tumors (41%) contained rearrangements at N-myc loci due to viral genomic integration. Six of these seven inserts affected N-myc2, and most of these were at the 5' end of the gene. In contrast, only two of seven GSHV-induced woodchuck HCCs expressed N-myc RNA, and only 1 of the 16 tumors (6%) contained a rearranged N-myc allele. The GSHV-associated HCCs all contained numerous viral insertions, so the low frequency of integration into N-myc loci by GSHV was not due to a general block to integration. Four of sixteen GSHV-induced tumors harbored amplified c-myc alleles, and five of seven GSHV tumors tested contained elevated c-myc RNA levels. By contrast, enhanced c-myc RNA levels were observed in only 2 of 13 WHV-induced HCC. We conclude that N-myc overexpression is a regular feature of WHV- but not GSHV-associated hepatocarcinogenesis in a common host. In contrast, c-myc transcriptional deregulation is rarely encountered in WHV-induced HCC but is frequent in GSHV-induced HCC.

Animals↗
PubMed1992

The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis.

Hepatitis B viruses (hepadnaviruses) replicate their DNA genomes by reverse transcription of an RNA intermediate. Efforts to examine the biochemical mechanism for viral DNA synthesis have been hampered by the failure to solubilize the reverse transcriptase from virions and to express the polymerase in heterologous systems in an enzymatically active form. Here, we demonstrate that the polymerase of a hepadnavirus synthesized in an in vitro translation reaction exhibits reverse transcriptase activity. Furthermore, our results show that the polymerase acts as a primer for DNA synthesis and remains covalently linked to nascent DNA, a feature that is not known to exist in any other RNA-directed DNA polymerases. Priming of DNA synthesis requires viral RNA but occurs independently of other viral components. The ability to express the hepadnavirus reverse transcriptase in an enzymatically active form will allow detailed biochemical and functional analyses of this complex enzyme, and may facilitate the identification of inhibitors required for antiviral therapy.

Base Sequence↗
PubMed1992

Replication of DHBV genomes with mutations at the sites of initiation of minus- and plus-strand DNA synthesis.

We have examined the consequences on duck hepatitis B virus DNA synthesis of deleting the 5' and 3' copies of the 12 base sequence, DR1, from the viral pregenome. With the wild-type virus, reverse transcription initiates at nt 2537 within the 3' copy of DR1. When this sequence was deleted, initiation of reverse transcription was found at two other sites located closer to the 3' end of the pregenome (nt 2576 and nt 2644). The 3-base motif UUA was the only sequence common to these sites as well as the wild-type initiation site in DR1. Deletion of the 5' copy of DR1 did not alter minus strand synthesis, but led to aberrant priming of plus strand synthesis to generate predominantly linear rather than relaxed circular, double-stranded viral DNA, in agreement with the recent report by Loeb et al. (EMBO J. 10, 3533-3540, 1991). A mutant lacking only the 3' copy of DR1 rapidly converted to wild type in transfected cells. This apparently occurred as a consequence of conversion of newly synthesized relaxed circular to covalently closed circular (CCC) DNA, which might then serve as a template for the synthesis of wild-type viral RNAs. A mutant lacking only the 5' copy of DR1 did not exhibit this behavior. These results support the conclusion that amplified CCC DNA serves as transcriptional template.

Animals↗
PubMed1991

Beat-to-beat variation of heart rate in diabetic patients with autonomic neuropathy and in completely cardiac denervated patients following orthotopic heart transplantation.

Dysfunction of the vagal nerve, an early symptom in the development of autonomic neuropathy, can be assessed reliably by the beat-to-beat variation in heart rate. Patients after a cardiac transplantation are a unique model to investigate the beat-to-beat variation of a completely denervated heart. Heart rate and the beat-to-beat variation during normal and deep respiration were investigated in diabetic subjects with an autonomic neuropathy (n = 10), age and sex matched healthy controls (n = 10) and cardiac transplanted patients (n = 10). Further studies during pharmacological blockade of the parasympathetic nervous system with atropine were performed. In the denervated heart the coefficient of variation of the beat-to-beat interval was 0.38 +/- 0.02% during normal respiration, compared to 1.32 +/- 0.13% (P less than 0.0001) and 2.56 +/- 0.13% (P less than 0.0001) in the diabetic and control subjects, respectively. Administration of atropine (2 mg intravenously) decreased the coefficient of variation of the RR-interval to 0.73 +/- 0.09% in the diabetic patients (P less than 0.0005) and to 0.67 +/- 0.07% in the controls (P less than 0.0001), whereas the coefficient of variation remained unaffected in the cardiac denervated patients (0.39 +/- 0.02%). In the three groups an almost parallel increase of the RR-variation was observed during deep respiration at a rate of 6 breaths/min (from 0.38 +/- 0.02% to 1.99 +/- 0.38% in cardiac transplanted patients, P less than 0.0025; from 1.32 +/- 0.13% to 3.10 +/- 0.43% in diabetic patients, P less than 0.0025; from 2.56 +/- 0.13% to 5.42 +/- 0.94% in healthy controls, P less than 0.005). We conclude that a beat-to-beat variation of heart rate is present in the completely denervated heart. This RR-variation can not be influenced by a pharmacological blockade of the parasympathetic nervous system with atropine. The beat-to-beat variation increases during deep respiration not only in healthy controls but also in diabetic patients with autonomic neuropathy (partially denervated hearts) and cardiac transplanted patients (completely denervated hearts). This indicates an intracardiac mechanism in the modulation of heart rate.

