Retroid virus genome replication.
Explore the source record for details and available documents.
DNA Replication↗
Biomedical subjects
W S Mason
Publications and source records attributed to W S Mason.
Initiation and termination of duck hepatitis B virus DNA synthesis during virus maturation.
We characterized a number of important features of the structure of the cohesive overlap region of the DNA genome of duck hepatitis B virus. The 5'-terminal nucleotide of minus-strand DNA was localized to nucleotide 2537, a G residue within the 12-base repeat sequence DR1. This G residue was shown to be the site of a covalent linkage to a protein, consistent with speculation that this protein is the primer of minus-strand synthesis, which occurs by reverse transcription. The 3' terminus of the minus strand was heterogeneous, being mapped to nucleotides 2530 and 2531, indicating that the minus strand is terminally redundant by seven or eight bases and ends at the putative 5' end of the transcribed RNA template (pregenome) for reverse transcription. We previously demonstrated that the presumptive RNA primer of plus-strand synthesis remains attached to plus-strand DNA during virus maturation; moreover, the sequence of this primer suggested an origin from the 5' end of the pregenome (J.-M. Lien, C. E. Aldrich, and W. S. Mason, J. Virol. 57:229-236, 1986). We show here that over 75% of plus-strand primers are capped, further supporting the idea that these primers are uniquely derived from the 5' end of the pregenome. Finally, we found that seemingly mature duck hepatitis B virus genomes are incomplete by at least 12 bases, in that the 12-base repeat sequence DR2 is not copied into plus-strand DNA during virus maturation. Since DR2 in virion DNA is duplexed with the RNA primer of plus-strand synthesis, it is possible that the failure to make complete plus strands is due to an inability of the viral DNA polymerase to carry out a displacement of the bound RNA primer.
Animals↗
Humoral immune responsiveness in duck hepatitis B virus-infected ducks.
Immunofluorescence assays with fixed tissue sections were used to characterize antibody reactivity in sera obtained from duck hepatitis B virus-infected ducks. Under conditions of experimental infection, antibody to core antigen but not to surface antigen was detectable. A majority of the ducks infected at 8 days after hatching and a minority of those infected at 1 day after hatching showed a transient anti-core antigen humoral response; this response was stronger in the antibody-positive ducks infected on day 8 than in those infected on day 1. Antiviral antibody was not detected in the sera of ducks congenitally infected with duck hepatitis B virus. Several of the infected ducks, but none of the uninfected ducks, exhibited autoantibody reactivity for alpha-islet-cell-associated antigen.
Animals↗
Viral antigen expression in the pancreas of DHBV-infected embryos and young ducks.
The time course of appearance of viral antigen-positive pancreatic cells was examined in both congenitally duck hepatitis B virus (DHBV)-infected duck embryos and experimentally DHBV-infected posthatch ducks. In the embryos, the earliest detectable viral antigen-positive pancreatic cells were localized to islets and identifiable as endocrine on the basis of hormone expression. Non-islet-associated, viral antigen-positive cells appeared at a late stage of embryogenesis, following the onset of chymotrypsinogen production by exocrine tissue; a number of these viral antigen-positive cells were directly identifiable as exocrine on the basis of chymotrypsinogen expression. By contrast, in the pancreas of experimentally infected posthatch ducks, the appearance of viral antigen-positive exocrine cells (chymotrypsinogen-positive) predated the appearance of antigen-positive islet cells. These results are consistent with the possibility that viral antigen expression in exocrine tissue is dependent on the state of cell maturation.
Animals↗
Evidence that a capped oligoribonucleotide is the primer for duck hepatitis B virus plus-strand DNA synthesis.
