Stepwise loss of fluorescent core protein V from human adenovirus during entry into cells.
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.
Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Daniel
D
Puntener
Martin F
MF
Engelke
Zsolt
Z
Ruzsics
Sten
S
Strunze
Corinne
C
Wilhelm
Urs F
UF
Greber
D000260:Adenoviruses, Human / Q000187:drug effects / Q000378:metabolism* / Q000472:pathogenicity; D045744:Cell Line, Tumor; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000187:drug effects / Q000378:metabolism; D014773:Virology / Q000379:methods; D053586:Virus Internalization
D005231:Fatty Acids, Unsaturated; D014758:Viral Core Proteins; D049452:Green Fluorescent Proteins; C038753:leptomycin B