Domain organization of the adenovirus preterminal protein

Webster, Ailsa; Leith, Ian; Hay, Ronald T.

doi:10.1128/jvi.71.1.539-547.1997

Cited by 22 publications

(38 citation statements)

References 46 publications

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“…Purification of pTP, Adpol, DBP, NFI, and NFIII. Ad2 pTP, Ad2 pol, Ad2 DNA binding protein (DBP), and NFI were purified from Sf9 cells infected with recombinant baculoviruses as described previously (5,42,45,51). NFIII was expressed as a fusion with glutathione-S-transferase and purified by affinity chromatography on glutathione-agarose and DNA-Sepharose columns (51).…”

Section: Methodsmentioning

confidence: 99%

“…Ad2 pTP, Ad2 pol, Ad2 DNA binding protein (DBP), and NFI were purified from Sf9 cells infected with recombinant baculoviruses as described previously (5,42,45,51). NFIII was expressed as a fusion with glutathione-S-transferase and purified by affinity chromatography on glutathione-agarose and DNA-Sepharose columns (51). Ad4 pTP-expressing baculovirus (38) was obtained from Michael Stanglmaier and Ernst Winnacker (Institut für Biochemie der Universität München, Munich, Germany), and Ad4 pTP was purified with a combination of phenyl Superose and DNA-Sepharose columns as described previously for Ad2 pTP (51).…”

Section: Methodsmentioning

confidence: 99%

“…Extensive mutagenesis of pTP has been carried out and the conclusions from such studies are that regions spanning the molecule are required for matrix attachment, DNA replication, and virus viability (15-17, 27, 33, 34). More recently, domain-mapping experiments reveal that an N-terminal protease-resistant fragment of pTP contacts DNA whereas multiple regions within the C-terminal two-thirds of pTP interact with Adpol (51). Furthermore, an extreme dependence on the conformation of pTP was demonstrated for both DNA and Adpol binding.…”

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Role of preterminal protein processing in adenovirus replication

Webster

Leith

Nicholson

et al. 1997

J Virol

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Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5 end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection.Adenovirus DNA replicates by a protein-priming mechanism in which the virus-encoded preterminal protein (pTP) and adenovirus DNA polymerase (Adpol) form a stable heterodimer and pTP serves as the protein primer (20,35,37,39). DNA replication is initiated by the covalent attachment of dCMP to pTP, following the binding of pTP-Adpol to the core origin of replication (20,35,42). Initiation is enhanced by two cellular factors, nuclear factor I (NFI or CTF1) and nuclear factor III (NFIII or Oct1), and by the virus-encoded, singlestranded DNA binding protein (3,5,6,8,10,12,25,26,31,40). In addition to the incoming pTP, the presence of terminal protein (TP), covalently attached to the 5Ј end of the DNA, stimulates initiation in in vitro DNA replication assays, a phenomenon due in part to an increased affinity of the incoming pTP-Adpol for TP-linked DNA (29,30). Initiation of DNA replication is followed by dissociation of NFI from the complex (9), elongation of the daughter strand by Adpol, and strand displacement. The nature of the molecular rearrangements at the origin of replication preceding elongation and the functional contributions of each of the protein components remain to be established. In particular, questions that elude us are whether pTP and Adpol remain associated throughout elongation, and the nature, if any, of the roles of the pTP moieties attached to the displaced and daughter strands (20,43).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Role of preterminal protein processing in adenovirus replication

Webster

Leith

Nicholson

et al. 1997

J Virol

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“…pTP is part of a complex initiating viral DNA replication. 23,26,28 Thus, its deletion blocks the viral life cycle at a very early stage after infection. 100K is a multifunctional protein that also is essentially involved in hexon-trimerization and in scaffolding during virus assembly and maturation.…”

Section: Generation and Characterization Of Ad Mutants δPtp And δ100kmentioning

confidence: 99%

“…Because this protein is essential for the initiation of Ad genome replication, its absence results in a very early block during the adenoviral life cycle. [23][24][25][26] Ad mutant 2 (Ad Δ100K) has a deletion in the L4 100K gene coding for a multifunctional protein that does not affect replication of the viral genome but is essential during the late phase of the viral life cycle. [27][28][29] 2 | MATERIALS AND METHODS…”

