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D Ganem

Publications and source records attributed to D Ganem.

At least 127 records · Page 7Linked to original sources
PubMed1988

Secreted hepatitis B surface antigen polypeptides are derived from a transmembrane precursor.

Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also independently secreted from infected cells as a lipoprotein particle. Secretion proceeds without signal sequence removal or cleavage of other segments of the polypeptide. We have examined the synthesis and transport of HBsAg in cultured cells expressing the cloned surface antigen gene. Our results show that HBsAg is initially synthesized as a integral membrane protein. This transmembrane form is slowly converted to a secreted lipoprotein complex in the lumen of the endoplasmic reticulum via a series of definable intermediates, after which it is secreted from the cell. This unusual export process shares many features with the assembly and budding reactions of conventional enveloped animal viruses. However, it differs importantly in its absence of a requirement for the participation of nucleocapsid or other viral proteins.

Animals↗
PubMed1988

Developmental regulation of the cysteine-rich outer-membrane proteins of murine Chlamydia trachomatis.

The developmental cycle of the obligate intracellular prokaryote Chlamydia trachomatis involves the serial alternation of two distinct morphological forms of the organism. To examine the basis of chlamydial differentiation we have searched for developmentally regulated gene products in this species. Chlamydia-infected cells were pulse-labelled with [35S] cysteine at various stages of development and the products of synthesis examined by SDS-PAGE. Our results indicate that the synthesis of the cysteine-rich outer-membrane proteins is developmentally regulated, occurring only late in the cycle during the conversion of reticulate bodies to elementary bodies. Both hydroxyurea and ampicillin block this conversion; as a result of this blockade the cysteine-rich outer-membrane proteins are not produced in the presence of either drug.

Ampicillin↗
PubMed1987

Homologous recombination between hepadnaviral genomes following in vivo DNA transfection: implications for studies of viral infectivity.

The cloned DNA of duck hepatitis B virus (DHBV) can initiate a productive infection of susceptible ducklings when presented by direct intrahepatic injection. We have examined the effects of the structure of the incoming DNA upon the outcome of this in vivo transfection. Plasmid-linked DHBV genomes of greater than unit length regularly give rise to infectious virus irrespective of the location of the plasmid insert on the transcriptional map. When mutant DHBV genomes are coinjected with subgenomic viral DNA fragments spanning the mutation, wild-type recombinants arise. These results indicate that, as in transfection of cultured cells, in vivo transfection of hepatocytes is regularly followed by homologous recombination involving incoming DNA molecules. These frequent recombination events will complicate efforts to use in vivo transfection for certain genetic analyses of hepadnaviruses.

DNA, Viral↗
PubMed1987

Chlamydial rRNA operons: gene organization and identification of putative tandem promoters.

We isolated and characterized the rRNA operons of murine Chlamydia trachomatis. By exhaustively screening a library of chlamydial DNA and by blot hybridization of genomic DNA, we showed that there are only two rRNA operons in C. trachomatis. S1 nuclease protection and primer extension analysis were used to map the 5' and 3' ends of the mature 16S and 23S transcripts in both rRNA cistrons and, additionally, to demonstrate the lack of intervening sequences in these genes. The 5' ends of the presumed primary rRNA transcript were located and found to originate at two tandem sites separated by 100 base pairs. The two tandem chlamydial rDNA transcripts were not differentially regulated. Their products were coordinately expressed and were detectable as early as 9 h postinfection. However, the upstream transcript was only 10% as abundant as the downstream transcript. The sequences surrounding the transcription initiation sites bore little homology with each other or with the classic Escherichia coli -10 and -35 promoter sequences. This finding suggests that chlamydial transcription signals may differ from those of previously studied procaryotes.

Base Sequence↗
PubMed1987

5'-terminal sequences influence the segregation of ground squirrel hepatitis virus RNAs into polyribosomes and viral core particles.

