WW domains: A globular NLS recognized by importin-α.

In this issue, the discovery by Yang et al. (https://doi.org/10.1083/jcb.202308013) that folded WW domains of YAP1 and other proteins bind to Impα introduces a new class of globular NLS, contrasting with the extensively studied linear NLS motifs. This finding underscores the versatility of importins in recognizing their cargo proteins.

Karyopherin α2 is a maternal effect gene required for early embryonic development and female fertility in mice.

The nuclear transport of proteins plays an important role in mediating the transition from egg to embryo and distinct karyopherins have been implicated in this process. Here, we studied the impact of KPNA2 deficiency on preimplantation embryo development in mice. Loss of KPNA2 results in complete arrest at the 2cell stage and embryos exhibit the inability to activate their embryonic genome as well as a severely disturbed nuclear translocation of Nucleoplasmin 2. Our findings define KPNA2 as a new maternal effect gene.

Structural basis for nuclear import of bat adeno-associated virus capsid protein.

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

A Kpna1-deficient psychotropic drug-induced schizophrenia model mouse for studying gene-environment interactions.

KPNA1 is a mediator of nucleocytoplasmic transport that is abundantly expressed in the mammalian brain and regulates neuronal differentiation and synaptic function. De novo mutations in Kpna1 have been identified using genome-wide association studies in humans with schizophrenia; however, it remains unclear how KPNA1 contributes to schizophrenia pathogenesis. Recent studies have suggested a complex combination of genetic and environmental factors that are closely related to psychiatric disorders. Here, we found that subchronic administration of phencyclidine, a psychotropic drug, induced vulnerability and behavioral abnormalities consistent with the symptoms of schizophrenia in Kpna1-deficient mice. Microarray assessment revealed that the expression levels of dopamine d1/d2 receptors, an RNA editing enzyme, and a cytoplasmic dynein component were significantly altered in the nucleus accumbens brain region in a gene-environment (G × E) interaction-dependent manner. Our findings demonstrate that Kpna1-deficient mice may be useful as a G × E interaction mouse model for psychiatric disorders and for further investigation into the pathogenesis of such diseases and disorders.

Importin α2 participates in RNA interference against bamboo mosaic virus accumulation in Nicotiana benthamiana via NbAGO10a-mediated small RNA clearance.

Karyopherins, the nucleocytoplasmic transporters, participate in multiple RNA silencing stages by transporting associated proteins into the nucleus. Importin α is a member of karyopherins and has been reported to facilitate virus infection via nuclear import of viral proteins. Unlike other RNA viruses, silencing of importin α2 (α2i) by virus-induced gene silencing (VIGS) boosted the titre of bamboo mosaic virus (BaMV) in protoplasts, and inoculated and systemic leaves of Nicotiana benthamiana. The enhanced BaMV accumulation in importin α2i plants was linked to reduced levels of RDR6-dependent secondary virus-derived small-interfering RNAs (vsiRNAs). Small RNA-seq revealed importin α2 silencing did not affect the abundance of siRNAs derived from host mRNAs but significantly reduced the 21 and 22 nucleotide vsiRNAs in BaMV-infected plants. Deletion of BaMV TGBp1, an RNA silencing suppressor, compromised importin α2i-mediated BaMV enhancement. Moreover, silencing of importin α2 upregulated NbAGO10a, a proviral protein recruited by TGBp1 for BaMV vsiRNAs clearance, but hindered the nuclear import of NbAGO10a. Taken together, these results indicate that importin α2 acts as a negative regulator of BaMV invasion by controlling the expression and nucleocytoplasmic shuttling of NbAGO10a, which removes vsiRNAs via the TGBp1-NbAGO10a-SDN1 pathway. Our findings reveal the hidden antiviral mechanism of importin α2 in countering BaMV infection in N. benthamiana.

Structural basis of nuclear transport for NEIL DNA glycosylases mediated by importin-alpha.

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/ß and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

Appropriate reduction of importin-α gene expression enhances yellow dwarf disease resistance in common wheat.

Barley yellow dwarf viruses (BYDVs) cause widespread damage to global cereal crops. Here we report a novel strategy for elevating resistance to BYDV infection. The 17K protein, a potent virulence factor conserved in BYDVs, interacted with barley IMP-α1 and -α2 proteins that are nuclear transport receptors. Consistently, a nuclear localization signal was predicted in 17K, which was found essential for 17K to be transported into the nucleus and to interact with IMP-α1 and -α2. Reducing HvIMP-α1 and -α2 expression by gene silencing attenuated BYDV-elicited dwarfism, accompanied by a lowered nuclear accumulation of 17K. Among the eight common wheat CRISPR mutants with two to four TaIMP-α1 and -α2 genes mutated, the triple mutant α1aaBBDD /α2AAbbdd and the tetra-mutant α1aabbdd /α2AAbbDD displayed strong BYDV resistance without negative effects on plant growth under field conditions. The BYDV resistance exhibited by α1aaBBDD /α2AAbbdd and α1aabbdd /α2AAbbDD was correlated with decreased nuclear accumulation of 17K and lowered viral proliferation in infected plants. Our work uncovers the function of host IMP-α proteins in BYDV pathogenesis and generates the germplasm valuable for breeding BYDV-resistant wheat. Appropriate reduction of IMP-α gene expression may be broadly useful for enhancing antiviral resistance in agricultural crops and other economically important organisms.

