Parvovirus B19 Uncoating Occurs in the Cytoplasm without Capsid Disassembly and It Is Facilitated by Depletion of Capsid-Associated Divalent Cations

Abstract

Human parvovirus B19 (B19V) traffics to the cell nucleus where it delivers the genome for replication. The intracellular compartment where uncoating takes place, the required capsid structural rearrangements and the cellular factors involved remain unknown. We explored conditions that trigger uncoating in vitro and found that prolonged exposure of capsids to chelating agents or to buffers with chelating properties induced a structural rearrangement at 4 °C resulting in capsids with lower density. These lighter particles remained intact but were unstable and short exposure to 37 °C or to a freeze-thaw cycle was sufficient to trigger DNA externalization without capsid disassembly. The rearrangement was not observed in the absence of chelating activity or in the presence of MgCl2 or CaCl2, suggesting that depletion of capsid-associated divalent cations facilitates uncoating. The presence of assembled capsids with externalized DNA was also detected during B19V entry in UT7/Epo cells. Following endosomal escape and prior to nuclear entry, a significant proportion of the incoming capsids rearranged and externalized the viral genome without capsid disassembly. The incoming capsids with accessible genomes accumulated in the nuclear fraction, a process that was prevented when endosomal escape or dynein function was disrupted. In their uncoated conformation, capsids immunoprecipitated from cytoplasmic or from nuclear fractions supported in vitro complementary-strand synthesis at 37 °C. This study reveals an uncoating strategy of B19V based on a limited capsid rearrangement prior to nuclear entry, a process that can be mimicked in vitro by depletion of divalent cations.

Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection

“…4B). In a recent study, we showed that a large proportion of B19V particles accumulating in the nuclear fraction of the infected cells have the viral DNA accessible and therefore are sensitive to nuclease digestion (26). As shown in Fig.…”

“…After three washes with PBS, the cells were resuspended in 500 l MEM containing 5% FCS and 2 U/ml EPO and incubated at 37°C. After 3 h, cells were pelleted and washed thrice with PBS, and the nuclei were extracted using the Nuclei EZ Prep kit (Sigma-Aldrich, MO), as previously described (26). Trypan blue staining was used to evaluate the integrity of the isolated nuclei.…”

Microtubules in Polyomavirus Infection

“…On the other hand, a recent study on another non enveloped virus, Parvovirus B19, pointed to the involvement of dynein in the transport of virus particles released from endosomes into the proximity of the nucleopore and, also, in the induction of conformation changes in virus particles occurring in the cytosol (facilitated by the depletion of capsid-associated divalent cations). The changes in conformation resulted in partial exposure of the genomic DNA prior to particle translocation to the nucleus [89].…”

Advances in the Development of Antiviral Strategies against Parvovirus B19

“…However, even a transient binding of a capsid to globoside can trigger conformational modifications leading to exposure of the VP1u region [12], allowing interaction of its N-terminal region with a specific, but yet uncharacterized receptor whose distribution in cells of erythroid lineage matches the susceptibility of cells to productive infection [13,14,15]. Following binding, internalization via clathrin-mediated endocytosis can occur, the phospholipase activity associated to the VP1u region consents escape from the endosome, and by subsequent coordinated intracellular transport and uncoating events, a single-stranded genome is finally delivered in the nuclear environment [16,17].…”