The plus strand of virion DNA of duck hepatitis B virus possessed, at its 5' terminus, a capped oligoribonucleotide 18 to 19 bases in length. This oligoribonucleotide had a unique 5' end, the heterogeneity in length reflecting two distinct junctions with plus-strand DNA that were 1 base apart. The sequence of the RNA differed from that predicted by the sequence of duck hepatitis B virus upstream of the 5' ends of plus-strand DNA but was identical to a downstream sequence corresponding to the 5' terminus of a major poly(A)+ viral RNA mapped by Büscher and co-workers (Cell 40:717-724, 1985). This RNA transcript is thought to serve as the template (i.e., the pregenome) for minus-strand synthesis via reverse transcription. The results suggest that the pregenome also donates a capped oligoribonucleotide that acts as the primer of plus-strand DNA synthesis, using the minus-strand DNA as template.
Base Sequence↗
Ablation of humoral immunity in 15I5 x 72 chickens is not predisposing to the formation of subgroup G virus-induced distal sarcomas.
Clone (cl.) 85 infection of 15I5 x 72 chickens at 4 weeks posthatch results in a lower frequency of distal sarcomas than does Prague (Pr)-B infection. As higher titers of virus-neutralizing antibody are generated in the cl.85-infected chickens, the possibility was addressed in the present study that the low frequency of cl.85-induced distal sarcomas is a direct consequence of the strong neutralizing response. Our observations indicate that ablation of humoral immunity by embryonic bursectomy does not serve to increase this frequency, implying that the antiviral humoral response is not required to limit formation of cl.85-induced distal sarcomas.
Animals↗
Sarcoma growth in 15I5 x 7(2) chickens infected with avian sarcoma viruses of subgroup B or G.
A comparative study was made of sarcoma growth in 15I5 x 7(2) chickens infected in the wing web at 4 weeks of age with strains of subgroup B or G avian sarcoma viruses. Infection with sarcoma viruses of either subgroup B or G resulted in the formation of progressive wing web sarcomas at the site of inoculation. The survival times of the subgroup G virus-infected chickens were generally at least twice as great as the survival times of the subgroup B virus-infected chickens, which averaged 6-9 weeks postinoculation. At 5 weeks postinfection, a significantly higher titer of virus neutralizing antibody was detected in the subgroup G virus-infected chickens. Necropsy indicated that a high percentage of subgroup B virus-infected chickens exhibited fibrosarcomas at sites distal to the primary wing web sarcomas, whereas only a small percentage of subgroup G virus-infected chickens exhibited distal sarcomas. The results further indicated that the viral env gene is a determinant of the pattern of distal sarcoma formation.
Animals↗
Individual cells in tissues of DHBV-infected ducks express antigens crossreactive with those on virus surface antigen particles and immature viral cores.
Double immunofluorescence assays of fixed sections of tissues from Pekin ducks congenitally infected with duck hepatitis B virus were carried out to score cells reactive to antiserum elicited either to viral DNA synthesis complexes (immature cores) purified from liver of infected ducks or to viral surface antigen particles and virions purified from sera of viremic ducks. Subpopulations of doubly fluorescent cells were detectable in liver (hepatocytes and bile duct epithelia), in kidney (proximal tubular epithelia), and in pancreas (acinar-associated cells and endocrine alpha and beta cells); all cells reactive with one antiserum were reactive with the second antiserum. Competition assays established that the cell-associated antigens recognized by the two antisera were mutually non-crossreactive. The cell-associated antigen recognized by the antiserum elicited to the purified immature cores appears to be crossreactive with a 35,000-Da polypeptide fraction associated with immature cores.
Animals↗
Viral antigen in endocrine cells of the pancreatic islets and adrenal cortex of Pekin ducks infected with duck hepatitis B virus.
Endocrine cells in the pancreas and adrenal glands of duck hepatitis B virus-infected ducks were examined for the presence of viral antigen. Analysis of pancreas tissue was based on double immunofluorescence assays in which anti-duck hepatitis B virus serum was used to detect viral antigen, and anti-glucagon and anti-insulin serum were used, respectively, to identify endocrine alpha and beta cells. Assays of pancreas from infected ducks ranging in age from 3 to 20 weeks indicated that subpopulations of both alpha and beta cells expressed viral antigen. A higher percentage of viral antigen-positive cells was observed in alpha-islets than in beta-islets. As assayed by immunofluorescence and immunoperoxidase staining with anti-viral serum, small clusters of viral antigen-positive cells were detected in the adrenal glands of young infected ducks. These cells were identified as cortical cells on the basis of histologic criteria.