To determine which of the major ground squirrel hepatitis virus RNAs serve as mRNAs and which serve as templates for reverse transcription of the genome, we analyzed the subcellular distribution of these RNAs in livers of infected ground squirrels. Both major classes of viral RNA, the 2.3- and 3.5-kilobase (kb) classes, are unspliced, are polyadenylated at a common position, and display heterogeneous 5' ends that can encode proteins with different amino termini (G.H. Enders, D. Ganem, and H. Varmus, Cell 42:297-308, 1985). Both of the 2.3-kb RNAs, which encode surface antigens, appear to be predominantly associated with polyribosomes. Of the three 3.5-kb RNAs, the two longer, which can encode a protein initiated from the first methionine codon in the core antigen gene, appear to be predominantly associated with polyribosomes, and a minority of the shortest 3.5-kb RNAs, which can encode a protein initiated from the second methionine in the core antigen gene, appears to be associated with polyribosomes. This last RNA is instead found predominantly within viral core particles, consistent with evidence that indirectly implicates it in two steps of viral DNA synthesis (C. Seeger, D. Ganem, and H.E. Varmus, Science 232:477-484, 1986). None of the other viral RNAs is detectably packaged into cores. These findings provide independent evidence that the shortest 3.5-kb RNA is the template for synthesis of the viral genome and reveal a novel selectivity in viral RNA packaging.

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↗
PubMed1987

Expression of the precore region of an avian hepatitis B virus is not required for viral replication.

The core-antigen-coding region of all hepadnaviruses is preceded by a short, in-phase open reading frame termed precore whose expression can give rise to core-antigen-related polypeptides. To explore the functional significance of precore expression in vivo, we introduced a frameshift mutation into this region of the duck hepatitis B virus (DHBV) genome and examined the phenotype of this mutant DNA by intrahepatic inoculation into newborn ducklings. Animals receiving mutant DNA developed DHBV infection, as judged by the presence in hepatocytes of characteristic viral replicative intermediates; molecular cloning and DNA sequencing confirmed that the original mutation was present in the progeny genomes. Infection could be efficiently transmitted to susceptible ducklings by percutaneous inoculation with serum from mutant-infected animals, indicating that infectious progeny virus was generated. These findings indicate that expression of the precore region of DHBV is not essential for genomic replication, core particle morphogenesis, or intrahepatic viral spread.

Animals↗
PubMed1987

The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid.

The preS/S coding region of hepatitis B virus encodes two polypeptides (preS1 and preS2) that are larger in size but less abundant than the major viral surface antigen (S) protein. Unlike the preS2 and S proteins, the preS1 protein is preferentially localized on circulating virus particles but is not efficiently secreted from mammalian cells in culture. To search for differences in protein processing that might relate to these properties, we determined whether any of the hepatitis B virus surface proteins are acylated with long-chain fatty acids. Transfected COS cells expressing all three proteins were incubated with 3H-palmitate or 3H-myristate, and the cell extracts were examined by immunoprecipitation. While none of these proteins was labeled with 3H-palmitate, the preS1 protein but not the preS2 or S protein incorporated 3H-myristate via a hydroxylamine-resistant amide linkage. Comparison of the N-terminal amino acid sequences of hepadnaviral preS1 proteins with those of known myristylated proteins suggests that this unusual modification may be a common feature of all hepadnaviral preS1 proteins.

Acylation↗
PubMed1987

Multiple topogenic sequences determine the transmembrane orientation of the hepatitis B surface antigen.

To investigate the mechanism by which complex membrane proteins achieve their correct transmembrane orientation, we examined in detail the hepatitis B surface antigen for sequences which determine its membrane topology. The results demonstrated the presence of at least two kinds of topogenic elements: an N-terminal uncleaved signal sequence and an internal element containing both signal and stop-transfer function. Fusion of reporter groups to either end of the protein suggested that both termini are translocated across the membrane bilayer. We propose that this topology is generated by the conjoint action of both elements and involves a specifically oriented membrane insertion event mediated by the internal sequence. The functional properties of each element can be instructively compared with those of simpler membrane proteins and may provide insight into the generation of other complex protein topologies.

Biological Transport↗
PubMed1986

Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide.

The presurface (preS) proteins of hepatitis B virus are structural components of the viral envelope that may play important roles in virion assembly and infectivity. They are specified by a large open reading frame that includes the coding region for the major surface (S) protein in its 3' half. Translation of the preS proteins initiates upstream from the S region, giving rise to proteins that are composed of the S domain and an additional 163 (preS1) or 55 (preS2) amino acids. Little is known about the biosynthesis and assembly of these proteins. The expression of the S and preS1 proteins was examined by transfecting cultured mammalian cells with viral DNA and injecting synthetic messenger RNA's into Xenopus oocytes. In contrast to the proteins encoded by the S region, the preS1 proteins are not detectably secreted into the culture medium. Furthermore, when the S and preS1 proteins are synthesized together, secretion of the S proteins is specifically and strongly inhibited. The results suggest a unique molecular interaction during secretion of the S and preS proteins that may be important for virus assembly.

