Biomedical subjects

D Ganem

Publications and source records attributed to D Ganem.

At least 73 records · Page 4Linked to original sources

PubMed1996

The size and conformation of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) DNA in infected cells and virions.

The genome of a novel human herpesvirus has been detected in specimens of Kaposi's sarcoma (KS) and in several AIDS-related lymphoproliferative disorders. Here we examine the size and genomic conformation of the DNA of this virus (known as KS-associated herpesvirus or human herpesvirus 8) in latently and lytically infected cells and in virions. Pulsed-field gel electrophoresis of viral DNA shows that the viral genome is similar in size to those of other gammaherpesviruses (160 to 170 kb). As with Epstein-Barr virus, KS-associated herpesvirus DNA is stably maintained in latently infected B cells as episomal monomer circles and induction from latency is associated with the selective accumulation of linear genomic forms.

Cell Line↗

PubMed1996

Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II.

Induction of hepatocellular carcinoma in woodchucks by woodchuck hepatitis virus is associated with the activation of N-myc gene expression, usually by viral DNA integration in cis to the N-myc locus. We have examined the consequences of N-myc up-regulation in rodent hepatic cells in culture. Mouse alpha ML hepatocytes infected with a retroviral vector overexpressing the woodchuck N-myc2 gene display a higher proliferation rate than parental alpha ML cells but are morphologically unchanged and do not form colonies in soft agar. However, they display an increased propensity to undergo apoptosis, an effect that is markedly augmented by serum deprivation. Expression of the woodchuck hepatitis virus X gene in alpha ML cells does not alter the growth phenotype of the cells and has no effect upon N-myc-dependent apoptosis. However, apoptosis in N-myc2-expressing alpha ML cells is strongly inhibited by insulin-like growth factor II (IGF II). IGF II gene expression is also strongly up-regulated during hepatic carcinogenesis in vivo in virally infected animals and has been speculated to be part of an autocrine growth-stimulatory pathway. Our results suggest that IGF II may play another role in the development of virus-induced hepatoma: the prevention of programmed cell death triggered by deregulated N-myc expression.

Animals↗

PubMed1996

Cellular factors controlling the activity of woodchuck hepatitis virus enhancer II.

Woodchuck hepatitis virus (WHV) efficiently induces hepatocellular carcinoma in chronically infected hosts. A key step in hepatocarcinogenesis by WHV is insertional activation of the cellular N-myc gene by integrated viral DNA. WHV enhancer II (En II) is the major cis-acting element involved in this activation. Here we characterize this viral enhancer element and define the cellular factors involved in its activity. WHV En II activity is strongly liver specific and maps to an 88-nucleotide DNA segment (nucleotides 1772 to 1859) located 5' to the pregenomic RNA start site. Genetic analyses and electrophoretic mobility shift assays indicate that the enhancer contains three subregions important to its activity. The core elements of the enhancer are recognition sites for the liver-enriched factors HNF1 and HNF4; together, these signals account for the bulk of En II activity as well as its strong liver specificity. Multimerization of either recognition site produced strong activity even in the absence of other En II sequences. 5' to these elements is a binding site for the ubiquitous Oct-1 transcription factor, which further augments enhancer activity ca. twofold.

3T3 Cells↗

PubMed1996

Evidence for activation of the hepatitis B virus polymerase by binding of its RNA template.

The hepatitis B viruses replicate by reverse transcription of an RNA pregenome by using a virally encoded polymerase. A key early step in replication is binding of the polymerase to an RNA stem-loop (epsilon) of the pregenome; epsilon is both the RNA encapsidation signal and the origin of reverse transcription. Here we provide evidence that this interaction is also key to the development of enzymatic activity during biosynthesis of the polymerase. Duck hepatitis B virus polymerase expressed in Saccharomyces cerevisiae can synthesize DNA from epsilon-containing RNAs and can also end label other small RNAs. Expression of functional polymerase in S. cerevisiae requires interaction between the polymerase and epsilon during or shortly after translation for it to develop any enzymatic activity; if epsilon is absent during expression, the polymerase is inactive on RNAs both with and without epsilon. Functional duck polymerase can also be produced by in vitro translation, and synthesis of the polymerase in the presence of epsilon induces resistance in the polymerase to proteolysis by papain, trypsin, and bromelain. Induction of the resistance is specific for epsilon sequences that can support RNA encapsidation and initiation of DNA synthesis. Induction of the resistance precedes initiation of DNA synthesis and is reversible by degradation of epsilon. These two sets of data (i) support a model in which binding of epsilon to the polymerase induces a structural alteration of the polymerase prior to the development of enzymatic activity and (ii) suggest that this alteration may be required for the polymerase to mature to an active form.

Animals↗

PubMed1996

Relationship between viral DNA synthesis and virion envelopment in hepatitis B viruses.