Adrenal Cortex↗
Mapping of the cohesive overlap of duck hepatitis B virus DNA and of the site of initiation of reverse transcription.
The hepatitis B-like viruses have a approximately 3.2 kilobase, partially double-stranded DNA genome that is held in a circular conformation by a cohesive overlap between the 5' ends of the two strands. In addition, a protein is covalently bound to the 5' end of the minus strand of virion DNA. The sequence of the cohesive overlap region and its location relative to open reading frames and to the initiation site for minus-strand DNA synthesis, which occurs by reverse transcription of viral RNA, were investigated in duck hepatitis B virus. The 5' ends of virion DNA were mapped by restriction endonuclease analysis of labeled virion DNA, S1 nuclease digestion, and primer extension, using avian myeloblastosis virus DNA polymerase. The cohesive overlap region was shown to be 69 +/- 4 base pairs in length. It contained a 10-base pair inverted repeat in approximately the middle and a 12-base pair direct repeat near each end. The apparent initiation site of reverse transcription was determined by partial sequence analysis of dideoxynucleotide-truncated minus-strand DNA intermediates and comparison of their lengths with the length of a known DNA sequence. It mapped within two to four nucleotides of the 5' end of the minus strand of virion DNA. The results are consistent with the interpretation that the 5' end of the minus strand of virion DNA is the origin of reverse transcription and that the protein covalently bound to virion DNA is the primer of reverse transcription.
Animals↗
Differential reactivity of serum antibody from tumor-bearing 15I5 X 7(2) chickens for cross-reactive species of endogenous retroviral envelope glycoprotein.
Experiments were undertaken to determine if the sera of avian sarcoma virus-infected 15I5 X 7(2) chickens exhibit antibody reactivity for species of endogenous retroviral envelope glycoprotein expressed in 15I5 X 7(2) fibroblasts. Two viruses were used for infection of the 15I5 X 7(2) chickens, Pr-A and cl. 85; the envelope glycoprotein of Pr-A, but not of cl. 85, is antigenically cross-reactive with 15I5 X 7(2) endogenous retroviral envelope glycoprotein. Both the Pr-A and cl. 85-infected 15I5 X 7(2) chickens exhibited serum antibody reactivity for the envelope glycoprotein of the endogenous retrovirus RAV-O. In contrast, neither group of infected chickens exhibited detectable serum antibody reactivity for a distinct species of endogenous retroviral envelope glycoprotein, one which though antigenically cross-reactive with the envelope glycoprotein of RAV-O is expressed to much higher levels in 15I5 X 7(2) fibroblasts. Possible mechanisms to account for the observed pattern of antibody reactivity are discussed.
Animals↗
Experimental transmission of duck hepatitis B virus.
Susceptibility to experimental infection with duck hepatitis B virus (DHBV) was explored, with the objective of defining procedures that were both rapid and reproducible. For the purpose of these experiments, a small flock of DHBV-free breeders was established as a source of susceptible eggs and ducklings, since ca. 10% of the ducks (all ages) from commercial flocks were DHBV infected. Intravenous inoculation of DHBV into 15-day duck embryos from the DHBV-free flock produced a persistent infection, with a high-titer viremia, in at least 80% of the injected animals. The tissue tropism of DHBV in these experimentally infected animals was similar to that associated with natural, congenital infections from viremic ducks to their progeny. Virus antigen was found not only in hepatocytes and bile duct epithelium of liver, but also in cells associated with exocrine and endocrine pancreas, and in proximal convoluted tubular epithelium of kidney. Infection of embryonic liver was rapid, as evidenced by active synthesis of DHBV-DNA by reverse-transcription of RNA by 24 hr postinjection. During this latter analysis, formation of supercoiled viral DNA appeared to precede the reverse-transcription phase of viral DNA synthesis, suggesting that this species may be important in initiation of infection.
Animals↗
Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus.
