Human parvovirus B19 (B19V) is a major human pathogen causing a variety of diseases, characterized by a selective tropism to human progenitor cells in bone marrow. In similar fashion to all Parvoviridae members, the B19V ssDNA genome is replicated within the nucleus of infected cells through a process which involves both cellular and viral proteins. Among the latter, a crucial role is played by non-structural protein (NS)1, a multifunctional protein involved in genome replication and transcription, as well as modulation of host gene expression and function. Despite the localization of NS1 within the host cell nucleus during infection, little is known regarding the mechanism of its nuclear transport pathway. In this study we undertake structural, biophysical, and cellular approaches to characterize this process. Quantitative confocal laser scanning microscopy (CLSM), gel mobility shift, fluorescence polarization and crystallographic analysis identified a short sequence of amino acids (GACHAKKPRIT-182) as the classical nuclear localization signal (cNLS) responsible for nuclear import, mediated in an energy and importin (IMP) α/β-dependent fashion. Structure-guided mutagenesis of key residue K177 strongly impaired IMPα binding, nuclear import, and viral gene expression in a minigenome system. Further, treatment with ivermectin, an antiparasitic drug interfering with the IMPα/β dependent nuclear import pathway, inhibited NS1 nuclear accumulation and viral replication in infected UT7/Epo-S1 cells. Thus, NS1 nuclear transport is a potential target of therapeutic intervention against B19V induced disease.

SOX proteins are a family of transcription factors (TFs) that play critical functions in sex determination, neurogenesis, and chondrocyte differentiation, as well as cardiac, vascular, and lymphatic development. There are 20 SOX family members in humans, each sharing a 79-residue L-shaped high mobility group (HMG)-box domain that is responsible for DNA binding. SOX2 was recently shown to interact with long non-coding RNA and large-intergenic non-coding RNA to regulate embryonic stem cell and neuronal differentiation. The RNA binding region was shown to reside within the HMG-box domain; however, the structural details of this binding remain unclear. Here, we show that all SOX family members, except group H, interact with RNA. Our mutational experiments demonstrate that the disordered C-terminal region of the HMG-box domain plays an important role in RNA binding. Further, by determining a high-resolution structure of the HMG-box domain of the group H family member SOX30, we show that despite differences in RNA binding ability, SOX30 shares a very similar secondary structure with other SOX protein HMG-box domains. Together, our study provides insight into the interaction of SOX TFs with RNA.

Anelloviridae members are ubiquitous viruses with a small, negative sense, single-stranded DNA genome which is replicated by host cell DNA polymerases. Anelloviruses are postulated to interact with the host cell nuclear transport machinery, however, the lack of reliable cell culture models strongly limits our knowledge regarding Anelloviridae-host interactions. In particular, capsid nuclear import is a largely uncharacterized process. We addressed this by investigating the relationship between host cell nuclear transport receptors (NTRs) and ORF1, the putative capsid protein from torque teno douroucouli virus (TTDoV). We identified the subcellular targeting signals and NTRs responsible for its nucleolar and nuclear localization, and characterized their relative contribution to ORF1 subcellular localization. In the absence of other viral proteins, ORF1 accumulated in the nucleoli. Bioinformatics analysis revealed a putative classical nuclear localization signal (cNLS) within the highly conserved N-terminal arginine rich motif (ARM) (‘NLSn’, 27-RRWRRRPRRRRRPYRRRPYRRYGRRRKVRRR-57), and an additional C-terminal cNLS (‘NLSc’, 632-LPPPEKRARWGF-643), which has been specifically acquired by Anelloviridae capsids with larger projection domains. Such NLSs play distinct roles in ORF1 subcellular localization by interacting with specific NTRs. NLSn, a non-classical NLS, features broad importin (IMP) binding affinity yet plays a minor role in nuclear import, being responsible for nucleolar targeting likely through interaction with nucleolar components. NLSc, a bona fide cNLS, specifically interacts with IMPα and is the main driver of active nuclear transport in an IMPα/β1-dependent fashion. These findings suggest an evolutionary correlation between the acquisition of progressively larger projection domains and the presence of additional cNLSs in Anelloviridae capsids, aimed at maximizing IMPα/β1-mediated nuclear import.

Generation of diverse neural cell types through direct conversion