Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-39494
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.
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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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|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||07 Faculty of Science > Institute of Molecular Life Sciences|
|DDC:||570 Life sciences; biology|
|Deposited On:||15 Jan 2011 16:01|
|Last Modified:||27 Nov 2013 16:19|
|Publisher:||American Society for Microbiology|
|Free access at:||PubMed ID. An embargo period may apply.|
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