While the intracellular pool of encapsidated hepatitis B viral DNA contains genomes in all stages of DNA replication, serum-derived virions contain predominantly mature, partially duplex, circular DNA genomes. To account for this finding, Summers and Mason proposed in 1982 that virion envelopment is somehow linked to the state of genomic maturation (J. Summers and W.S. Mason, Cell 29:403-415, 1982). Core gene mutations with phenotypes consistent with this concept have previously been identified in the duck hepatitis B virus (DHBV). Here we show that DHBV polymerase mutants with altered DNA synthesis also display defects in envelopment, and we provide quantitative estimates of the magnitude of the preference for the envelopment of mature DNA. In cells transfected with wild-type DHBV DNA, immature minus-strand DNA represents 18% of the intracellular pool but only 4% of extracellular virion DNA. A point mutation in the C-terminal domain of the polymerase strongly and selectively impairs plus-strand synthesis; in this mutant, the ratio of immature to mature DNA in the intracellular pool rises to 6:1 but is reduced to 1.5:1 in released virions. A missense mutation in the polymerase active site inactivates all viral DNA synthesis but still allows efficient RNA encapsidation; in this mutant, no detectable viral nucleic acid is enveloped and released. Thus, viral DNA synthesis is absolutely required for envelopment and export, and a strong further bias exists in favor of the export of genomes that have completed minus-strand synthesis and at least initiated plus-strand synthesis. These results imply that events within the interior of the nucleocapsid can powerfully influence its interactions with external viral envelope glycoproteins.

Amino Acid Sequence↗

PubMed1995

The pre-S domain of the large viral envelope protein determines host range in avian hepatitis B viruses.

In addition to their well-recognized hepatotropism, all hepatitis B viruses (HBVs) display marked species specificity, growing poorly or not at all in species other than those closely related to their natural hosts. We have examined the molecular basis for this narrow host range, using duck HBV (DHBV) and heron HBV (HHBV) as a model system. HHBV virions will not infect ducks in vivo and infect cultured duck hepatocytes extremely inefficiently in vitro. Mutant HHBV genomes lacking all viral envelope proteins (HHBV env-) can be complemented in trans with DHBV envelope proteins; the resulting pseudotyped virions can efficiently infect duck hepatocytes. Further complementation analysis reveals that of the two viral surface proteins (L and S), it is the L protein that determines host range. Pseudotyping of HHBV env- with DHBV/HHBV chimeric envelope proteins reveals that replacement of as few as 69 amino acids of the pre-S domain of the HHBV L protein by their DHBV counterparts is sufficient to permit infection of duck hepatocytes. These studies indicate that the species-specificity of hepadnaviral infection is determined at the level of virus entry and is governed by the pre-S domain of the viral L protein.

Amino Acid Sequence↗

PubMed1995

gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family.

Duck hepatitis B virus particles bearing the L and S envelope proteins bind a cellular glycoprotein of 180 kDa (gp180) with high affinity and specificity. Binding is mediated by the pre-S region of the L protein and is blocked by neutralizing but not by non-neutralizing monoclonal antibodies to the virus. These and other properties have suggested that gp180 may be a component of the viral entry machinery. Here we report the purification of gp180 from duck liver and the isolation and characterization of cDNA encoding it. DNA sequence analysis of this cDNA indicates that gp180 is a novel member of the basic carboxypeptidase gene family.

Amino Acid Sequence↗

PubMed1995

AIDS. Viruses, cytokines and Kaposi's sarcoma.

HIV infection strongly predisposes people to Kaposi's sarcoma, a complex proliferative lesion driven by autocrine and paracrine cytokine expression; new evidence implicates a second virus in the etiology of the disease.

Animals↗

PubMed1995

Sequences in the preC region of duck hepatitis B virus affect pregenomic RNA accumulation.

The pregenomic RNA of hepadnaviruses serves as both the mRNA for the core and polymerase proteins and the RNA template for reverse transcription. We have identified a region in the duck hepatitis B virus pregenomic RNA transcription unit that is critical for the accumulation of this transcript. This 85-nt region, termed alpha, is located within the preC region; deletion of alpha results in drastically reduced steady-state levels of pregenomic RNA. This effect is not due to reduction in transcription initiation or to enhancement of premature polyadenylation at the 5' copy of the viral poly(A) signal. However, this phenotype is suppressed by deletion of a second, larger region (beta) located ca. 1 kb downstream. The activity of the alpha element is tissue- and species-nonspecific; however, it displays absolute orientation-dependence and its activity is influenced by its position within the transcript. Models for its action are discussed.

Animals↗

PubMed1995

RNA sequences controlling the initiation and transfer of duck hepatitis B virus minus-strand DNA.

