LAUSR

Parvovirus particles are expected to be applied as nanoparticles in drug delivery systems. However, little is known about how nuclear-assembled B19 virus (B19V) virions are released from host cells. ABSTRACT B19 virus (B19V) is a pathogenic human parvovirus that infects erythroid progenitor cells. Because there are limited in vitro culture systems to propagate this virus, little is known about the molecular mechanisms by which it propagates in cells. In this study, we introduced a HiBiT peptide tag into various loops of VP2 located on the surface of B19V particles and evaluated their ability to form virus-like particles (VLPs). Three independent sites were identified as permissive sites for peptide tag insertion without affecting VLP formation. When the HiBiT tag was introduced into B19V clones (pB19-M20) and transfected into a semipermissive erythroleukemia cell line (UT7/Epo-S1), HiBiT-dependent luciferase activities (HiBiT activities) increased depending on helicase activity of viral NS1. Furthermore, we used a GFP11 tag-split system to visualize VLPs in the GFP1-10-expressing live cells. Time-lapse imaging of green fluorescent protein (GFP)-labeled VLPs revealed that nuclear VLPs were translocated into the cytoplasm only after cell division, suggesting that the breakdown of the nuclear envelope during mitosis contributes to VLP nuclear export. Moreover, HiBiT activities of culture supernatants were dependent on the presence of a detergent, and the released VLPs were associated with extracellular vesicles, as observed under electron microscopy. Treatment with an antimitotic agent (nocodazole) enhanced the release of VLPs. These results suggest that the virions accumulated in the cytoplasm are constitutively released from the cell as membrane-coated vesicles. These properties are likely responsible for viral escape from host immune responses and enhance membrane fusion-mediated transmission. IMPORTANCE Parvovirus particles are expected to be applied as nanoparticles in drug delivery systems. However, little is known about how nuclear-assembled B19 virus (B19V) virions are released from host cells. This study provides evidence of mitosis-dependent nuclear export of B19V and extracellular vesicle-mediated virion release. Moreover, this study provides methods for modifying particle surfaces with various exogenous factors and contributes to the development of fine nanoparticles with novel valuable functions. The pB19-M20 plasmid expressing HiBiT-tagged VP2 is a novel tool to easily quantify VP2 expression. Furthermore, this system can be applied in high-throughput screening of reagents that affect VP2 expression, which might be associated with viral propagation.