DNA Genome Size Affects the Stability of the Adenovirus Virion

Smith, Adam C.; Poulin, Kathy L.; Parks, Robin J.

doi:10.1128/jvi.01644-08

Cited by 33 publications

(36 citation statements)

References 32 publications

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“…Furthermore, normal and pIX-deficient hdAds were significantly less stable than wild-type Ad, which showed no drop in titer after incubation at 47 °C for 30 minutes. 22 The hdAd generated with the pIX + helper virus had virion protein constituents that were essentially identical to those of the parental helper virus and wild-type Ad, suggesting that the defect underlying the capsid instability was not due to an overt deficiency in a capsid protein, as was previously observed in virions with very small genomes (<12 kb; refs. 23,24).…”

mentioning

confidence: 80%

Adenovirus Virion Stability and the Viral Genome: Size Matters

Kennedy

Parks

2009

Molecular Therapy

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mentioning

confidence: 80%

Adenovirus Virion Stability and the Viral Genome: Size Matters

Kennedy

Parks

2009

Molecular Therapy

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“…hdAd-PGK-mSEAP, hdAd-lacZ, and hdAd⌬28E4 were amplified with the helper virus AdNG163 (46) or AdJR46 using 116 cells and standard techniques (46,49). AdJR46 is similar in structure to AdRP2050 (6) but is engineered to contain a FLAG epitope tag on the C terminus of core protein VII (67). The presence of the FLAG tag on core VII has no discernible deleterious effect on virus viability.…”

Section: Methodsmentioning

confidence: 99%

Assembly of Helper-Dependent Adenovirus DNA into Chromatin Promotes Efficient Gene Expression

Ross

Kennedy

Christou

et al. 2011

J Virol

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Helper-dependent adenovirus (hdAd) vectors have shown tremendous potential in animal models of human disease in numerous preclinical studies. Expression of a therapeutic transgene can be maintained for several years after a single administration of the hdAd vector. However, despite the long-term persistence of hdAd DNA in the transduced cell, little is known of the fate and structure of hdAd DNA within the host nucleus. In this study, we have characterized the assembly of hdAd DNA into chromatin in tissue culture. Eviction of the Ad DNA-packaging protein VII, histone deposition, and vector-associated gene expression all began within 2 to 6 h of host cell transduction. Inhibition of transcription elongation through the vector DNA template had no effect on the loss of VII, suggesting that transcription was not necessary for removal of the majority of protein VII. Vector DNA assembled into physiologically spaced nucleosomes within 6 h. hdAd vectors incorporated the histone H3 variant H3.3, which was dependent on the histone chaperone HIRA. Knockdown of HIRA reduced hdAd association with histones and reduced expression of the vector-carried transgene by 2-to 3-fold. Our study elucidates an essential role for hdAd DNA chromatinization for optimal vector gene expression.

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“…Binding of the primary antibody was detected using a goat anti-mouse (Bio-Rad, Hercules, CA) or rabbit anti-goat (Sigma) secondary antibody conjugated to horseradish peroxidase and visualized by a chemiluminescent reaction (Amersham ECL Plus) and autoradiography. Preparation and analysis of protein constituents of purified Ad virions were as previously described (47,70).…”

Section: Methodsmentioning

confidence: 99%

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Poulin

Lanthier

Smith

et al. 2010

J Virol

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Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

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DNA Genome Size Affects the Stability of the Adenovirus Virion

Cited by 33 publications

References 32 publications

Adenovirus Virion Stability and the Viral Genome: Size Matters

Adenovirus Virion Stability and the Viral Genome: Size Matters

Assembly of Helper-Dependent Adenovirus DNA into Chromatin Promotes Efficient Gene Expression

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

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