Atropine↗
PubMed1991

Identification of a signal necessary for initiation of reverse transcription of the hepadnavirus genome.

Reverse transcription of the hepadnavirus genome initiates near the 3' end of the RNA template and has previously been shown to depend on sequences flanking the initiation site for DNA synthesis (C. Seeger and J. Maragos, J. Virol. 64:16-23, 1990). DNA synthesis leads to the covalent attachment of a protein to the 5' end of minus-strand DNA, and it is generally believed that this protein serves as the primer for reverse transcription. To examine priming in more detail, we have carried out a detailed genetic analysis of the nucleotide sequences at the origin of minus-strand DNA synthesis characterized in our earlier study. This mutational analysis has led to the identification of a short, four-nucleotide-long sequence as the signal for initiation of reverse transcription. This signal is a UUUC sequence motif flanking the position of the 5' end of minus-strand DNA, which alone is not sufficient for DNA synthesis, indicating that positional effects are also important to specify the origin of DNA synthesis.

Base Sequence↗
PubMed1991

Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host.

Chronic infection with hepatitis B viruses (hepadnaviruses) is a major cause of hepatocellular carcinoma (HCC), but the incubation time varies from 1 to 2 years to several decades in different host species infected with indigenous viruses. To discern the influence of viral and host factors on the kinetics of induction of HCC, we exploited the recent observation that ground squirrel hepatitis virus (GSHV) is infectious in woodchucks (C. Seeger, P. L. Marion, D. Ganem, and H. E. Varmus, J. Virol. 61:3241-3247, 1987) to compare the pathogenic potential of GSHV and woodchuck hepatitis virus (WHV) in chronically infected woodchucks. Chronic GSHV infection in woodchucks produces mild to moderate portal hepatitis, similar to that observed in woodchucks chronically infected with WHV. However, HCC developed in GSHV carriers about 18 months later than in WHV carriers. Thus, although both viruses are oncogenic in woodchucks, GSHV and WHV differ in oncogenic determinants that can affect the kinetics of appearance of HCC in chronically infected animals.

Animals↗
PubMed1990

Identification and characterization of the woodchuck hepatitis virus origin of DNA replication.

Replication of the woodchuck hepatitis virus (WHV) genome requires the synthesis of minus-strand DNA from an RNA template, the pregenome, by reverse transcription. During this reaction, the 5' end of minus-strand DNA becomes covalently linked to a protein. The position of the 5' end of minus-strand DNA was identified previously, but the initiation site for DNA synthesis on pregenomic RNA remained ambiguous because of a sequence repetition at the termini of the RNA template for reverse transcription. Employing a recently designed expression vector for the production of infectious WHV, we localized the origin of minus-strand DNA synthesis to the 3' end of pregenomic RNA. In addition, we identified the nucleotide sequences on pregenomic RNA that provide the signal for the initiation of reverse transcription. Removal of this signal sequence from pregenomic RNA abolished minus-strand DNA synthesis. Insertion of a DNA oligomer bearing this signal sequence at the 3' end of pregenomic RNA restored the production of minus-strand DNA joined to protein. Our results support a model in which protein is the primer for reverse transcription of minus-strand DNA of WHV.

Animals↗
PubMed1989

In vitro infection of woodchuck hepatocytes with woodchuck hepatitis virus and ground squirrel hepatitis virus.

Primary cultures of woodchuck hepatocytes were demonstrated to be susceptible to in vitro infection by both woodchuck hepatitis virus and ground squirrel hepatitis virus, as evidenced by the appearance of DNA species characteristic of hepadnavirus replication. Initiation of infection by woodchuck hepatitis virus was blocked by the presence of suramin, polybrene, or dideoxycytidine. Viral CCC DNA, the putative template for viral RNA transcription, was detected at 2 days postinfection. Accumulation of intracellular intermediates in virion DNA synthesis was negligible until 7-10 days postinfection, but these DNA intermediates then increased dramatically in amount over the next few weeks. Results were obtained which suggested that the prolonged accumulation of intermediates in virion DNA synthesis was an intrinsic property of the infection of individual cells, and not the result of a slow spread of virus through the cultures.

Animals↗
PubMed1989

Expression of infectious woodchuck hepatitis virus in murine and avian fibroblasts.

