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

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.