Animals↗
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↗
PubMed1986

Antibodies to pre-S and X determinants arise during natural infection with ground squirrel hepatitis virus.

The DNA sequence of the ground squirrel hepatitis virus (GSHV) genome predicts the existence of several proteins in addition to the major surface (S) and core antigens. These include the pre-S1 and pre-S2 proteins, initiated at sites within the open reading frame preceding and continuous with the coding region for the S gene product, and the X protein, the putative product of an independent reading frame. Using an antibody directed against a peptide predicted by codons 130 to 143 of the pre-S1 reading frame, we identified a 43-kilodalton product of the pre-S1 coding region in preparations of GSHV surface antigen purified from the sera of infected animals. In addition, by immunoprecipitation of S- and pre-S-specific in vitro translation products with ground squirrel sera obtained after GSHV infection, we determined that antibodies arise to both S and pre-S determinants. The antibody response to pre-S includes, in some cases, reactivity to pre-S1-specific domains and is not always associated with an anti-S response. Similarly, by production of the viral X gene product in vitro followed by immunoprecipitation with ground squirrel sera, we showed that antibodies to this viral gene product also arise during infection, indicating that X antigenic determinants are synthesized during viral infection and are recognized by the host immune system.

Animals↗
PubMed1986

Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide.

Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also secreted from cells as a subviral particle, without concomitant cleavage of N-terminal amino acid sequences. We examined this unusual export process in a cell-free system and showed that the initial product of HBsAg biosynthesis is an integral transmembrane protein, with most or all of its C-terminal half on the lumenal side of the endoplasmic reticulum membrane. To study the nature of its topogenic signals, we synthesized fusion proteins between HBsAg and the nonsecreted protein alpha-globin. Fusion proteins in which approximately 100 amino acids of globin preceded all HBsAg sequences were successfully translocated in vitro; the same domain as in the wild-type HBsAg was transported into the vesicle lumen. Fusions in which the entire globin domain was C terminal were able to translocate both the C-terminal region of HBsAg and its attached globin domain. Thus, uncleaved signal sequences in p24s function to direct portions of the molecule across the membrane and are able to perform this function even when positioned in an internal protein domain.

DNA Restriction Enzymes↗
PubMed1985

Mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant.

The ground squirrel hepatitis virus (GSHV) belongs to a recently defined class of viruses with open circular DNA genomes that encode proteins in extensively overlapping reading frames and appear to replicate via RNA intermediates. We have determined the primary structure of the major GSHV transcripts in the livers of infected ground squirrels. Both major classes of transcripts, 2.3 kb and 3.5 kb, are plus-stranded, unspliced, polyadenylated at a common position, and display heterogeneous 5' ends that can encode proteins with different amino termini. The 2.3 kb transcripts, like their structural analogs transcribed from human hepatitis B virus DNA, are likely mRNAs for products of the major surface antigen and presurface coding domains. The 3.5 kb transcripts are likely mRNAs for one or more products of the core antigen reading frame; these transcripts also encompass the entire genome and contain terminal redundancies of 130-160 nucleotides that include a putative initiation site for reverse transcription.

Animals↗
PubMed1985

A frameshift mutation in the pre-S region of the human hepatitis B virus genome allows production of surface antigen particles but eliminates binding to polymerized albumin.

The coding region for the major polypeptide (p24S) of hepatitis B surface antigen (HBsAg) is preceded by an in-phase open reading frame termed pre-S. The coding potential of the pre-S region was examined in mouse L cells transformed with cloned hepatitis B virus DNA. Such cells produce three HBsAg-related polypeptides of Mr 24,000, 27,000, and 35,000 organized into complex particles of 22 nm diameter. These HBsAg particles bind to polymerized human albumin, but not to polyalbumins of several other species. In contrast, cells transformed with hepatitis B virus DNA bearing a frameshift mutation near the 3' end of the pre-S region secrete immunoreactive HBsAg particles containing only the 24,000 and 27,000 Mr species. These mutant particles, which lack the 35,000 Mr species, are unable to bind polymerized human albumin. These studies indicate that the pre-S region encodes the 35,000 Mr species, that this product accounts for the known polyalbumin-binding activity of HBsAg but is not required for assembly and secretion of HBsAg 22-nm particles, and that the major polypeptide of HBsAg is not derived primarily by cleavage of larger precursors encoded by the pre-S region.

DNA, Viral↗
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↗