Adenovirus vectors deleted for genes essential for viral DNA replication

Schaack, Jerome

doi:10.2741/1607

Cited by 23 publications

(18 citation statements)

References 91 publications

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“…Both of these DNA structures could stimulate a DNA damage response. Unlike adenoviruses with E1 deleted, which can replicate the viral genome under some circumstances (8,25,49), deletion of the preterminal protein gene renders the ⌬pTP mutant virus completely unable to direct genome replication (47). Thus, this mutant virus provides an efficient means of delivering viral DNA at high multiplicities without the complication of viral DNA replication.…”

Section: Resultsmentioning

confidence: 99%

“…To determine whether the H2AX phosphorylation observed is a response to viral DNA replication and possibly single-stranded DNA intermediates, rather than the presence and structure of mature viral genomes, cells were infected with either the wild-type adenovirus or a mutant virus, H5wt300⌬pTP (⌬pTP). ⌬pTP contains a deletion of the preterminal protein gene and thus is unable to direct the synthesis of viral DNA (47). Infection of HeLa cells with the ⌬pTP mutant virus resulted in a diffuse nuclear distribution of DBP characteristic of that seen at early times of infection.…”

Section: Resultsmentioning

confidence: 99%

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Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication

Nichols

Schaack

Ornelles

2009

J Virol

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Adenovirus infection activates cellular DNA damage response and repair pathways. Viral proteins that are synthesized before viral DNA replication prevent recognition of viral genomes as a substrate for DNA repair by targeting members of the sensor complex composed of Mre11/Rad50/NBS1 for degradation and relocalization, as well as targeting the effector protein DNA ligase IV. Despite inactivation of these cellular sensor and effector proteins, infection results in high levels of histone 2AX phosphorylation, or γH2AX. Although phosphorylated H2AX is a characteristic marker of double-stranded DNA breaks, this modification was widely distributed throughout the nucleus of infected cells and was coincident with the bulk of cellular DNA. H2AX phosphorylation occurred after the onset of viral DNA replication and after the degradation of Mre11. Experiments with inhibitors of the serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK), the kinases responsible for H2AX phosphorylation, indicate that H2AX may be phosphorylated by ATR during a wild-type adenovirus infection, with some contribution from ATM and DNA-PK. Viral DNA replication appears to be the stimulus for this phosphorylation event, since infection with a nonreplicating virus did not elicit phosphorylation of H2AX. Infected cells also responded to high levels of input viral DNA by localized phosphorylation of H2AX. These results are consistent with a model in which adenovirus-infected cells sense and respond to both incoming viral DNA and viral DNA replication.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication

Nichols

Schaack

Ornelles

2009

J Virol

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“…Since we observed rescue using Ad vectors with E1 deleted as "helpers" in Mo7e cells, we formally excluded the possibility that E1 proteins support rescue. We obtained or generated plasmids that express DNA-binding protein (DBP) (39), polymerase (26), or E4orf 6 (25) under the control of the CMV promoter. We also used the pHelper plasmid from Stratagene (35), which expresses a combination of E4, E2A, and VA RNA genes (Fig.…”

Section: Construction and Characterization Ofmentioning

confidence: 99%

A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette

Wang

Lieber

2006

J Virol

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Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCRrep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.For gene therapy applications, the development of vectors that target transgene integration to specific sites in the host genome is a major focus (7, 32). In this context, the ability of adeno-associated virus (AAV) to integrate into the host genome at a preferred site on chromosome 19q13.4q is of interest (13). This site, called AAVS1, is about 4 kb long and has been mapped to the first exon of myosin binding subunit 85 of the protein phosphatase 1. The AAVS1 site appears to be in an open chromatin conformation in the cell lines tested (31), which is thought to help AAV integration and viral-gene expression (15). Integration into this site is mediated by the large AAV Rep protein Rep68 or Rep78. Rep68/78 demonstrate sequence-specific endonuclease activity and ATP-dependent helicase activity and can mediate rescue/excision of DNA flanked by AAV ITRs in the presence of adenovirus (Ad) superinfection (24). Rep-mediated rescue requires the presence of both a Rep binding site (R...

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“…The virus dl 327 bears a deletion in the immunomodulatory E3 region (Ginsberg et al , 1989, Ginsberg & Prince, 1994) and is otherwise isogenic with wt 300. The H5 wt 300ΔpTP virus bears a deletion in the terminal protein gene, rendering it completely incapable of replicating (Schaack, 2005). The H5 wt 300ΔpTP virus was grown in 293 cells expressing the E2B gene.…”

Section: Methodsmentioning

confidence: 99%

Replication of type 5 adenovirus promotes middle ear infection byStreptococcus pneumoniaein the chinchilla model of otitis media

et al. 2014

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Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyper-inflammatory adenovirus mutant dl327 and the non-replicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by Streptococcus pneumoniae as compared to non-adenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the non-replicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease.

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Adenovirus vectors deleted for genes essential for viral DNA replication

Cited by 23 publications

References 91 publications

Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication

Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication

A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette

Replication of type 5 adenovirus promotes middle ear infection byStreptococcus pneumoniaein the chinchilla model of otitis media

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