The liver is the primary site for replication of the hepadnavirus genome. We asked whether the posttranscriptional phase of the viral replication cycle would depend on hepatocyte-specific functions. For this purpose, we assayed a previously constructed chimera between sequences of the cytomegalovirus immediate-early promoter-enhancer region and woodchuck hepatitis virus (WHV) (C. Seeger and J. Maragos, J. Virol. 63:1907-1915, 1989) for its ability to direct the synthesis of infectious WHV in hepatoma cells and in murine and avian fibroblast cells. Viruslike particles containing WHV DNA were produced transiently in transfected hepatoma cells and in fibroblasts. Inoculation of woodchucks with culture medium from hepatoma cells or fibroblasts transfected with viral DNA led to productive WHV infection, as observed following infection of woodchucks with serum from WHV-infected animals. These results demonstrate that posttranscriptional events of the hepadnavirus replication cycle are not dependent on hepatocyte-specific functions.

Animals↗
PubMed1989

Molecular analysis of the function of direct repeats and a polypurine tract for plus-strand DNA priming in woodchuck hepatitis virus.

The replication of the hepadnavirus DNA genome is initiated by reverse transcription of pregenome RNA into minus-strand DNA followed by plus-strand DNA synthesis. The priming of plus-strand DNA requires the transfer of an RNA primer from pregenome RNA to the primer-binding site on minus-strand DNA. Annealing of the primer to the primer-binding site is facilitated by short direct repeats, DR1 and DR2. To investigate the mechanism of plus-strand primer formation, we have introduced specific mutations into DR1 and DR2 and measured the effect of these mutants on initiation of plus-strand DNA synthesis. To facilitate such an analysis, we have constructed a vector for the efficient expression of woodchuck hepatitis virus in cultured cells. Our results suggest that the 3' end of the RNA primer is determined prior to its transfer to the primer-binding site and that the determination of the 3' end of the primer does not depend on a specific sequence motif at the cleavage site. In addition, we have identified an alternative initiation site for plus-strand DNA synthesis at a purine-rich sequence between DR1 and DR2. Initiation at this site occurs by a mechanism that is independent of the direct repeats and does not require the transfer of an RNA primer to the primer-binding site.

Animals↗
PubMed1987

In vitro recombinants of ground squirrel and woodchuck hepatitis viral DNAs produce infectious virus in squirrels.

Hepatitis B viruses of humans, woodchucks, ground squirrels, and ducks are similar biochemically but differ with respect to host range and pathogenicity. To pursue the genetic basis of these properties in the absence of a cell culture system for virus growth, we exploited the demonstrated infectivity of cloned hepatitis B virus DNA in whole animals. We constructed several recombinant molecules in vitro between cloned infectious genomes of woodchuck hepatitis virus (WHV) and ground squirrel hepatitis virus (GSHV) and assayed the recombinants for infectivity after intrahepatic injection in ground squirrels, which support growth of GSHV but not WHV. Two of the recombinants molecules initiated productive infection; in one recombinant genome, 76% of the coding region for the major surface glycoprotein of GSHV and for the overlapping portion of the presumptive gene for DNA polymerase was replaced by WHV DNA; in the other, 29% of the same coding domain was replaced by WHV DNA. These findings demonstrate the feasibility of generating viable recombinants of hepatitis B viruses from different animal species and suggest that the major host range determinants are not encoded within the surface antigen gene of these viruses.

Amino Acid Sequence↗
PubMed1986

Biochemical and genetic evidence for the hepatitis B virus replication strategy.

Hepatitis B viruses synthesize their open circular DNA genomes by reverse transcription of an RNA intermediate. The details of this process have been examined with the use of mammalian hepatitis B viruses to map the sites for initiation and termination of DNA synthesis and to explore the consequences of mutations introduced at short, separated direct repeats (DR1 and DR2) implicated in the mechanisms of initiation. The first DNA strand to be synthesized is initiated within DR1, apparently by a protein primer, and the completed strand has a short terminal redundancy. In contrast, the second DNA strand begins with the sequence adjacent to DR2, but its 5' end is joined to an oligoribonucleotide that contains DR1; thus the putative RNA primer has been transposed to the position of DR2. It is now possible to propose a detailed strategy for reverse transcription by hepatitis B viruses that can be instructively compared with that used by retroviruses.

Animals↗
PubMed1984

The cloned genome of ground squirrel hepatitis virus is infectious in the animal.

The lack of an in vitro infectivity assay for hepatitis B viruses has impeded the analysis of their genetic organization. To examine the feasibility of generating mutant and recombinant viruses after manipulation of cloned viral DNA in vitro, we have tested the infectivity of the cloned genome of ground squirrel hepatitis virus (GSHV) in virus-free Beechey ground squirrels. We demonstrate that cloned GSHV DNA is infectious when injected directly into the liver in the form of trimeric, head-to-tail recombinant clones and recircularized monomeric molecules but not when injected into the portal vein. Infections established in all four recipients of intrahepatic injections of cloned GSHV DNA exhibited the characteristics observed after administration of virus: GSHV surface antigen and viral DNA appeared in the serum 14-22 weeks after inoculation, and both circular and heterogeneous protein-linked forms of viral DNA were found in liver biopsy samples. Furthermore, virus present in the sera of these animals can be transmitted to other ground squirrels. These findings imply that any function of virion proteins in the initiation of infection by hepatitis B viruses can be bypassed with the use of cloned viral DNA and that this animal model is suitable for testing mutant genomes.

Animals↗
PubMed1984

Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.

Amino Acid Sequence↗