Liver, pancreas, and kidney from Pekin ducks infected with duck hepatitis B virus (DHBV) were assayed for the presence of both viral antigen and replication-specific forms of viral nucleic acid. In young congenitally infected ducks, antigen was detectable in hepatocytes and bile duct epithelia, in kidney glomeruli and tubular epithelia, and in cells localized to pancreatic acini. In older experimentally infected ducks, antigen was detectable in hepatocytes, in glomeruli and tubular epithelia, and in cells localized to presumptive pancreatic alpha-islets. All but the glomeruli-associated viral antigen appeared to be localized to the cytoplasm of antigen-positive cells. Much of the glomeruli-associated antigen appeared to be extracellular and was detected in glomeruli that were positive for the accumulation of immunoglobulin, observations suggestive of the deposition of viral antigen-antibody complexes. As analyzed with bulk tissue, replication-specific forms of viral nucleic acid were detectable in liver and pancreas from the young congenitally infected ducks and in liver and kidney from the older experimentally infected ducks.
Animals↗
Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus.
Analysis of duck hepatitis B viral DNA by gel electrophoresis, Southern blotting, and binding to benzoylated naphthoylated DEAE-cellulose showed that a protein is bound to the minus-strand virion DNA as well as to the full-length single strand, minus-strand species, and minus-strand DNA intermediates isolated from replicating complexes present in infected duck liver. By utilizing a modified dideoxynucleotidyl sequencing method, it was shown that the protein is covalently bound to the smallest detectable growing strands (ca. 30 bases) and that minus-strand synthesis begins at a unique site. These results support the notion that the protein may function as a primer for synthesis of the minus-strand DNA.
Animals↗
Alterations in the genomes of avian sarcoma viruses.
Explore the source record for details and available documents.
Alpharetrovirus↗
Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate.
Duck hepatitis B virus, a DNA virus closely related to human hepatitis B virus, was studied in infected duck liver. Subviral particles resembling the viral nucleocapsid cores were isolated from persistently infected liver and shown to have a DNA polymerase activity that utilizes an endogenous template and synthesizes both plus- and minus-strand viral DNA. Synthesis of the viral minus-strand DNA utilized an RNA template that was degraded as it was copied. Viral plus-strand synthesis occurred on the completed minus-strand DNA. A pathway for the replication of the DNA genome of hepatitis B-like viruses by reverse transcription of an RNA intermediate is proposed.
Animals↗
Asymmetric replication of duck hepatitis B virus DNA in liver cells: Free minus-strand DNA.
In order to study the replication of the DNA genome of duck hepatitis B virus, an avian virus related to human hepatitis B virus, we have characterized viral DNAs present in the livers of viremic ducks by agarose gel electrophoresis and the Southern blot procedure. In addition to relaxed circular DNA similar to virion DNA, livers contained a heterogeneous population of rapidly migrating species. The conformation of the rapidly migrating species was markedly sensitive to salt, suggesting that these species were largely single stranded. The largest major rapidly migrating species was shown to have an electrophoretic mobility that was insensitive to preheating of the DNA to 100 degrees C and was similar to that of denatured virus DNA 3 kilobases long, suggesting that this DNA was a single-stranded copy of the entire virus genome. Hybridization with strand-specific probes demonstrated that this 3-kilobase species, as well as more rapidly migrating DNAs, were predominantly minus strands.
Animals↗
Tandem duplication of the proviral DNA in an avian sarcoma virus-transformed quail clone.
Previous study has shown that an avian sarcoma virus-transformed quail clone, Q-B77-11, apparently contains full-size proviral DNA and releases virus particles which contain polymerase activity, but are unable to form foci. Recently, we have found that after extended tissue culture of Q-B77-11, the predominant cell type changed from one in which there was a single copy of the integrated viral DNA to one with two copies in tandem. There was approximately one copy of the large terminal redundancy between these two tandem proviruses. Polyadenylic acid-containing RNA species of twice the size of the viral genome were detected in these cells and are interpreted as transcripts of the tandem provirus.
Animals↗