The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo (original) (raw)
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Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins
Proceedings of the National Academy of Sciences, 1983
A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase a, ,B, and -y activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor H complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-basepair Ad DNA.
Journal of General Virology, 1986
A series of human adenovirus type 2 genomes with deletions in the left inverted terminal repeat (ITR) have been constructed. Viral genomes that contained a minimum of 45 base pairs (bp) from the terminus of the genome were fully infectious and gave rise to progeny virus which maintained the deletion. In contrast, genomes containing 36 bp or less from the termini of the genome were not infectious. The boundary of a cis-acting element required for viral replication is therefore between 36 and 45 bp from the adenovirus termini and corresponds to the previously identified viral origin of replication, defined using a transfection assay to measure ori activity in vivo. The growth parameters of viruses with deletions in the left ITR were examined. These deletions had no measurable effect on plaque formation or morphology, viral DNA synthesis or early viral mRNA synthesis. Thus, it appears that DNA sequences in the left ITR, outside the replication origin, are completely dispensable for lyric viral growth in tissue culture cells.
Current Topics in Microbiology and Immunology, 1995
The adenovirus genome can be replicated in vitro by the combined action of three viral proteins, two cellular transcription factors, and topoisomerase I (Table 1). Early viral transcription results in expression of three viral genes, preterminal protein (pTP), DNA polymerase (Ad pol), and DNA-binding protein (DBP), that genetic evidence indicates are required for viral DNA replication. With the development of in vitro systems (Challberg and Kelly 1979), it was soon shown that cellular factors were required in addition to the three viral proteins. One of these proteins could be replaced by calf thymus topoisomerase and was required for complete elongation of the genome (Nagata et al. 1982). In contrast, the other factors were both sequence-specific DNA-binding proteins that recognized DNA within the inverted terminal repeats (ITRs) of the adenovirus genome (Nagata et al. 1983; Pruijn et al. 1986). Because all of these proteins have been cloned, overexpressed, and purified, the adenovirus genome represents one of the few eukaryotic systems in which all the components for DNA replication have been precisely defined. Within virions, the adenovirus genome is maintained in a highly condensed form by small basic proteins, but after infection of susceptible cells, the virion is uncoated and viral DNA is released into the cell nucleus. Adenovirus genomes are linear double-stranded DNA molecules of 35-36 kb with ITRs of about 100 bp, the exact size depending on serotype. DNA replication initiates at either end of the linear 36-kbp viral genome by a protein-priming mechanism in which a covalent linkage is formed between the a-phosphoryl group of the terminal residue, dCMP, and the P-OH group of a serine residue in pTP. This reaction is catalyzed by Ad pol, which then utilizes the 3 '-OH group of the pTP/dCMP complex as a primer for synthesis of the nascent strand. DNA synthesis is accompanied by displacement of the non-template strand, which can form a partial duplex by base-pairing of the ITRs on which a second round of DNA synthesis may be initiated (Hay et al. 1984; Stow 1982). Alterna-DNA Replication in Eukaryotic CeNs
Journal of virology, 1998
We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded...
The Role of Sequence-Specific DNA-Binding Proteins in Adenovirus DNA Replication
Journal of Cell Science, 1987
In prokaryotes it is well established that proteins which recognise defined D N A sequences are involved in the control of gene expression and replication. Cellular proteins in eukaryotes which may perform a similar function have been identified by their interactions with control regions of the human adenovirus genome.
Nucleic Acids Research, 1979
An Adenovirus (Ad) DNA replication complex extracted from infected HeLa nuclei could be purified free of the bulk of intracellular DNA polymerase activity by serii mention in neutral sucrose gradients. However, the replication complex still retained some a and y DNA polymerase activity. Since this complex is inhibited by 2', 3 1 dideoxythymidine-5'-triphosphate (ddTTP), an inhibitor of ENA polymerase y, a functional role for this enzyme in Ad DNA replication is suggested. Similar inhibition by ddTTP in intact Ad infected nuclei and comparable inhibition of Ad DNA synthesis in whole cells by dideoxythymidine (ddThy) are consistent with a role for DNA polymerase y. Dninfected HeLa nuclei or whole cells are not similarly inhibited by ddTTP or ddThy respectively. Such data does not rule out an additional functional role for other DNA polymerases, and recent experiments from this laboratory (1) suggest that DNA polymerase a is also involved in Ad DNA synthesis.
Journal of Virology
The adeno-associated virus type 2 (AAV) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. However, following rescue, the AAV genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector DNA sequences occur. In view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the HP formation, we evaluated the involvement of the individual ITRs as well as their components in the selective viral DNA replication and encapsidation. A number of recombinant AAV plasmids that contained deletions-substitutions in different regions of the individual ITRs were constructed and examined for their potential to allow rescue, replication, and/or packaging in adenovirus-infected human cells in vivo. The results reported here document that (i) two HP structures and one D-sequence are sufficient for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA sequences, (iv) one HP structure alone or two D-sequences, but not one D-sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs in the absence of the HP structures and the D-sequences, (vi) in the absence of the D-sequences, the HP structures are insufficient for successful encapsidation of the AAV genomes, and (vii) the AAV genomes containing only one ITR structure can be packaged into biologically active virions. Thus, the D-sequence plays a crucial role in the efficient rescue and selective replication and encapsidation of the AAV genome. Furthermore, the D-sequence specifically interacts with a hitherto unknown host-cell protein that we have designated the D-sequence-binding protein (D-BP). These studies illustrate that the D-sequence-D-BP interaction constitutes an important step in the AAV life cycle.
Journal of virology, 1996
Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination ...
Replication of adenovirus DNA in vitro is ATP-independent
FEBS Letters, 1994
Using a reconstituted system for adenovirus DNA replication we tested the requirements for ATP and divalent cations. At the standard M$ concentration ATP stimulated initiation 5 to lo-fold. However, this effect was caused by complexing Mg2'. At the optimal Mg2' concentration ATP was not required for initiation or elongation. Besides Mg'+ also Mn", Ca2+ and Ba*' were shown to support initiation whereas for elongation only Mg2' was accepted. Since Mn2+ could efficiently be used for DNA chain elongation on synthetic templates we hypothesize that M$ is essential for the transition of initiation to elongation.