Parvoviruses package a linear single-stranded DNA genome with hairpin structures at both ends. It has been thought that terminal hairpin sequences are indispensable for viral DNA replication. Here, we provide evidence that the hairpin-deleted duplex genomes of human bocavirus 1 (HBoV1) replicate in human embryonic kidney (HEK) 293 cells. We propose an alternative model for HBoV1 DNA replication in which the leading strand can initiate strand-displacement without “hairpin-transfer.” The transfection of the HBoV1 duplex genomes that retain a minimal replication origin at the right-end (
OriR
), but with extensive deletions in the right-end hairpin (REH), generated viruses in HEK293 cells at a level 10-20 times lower than the wild-type (WT) duplex genome. Importantly, these viruses that have a genome with various deletions after the
OriR
, but not the one retaining only the
OriR
, replicated in polarized human airway epithelia. We discovered that the 18-nt sequence (nt 5,403-5,420) beyond the
OriR
was sufficient to confer virus replication in polarized human airway epithelia, although its progeny virus production was ∼5 times lower than that of the WT virus. Thus, our study demonstrates that hairpin transfer-independent productive parvovirus DNA replication can occur.
Importance
Hairpin transfer-independent parvovirus replication was modeled with human bocavirus 1 (HBoV1) duplex genomes whose 5’ hairpin structure was ablated by various deletions. In HEK293 cells, these duplex viral genomes with ablated 5’/hairpin sequence replicated efficiently and generated viruses that productively infected polarized human airway epithelium. Thus, for the first time, we reveal a previously unknown phenomenon that the productive parvovirus DNA replication does not depend on the hairpin sequence at REH to initiate “rolling hairpin” DNA replication. Notably, the intermediates of viral DNA replication, as revealed two-dimensional electrophoresis, from transfections of hairpin sequence-deleted duplex genome and full-length genome in HEK293 cells, as well as from virus infection of polarized human airway epithelia are similar. Thus, the establishment of the hairpin transfer-independent parvoviral DNA replication deepens our understanding in viral DNA replication and may have implications in development of parvovirus-based viral vectors with alternative properties.
Iota-carrageenan extracted from red algae is a potent inhibitor of SARS‐CoV-2 infection in reconstituted human airway epithelia