Hepadnaviruses replicate by reverse transcription of an RNA pregenome. Reverse transcription initiates within the stem-loop (SL) of the epsilon RNA packaging signal and is discontinuous: the nascent minus-polarity DNA is transferred to direct repeat 1 (DR1) at the 3' end of the pregenomic RNA prior to extensive elongation. In this study we analyzed the initiation and transfer of duck hepatitis B virus minus-strand DNA by using functional viral polymerase expressed in yeast cells. We extensively mutagenized both DR1 and the SL and observed the effects on reverse transcription initiation and on the transfer and subsequent extension of minus-strand DNA. Our results indicate that sequences throughout the SL affect initiation and that minus-strand DNAs initiated at three locations within the SL are competent for transfer to DR1. A short region of homology between the 5' end of minus-strand DNA and DR1 was necessary but not sufficient to direct the transfer and subsequent extension reactions. This homology was tolerant of minor substitutions, and 2 nucleotides of homology mediated transfer accurately. Mutations had greater detrimental effects on transfer and subsequent extension of minus-strand DNA when they were placed in DR1 than when they were placed in the SL. Efficient transfer of minus-strand DNA from a mutant SL to DR2 was observed in the yeast system. The hexanucleotide AAUUAC was identified as the primary cis element of the transfer acceptor, but this element was also insufficient to independently specify the acceptor location. Therefore, additional information, possibly positional context or unrecognized RNA secondary structure, is required.

Base Sequence↗

PubMed1994

Analysis of the binding of a host cell surface glycoprotein to the preS protein of duck hepatitis B virus.

We have previously identified a 180-kDa host cell glycoprotein (gp180) that specifically binds the surface envelope of duck hepatitis B virus (DHBV) and whose binding is inhibited by neutralizing antiviral monoclonal antibodies. Here we map the viral determinants required for gp180 binding to a 66-amino acid region within the preS domain of the envelope coding region. This region includes both major neutralizing preS epitopes previously defined by monoclonal antibodies. Examination of a series of linker-substitution mutations throughout preS indicates that all mutations that block gp180 binding ablate virus infectivity. Interestingly, two mutations that do not prevent binding can also impair infectivity.

Amino Acid Sequence↗

PubMed1994

A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis.

The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produced by alternative translation initiation in a single coding region. The smallest of these, the S protein, is a 24 kDa glycoprotein with multiple transmembrane domains. The M and L proteins contain the entire S domain at their C-termini, but harbor at their N-terminal additional (preS) domains of 55 or 174 amino acids, respectively. Most of these preS residues are displayed on the surface of mature virions and hence would be expected to be translocated into the endoplasmic reticulum (ER) lumen during biosynthesis. Using a coupled, in vitro translation/translocation system we now demonstrate that, contrary to expectation, virtually all preS residues of the L protein are cytoplasmically disposed in the initial translocation product. This includes some preS sequences which in the M protein are indeed translocated into the ER lumen. Since preS sequences are found on the external surface of the virion envelope, our results indicate that during or following budding a dramatic reorganization of either the envelope proteins or the lipid bilayer (or both components) must occur to allow surface display of these sequences. These findings imply that some membrane budding events can have remarkable and previously unsuspected topological consequences.

Base Sequence↗

PubMed1994

A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle.

We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcription-linked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron microscopic appearance. Production of unassembled core polypeptides can be uncoupled from production of capsid particles by decreasing core mRNA concentration. Addition of excess unlabeled core polypeptides allows the chase of the unassembled polypeptides into mature capsids. Using this cell-free system, we demonstrate that assembly of capsids proceeds by way of a novel high molecular weight intermediate. Upon isolation, the high molecular weight intermediate is productive of mature capsids when energy substrates are manipulated. A 60-kD protein related to the chaperonin t-complex polypeptide 1 (TCP-1) is found in association with core polypeptides in two different assembly intermediates, but is not associated with either the initial unassembled polypeptides or with the final mature capsid product. These findings implicate TCP-1 or a related chaperonin in viral assembly and raise the possibility that eukaryotic cytosolic chaperonins may play a distinctive role in multimer assembly apart from their involvement in assisting monomer folding.

Amino Acid Sequence↗

PubMed1994

A cell surface protein that binds avian hepatitis B virus particles.

We have identified a 180-kDa cellular glycoprotein (gp180) that binds with high affinity to duck hepatitis B virus (DHBV) particles. The protein was detected by coprecipitating labeled duck hepatocyte proteins with virions or recombinant DHBV envelope proteins, using nonneutralizing monoclonal antibodies to the virion envelope. Binding of gp180 requires only the pre-S region of the viral large envelope protein, since recombinant fusion proteins bearing only this region efficiently coprecipitate gp180. The DHBV-gp180 interaction is blocked by two independent neutralizing monoclonal antibodies. The protein is found on both internal and surface membranes of the cell, and the species distribution of gp180 binding activity mirrors the known host range of DHBV infection. Functional gp180 is expressed in a wide variety of tissues in susceptible ducks.

