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Stepwise loss of fluorescent core protein V from human adenovirus during entry into cells


Püntener, D; Engelke, M F; Ruzsics, Z; Strunze, S; Wilhelm, C; Greber, U F (2011). Stepwise loss of fluorescent core protein V from human adenovirus during entry into cells. Journal of Virology, 85(1):481-496.

Abstract

Human adenoviruses (HAdVs, short Ad) replicate and assemble particles in the nucleus. They organise a linear double-strand DNA-genome into a condensed core with about 180 nucleosomes by the viral protein VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics we generated a novel, non-conditionally 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, about 25% of Ad2 pV. GFP-pV cofractionated with the DNA-core like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from 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, entry into the cytosol, and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation specific anti-hexon 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.

Human adenoviruses (HAdVs, short Ad) replicate and assemble particles in the nucleus. They organise a linear double-strand DNA-genome into a condensed core with about 180 nucleosomes by the viral protein VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics we generated a novel, non-conditionally 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, about 25% of Ad2 pV. GFP-pV cofractionated with the DNA-core like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from 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, entry into the cytosol, and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation specific anti-hexon 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.

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29 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Molecular Life Sciences
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2011
Deposited On:15 Jan 2011 16:01
Last Modified:05 Apr 2016 14:26
Publisher:American Society for Microbiology
ISSN:0022-538X
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:10.1128/JVI.01571-10
PubMed ID:21047958
Permanent URL: http://doi.org/10.5167/uzh-39494

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