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Evaluation of life cycle defective adenovirus mutants for production of adeno‐associated virus vectors

Krüger-Haag

Lehmann

Schmidt

et al. 2019

The Journal of Gene Medicine

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Background Adeno‐associated virus‐based vectors are efficient and safe drug candidates for different in vivo gene therapy applications. With increasing numbers of clinical studies based on AAV2 vectors that include not only rare, but also common diseases as a therapeutic target, there is an increased demand for the development of improved production technologies. Methods In the present study, we compared two life cycle defective adenovirus mutants as helper viruses for AAV2 vector production. They had deletions either in the gene coding for the preterminal protein (pTP) that is expressed early in the viral life cycle and is essential for genome replication or in the gene coding for the 100K protein, a protein with many functions, one of which is involved in virus assembly. AAV2 vector production efficiencies were evaluated by analyzing genome‐containing particles using a real‐time polymerase chain reaction and functional units were investigated by transduction assays. Results Somewhat contrary to our expectations, the ∆100K mutant virus showed only a moderate efficiency as a helper virus for AAV2 vector production, whereas the replication‐deficient ∆pTP mutant supported AAV2 production almost as efficiently as adenovirus wild‐type. We also showed that a temperature shift to 32°C together with extended incubation times improved AAV2 vector productivity. Conclusions The present study indicates the advantages of using a ∆pTP mutant adenovirus rather than adenovirus wild‐type as a helper virus for AAV2 production and also indicates that temperature shifts to lower temperatures may improve AAV2 vector production rates.

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Imaging the adenovirus infection cycle

Pied

Wodrich

2019

FEBS Letters

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Edited by Urs GreberIncoming adenoviruses seize control of cytosolic transport mechanisms to relocate their genome from the cell periphery to specialized sites in the nucleoplasm. The nucleus is the site for viral gene expression, genome replication, and the production of progeny for the next round of infection. By taking control of the cell, adenoviruses also suppress cell-autonomous immunity responses. To succeed in their production cycle, adenoviruses rely on wellcoordinated steps, facilitated by interactions between viral proteins and cellular factors. Interactions between virus and host can impose remarkable morphological changes in the infected cell. Imaging adenoviruses has tremendously influenced how we delineate individual steps in the viral life cycle, because it allowed the development of specific optical markers to label these morphological changes in space and time. As technology advances, innovative imaging techniques and novel tools for specimen labeling keep uncovering previously unseen facets of adenovirus biology emphasizing why imaging adenoviruses is as attractive today as it was in the past. This review will summarize past achievements and present developments in adenovirus imaging centered on fluorescence microscopy approaches.Adenoviruses (Ads) are nonenveloped icosahedral viruses with a diameter of~90 nm with slight structural differences between genotypes. The majority of the Ad capsid shell is composed of 240 hexon trimers, and each of the 12 vertices of the icosahedron is occupied by a penton. Pentons are involved in cell attachment and receptor recognition and are composed of the pentameric penton base from which the trimeric fiber molecule extends. Minor capsid proteins IIIa, VI, VIII, and IX are embedded in the capsid and contribute to capsid stability. Protein VI is located at the inner surface of the capsid, and biochemical data suggest that it may connect the capsid to the core containing the viral genome via protein V. The genome is ã 36-kb double-stranded linear DNA molecule with the terminal protein (TP) covalently bound to each 5 0end. Adenovirus genomes are highly condensed and organized into chromatin by several hundred copies of Abbreviations ADP, adenovirus death protein

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Domain organization of the adenovirus preterminal protein

Cited by 22 publications

References 46 publications

Role of preterminal protein processing in adenovirus replication

Role of preterminal protein processing in adenovirus replication

Evaluation of life cycle defective adenovirus mutants for production of adeno‐associated virus vectors

Imaging the adenovirus infection cycle

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