Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles

Walkiewicz, Marcin P.; Morral, Núria; Engel, Daniel A.

doi:10.1016/j.jviromet.2009.04.010

Cited by 20 publications

(18 citation statements)

References 32 publications

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“…5A). As previously described (53,58), incoming protein VII is seen as discrete dots over the nuclear area (Fig. 5B, nuclear area defined by DAPI signal).…”

Section: Resultssupporting

confidence: 75%

A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

Suomalainen

Luisoni

Boucke

et al. 2013

J Virol

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Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.

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“…5A). As previously described (53,58), incoming protein VII is seen as discrete dots over the nuclear area (Fig. 5B, nuclear area defined by DAPI signal).…”

Section: Resultssupporting

confidence: 75%

A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

Suomalainen

Luisoni

Boucke

et al. 2013

J Virol

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“…Since incoming pVII but not capsids is delivered into the nucleus 1 to 3 h p.i. (23, 55, 67) and pVII is released from the viral DNA in the nucleus by ongoing transcription (12), our data suggest that the nuclear pVII puncta represent infectious incoming genomes, in agreement with recent analyses (64). Remarkably, we did not find GFP-pV on pVII-positive puncta in the nucleus, suggesting that it was lost prior to or during nuclear import of the viral genome.…”

Section: Vol 85 2011supporting

confidence: 89%

“…The precise composition of the imported DNA is unknown (reviewed in reference 47). It is known, however, that pVII remains with the incoming viral genome in the nucleus during the early infection phase (64,67). pVII, together with pV, then assembles newly synthesized viral DNA into core structures that are packaged into virions and released upon nuclear disintegration (reviewed in reference 4).…”

mentioning

confidence: 99%

Stepwise Loss of Fluorescent Core Protein V from Human Adenovirus during Entry into Cells

Püntener

Engelke

Ruzsics

et al. 2011

J Virol

112

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Human adenoviruses (Ads) replicate and assemble particles in the nucleus. They organize a linear double-strand DNA genome into a condensed core with about 180 nucleosomes, by the viral proteins VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics, we generated a novel, nonconditionally replicating Ad reporter by inserting green fluorescent protein (GFP) at the amino terminus of pV. Purified Ad2-GFP-pV virions had an oversized complete genome and incorporated about 38 GFP-pV molecules per virion, which is about 25% of the pV levels in Ad2. GFP-pV cofractionated with the DNA core, like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from that of pV, indicating that GFP-pV is a valid reporter for pV. Ad2-GFP-pV completed the replication cycle, although at lower yields than Ad2. Incoming GFP-pV (or pV) was not imported into the nucleus. Virions lost GFP-pV at two points during the infection process: at entry into the cytosol and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation-specific antihexon antibody R70, were devoid of GFP-pV. The loss of GFP-pV was reduced by the macrolide antibiotic leptomycin B (LMB), which blocks nuclear export and adenovirus attachment to the nuclear pore complex. LMB inhibited the appearance of R70 epitopes on Ad2 and Ad2-GFP-pV, indicating that the loss of GFP-pV from Ad2-GFP-pV is an authentic step in the adenovirus uncoating program. Ad2-GFP-pV is genetically complete and hence enables detailed analyses of infection and spreading dynamics in cells and model organisms or assessment of oncolytic adenoviral potential.DNA viruses and retroviruses maintain and replicate their genomes in host cell nuclei by using histone-based nucleosomes, similar to chromatin, or they encode their own DNA binding and DNA-organizing proteins (34,45,47). They assemble and maintain their genomes in different chromatin states by packaging the nucleic acids into proteinaceous capsids and sometimes lipid envelopes and thereby traffic their genome within and transmit it between cells (8, 41). The simian virus 40 (SV40) polyomavirus, for example, packages its virion DNA with cellular core histones and uses histones to replicate in infected nuclei (19). Herpesviruses, on the other hand, condense their double-strand DNA in particles with the help of polyamines and use histones during latent residence within infected nuclei or use irregularly spaced nucleosomes during productive phases of infection (45).Adenoviruses (Ads) replicate and assemble particles in the nucleus. They encode their own histone-like proteins to condense a linear double-strand DNA genome of about 36 kbp into a proteinaceous DNA core. Although it is unknown how the viral DNA is precisely organized in the virion, isolated cores of species C human adenovirus serotypes 2 and 5 (Ad2/5) contain six viral proteins, the basic proteins V (pV), pVII, and pX; the terminal protein covalently attached to the 5Ј ends of the DNA; and small numbe...

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“…A similar experimental result was found in other virus groups by Tannock et al (1987) and explained in terms of the disaggregation of clumped particles after reconstitution of lyophilized material. This is a very probable explanation here as well, as adenoviruses form aggregates very readily (Galdiero 1979;Walkiewicz et al 2009). Uncoating of herpesviruses and consequent loss of infectivity during lyophilization without high concentration of protectants, especially sugars, has already been described by Grose et al (1981), Hansen et al (2005) and Zhai et al (2004).…”

Section: Discussionmentioning

confidence: 67%

The influence of stabilizers and rates of freezing on preserving of structurally different animal viruses during lyophilization and subsequent storage

Malenovská

2014

J Appl Microbiol

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Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles

Cited by 20 publications

References 32 publications

A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

Stepwise Loss of Fluorescent Core Protein V from Human Adenovirus during Entry into Cells

The influence of stabilizers and rates of freezing on preserving of structurally different animal viruses during lyophilization and subsequent storage

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