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

Abstract

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...

The Role of Capsid Maturation on Adenovirus Priming for Sequential Uncoating

“…Neither pentons nor peripheral core components were released from immature ts1 particles under mild stress. Loss of peripheral core material is consistent with previous observations indicating release of some internal components in the early endosome, such as core polypeptide V and, more critically, the membrane disrupting polypeptide VI (12,13). Both V and VI are thought to occupy positions bridging the internal capsid surface to the core (4,5,62).…”

“…Elegant biophysics, cellular and molecular biology studies have given insight into the maturation process on the one hand (22,26,50) and the sequential uncoating on the other hand (8,9,12,13). The entry defect of ts1 had previously been related with increased stability (5,8,51).…”

“…The virus starts to disassemble at the plasma membrane, where upon binding to its receptor some fibers are released (10), and the penton base undergoes a conformational change that might result in weakening its interactions with the rest of the capsid (11). The virus is then internalized, and disassembly continues in the early endosome with release of some internal components, such as minor coat proteins IIIa, VI, VIII, and core polypeptide V (9,12). This is a crucial step for infection, as polypeptide VI plays the key role of altering the endosomal membrane to facilitate virus escape to the cytosol (13).…”

The nuclear export factor CRM1 controls juxta-nuclear microtubule-dependent virus transport

“…The incoming virion sheds accessory proteins in a stepwise manner during entry (Greber et al, 1993;Nakano et al, 2000). The cytosolic particles are leaky containers comprising the hexon protein and viral DNA, but lacking the pentons and some stabilizing proteins (Luisoni et al, 2015;Puntener et al, 2011;Wang et al, 2013). They are different from intact virions, which are regular icosahedral T=25 (where T is the triangular number, a standard way to describe the subunit number and organization of the facets of an icosahedron) particles of ∼90 nm in diameter (Liu et al, 2010;Reddy et al, 2010).…”

Uncoating of non-enveloped viruses

“…The uncoating cues are best characterized for two closely related species C serotypes, human adenoviruses type 2 (HAdV-2) and type 5 (HAdV-5) [47,[58][59][60][61].…”