Animals↗

PubMed1994

Hepadnavirus reverse transcription initiates within the stem-loop of the RNA packaging signal and employs a novel strand transfer.

Replication of the hepadnavirus genome occurs by reverse transcription of an RNA pregenome and is mediated by the viral polymerase; the polymerase is also required for packaging of the pregenome through interaction with the RNA packaging signal, epsilon. Previous work suggested that reverse transcription of minus-strand DNA initiates within the sequence element DR1 (direct repeat 1) and that disruption of DR1 activates a cryptic initiation site in a downstream copy of epsilon. However, using active duck hepatitis B virus polymerase expressed in a yeast Ty vector system, we demonstrate that synthesis of minus-strand DNAs with 5' ends at DR1 requires the stem-loop of epsilon, whereas the production of DNAs mapping to epsilon does not require DR1. Mutations at epsilon that remove homology between epsilon and DR1 eliminate reverse transcripts with 5' ends in DR1, and restoring homology at DR1 to a mutant epsilon partially restores DNAs mapping to DR1. Insertions of one nucleotide into the bulge region of the epsilon stem-loop increase the length of minus-strand DNA whose 5' ends map to DR1 by one nucleotide. Thus, very short minus-strand primers are initiated within epsilon, rather than in DR1 as previously supposed; they are then transferred to a four-nucleotide homology in DR1. Transfer was also observed in vivo during replication of duck hepatitis B virus in avian cells; in this case, transfer is from the 5' copy of epsilon to the 3' copy of DR1. This minus-strand transfer reaction is likely to be a general feature of all hepadnaviruses.

Animals↗

PubMed1994

Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential.

Hepadnaviruses encode a single core (C) protein which assembles into a nucleocapsid containing the polymerase (P) protein and pregenomic RNA during viral replication in hepatocytes. We examined the ability of heterologous hepadnavirus C proteins to cross-oligomerize. Using a two-hybrid assay in HepG2 cells, we observed cross-oligomerization among the core proteins from hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus. When expressed in Xenopus oocytes, in which hepadnavirus C proteins form capsids, the C polypeptides from woodchuck hepatitis virus and ground squirrel hepatitis virus, but not duck hepatitis B virus, can efficiently coassemble with an epitope-tagged HBV core polypeptide to form mixed capsids. However, when two different core mRNAs are coexpressed in oocytes the core monomers show a strong preference for forming homodimers rather than heterodimers. This holds true even for coexpression of two HBV C proteins differing only by an epitope tag, suggesting that core monomers are not free to diffuse and associate with other monomers. Thus, mixed capsids result from aggregation of different species of homodimers.

Animals↗

PubMed1994

Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis.

Hepatitis B viruses encode a polymerase (P) protein with key roles in both reverse transcription and genomic RNA encapsidation. Genetic analysis of cis-acting signals required for viral replication implicates an RNA stem-loop structure in both RNA packaging and the initiation of reverse transcription, a process in which P protein also serves as the primer. We now show that duck hepatitis B virus (DHBV) polymerase binds specifically and with high affinity to this RNA stem-loop structure. Mutational analysis indicates that all mutations in the RNA target that inhibit the P protein-RNA interaction inhibit both in vivo RNA packaging and in vitro DNA priming to comparable extents. However, certain mutations in the loop region of the RNA have minimal impact on P protein-RNA binding but are nonetheless severely defective for packaging and DNA synthesis. Thus, P protein-RNA complex formation is necessary but not sufficient to initiate these activities. In addition, examination of RNA binding by truncated P proteins indicates that the C terminus of the polymerase, although required for RNA encapsidation in vivo, is dispensable for RNA binding and DNA priming.

Base Sequence↗

PubMed1993

Expression of functional hepatitis B virus polymerase in yeast reveals it to be the sole viral protein required for correct initiation of reverse transcription.

Replication of hepatitis B viruses proceeds by reverse transcription of an RNA intermediate, a reaction catalyzed by the virus-encoded polymerase (P protein). The reaction product is a partially duplex DNA whose (-)-strand is covalently linked to the P protein. Efforts to understand the mechanism of the reaction have been severely retarded by an inability to express functional polymerase outside of viral particles. Here we report the successful expression of enzymatically active polymerase in yeast cells, by fusing the P gene to coding sequences of the retrotransposon Ty1. The enzyme initiates correctly on viral RNA in yeast cells in vivo, producing nascent DNA chains covalently linked to protein, exactly as found in virus-infected cells. Replication complexes isolated from these yeast are enzymatically active in vitro, synthesizing DNA in a reaction that is actinomycin D-resistant but sensitive to RNase pretreatment. These results indicate that P protein is the sole viral protein required for the correct priming of reverse transcription and establish a tractable system for the biochemical dissection of the reaction.

DNA Replication↗