A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α-dependent nuclear localization signals.

Nucleocytoplasmic transport regulates the passage of proteins between the nucleus and cytoplasm. In the best characterized pathway, importin (IMP) α bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPß1, which mediates transport through the nuclear pore complex. IMPα recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, while bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPα-cNLS interactions, our understanding of the evolution of cNLSs is limited. We combined bioinformatic, biochemical, functional, and structural approaches to study this phenomenon, using polyomaviruses (PyVs) large tumor antigens (LTAs) as a model. We characterized functional cNLSs from all human (H)PyV LTAs, located between the LXCXE motif and origin binding domain. Surprisingly, the prototypical SV40 monopartite NLS is not well conserved; HPyV LTA NLSs are extremely heterogenous in terms of structural organization, IMPα isoform binding, and nuclear targeting abilities, thus influencing the nuclear accumulation properties of full-length proteins. While several LTAs possess bipartite cNLSs, merkel cell PyV contains a hybrid bipartite cNLS whose upstream stretch of basic amino acids can function as an atypical cNLS, specifically binding to the IMPα minor site upon deletion of the downstream amino acids after viral integration in the host genome. Therefore, duplication of a monopartite cNLS and subsequent accumulation of point mutations, optimizing interaction with distinct IMPα binding sites, led to the evolution of bipartite and atypical NLSs binding at the minor site.

Distribution of importin-α isoforms in poultry species and their tissue- and age-related differences.

While importin-α is well studied in mammals, the knowledge in avian species is still limited. In this study, we compared the mRNA expression patterns of five importin-α isoforms in the respiratory tract, liver, and spleen of chickens, turkeys, and pekin ducks in two different age-groups. In addition, we determined the distribution of importin-α in selected tissue of conchae, trachea, and lung of post-hatch chickens at all cellular levels by immunohistochemical staining. Our results indicate that importin-α3 is the most abundant isoform in the respiratory tract of chickens, turkeys, and pekin ducks. Moreover, importin-α is expressed as a gradient with lowest mRNA levels in the conchae and highest levels in the lung. The mRNA expression levels of most isoforms were higher in tissues from post-hatch chickens and turkeys in comparison to the corresponding embryos. In contrast to that, duck embryos mostly show higher mRNA expression levels of importin-α than post-hatch ducks.

The role of nuclear pores and importins for herpes simplex virus infection.

Microtubule transport and nuclear import are functionally connected, and the nuclear pore complex (NPC) can interact with microtubule motors. For several alphaherpesvirus proteins, nuclear localization signals (NLSs) and their interactions with specific importin-α proteins have been characterized. Here, we review recent insights on the roles of microtubule motors, capsid-associated NLSs, and importin-α proteins for capsid transport, capsid docking to NPCs, and genome release into the nucleoplasm, as well as the role of importins for nuclear viral transcription, replication, capsid assembly, genome packaging, and nuclear capsid egress. Moreover, importin-α proteins exert antiviral effects by promoting the nuclear import of transcription factors inducing the expression of interferons (IFN), cytokines, and IFN-stimulated genes, and the IFN-inducible MxB restricts capsid docking to NPCs.

SIRT1 promotes the progression and chemoresistance of colorectal cancer through the p53/miR-101/KPNA3 axis.

INTRODUCTION: Sirtuin 1 (SIRT1) is a key modulator in several types of cancer, including colorectal cancer (CRC). Here, we probed into the molecular mechanism of SIRT1 regulating the development and chemoresistance of CRC. METHODS: Differentially expressed genes related to the growth, metastasis and chemoresistance of CRC were identified by bioinformatics analysis. The expression of SIRT1 in clinical tissues from CRC patients and CRC cell lines was detected by RT-qPCR. Interactions among SIRT1, p53, miR-101 and KPNA3 were analyzed. The effect of SIRT1 on the cell viability, migration, invasion, epithelial-mesenchymal transformation and chemoresistance to 5-FU was evaluated using loss-function investigations in CRC cells. Finally, a xenograft model of CRC and a metastasis model were constructed for further exploration of the roles of SIRT1 in vivo. RESULTS: SIRT1 was elevated in CRC tissues and cell lines. SIRT1 decreased p53 via deacetylation, and consequently downregulated the expression of miR-101 while increasing that of the miR-101 target gene KPNA3. By this mechanism, SIRT1 enhanced the proliferation, migration, invasion, epithelial-mesenchymal transformation, and resistance to 5-FU of CRC cells. In addition, in vivo data also showed that SIRT1 promoted the growth, metastasis and chemoresistance to 5-FU of CRC cells via regulation of the p53/miR-101/KPNA3 axis. CONCLUSIONS: In conclusion, SIRT1 can function as an oncogene in CRC by accelerating the growth, metastasis and chemoresistance to 5-FU of CRC cells through the p53/miR-101/KPNA3 axis.

ZNF143 facilitates the growth and migration of glioma cells by regulating KPNA2-mediated Hippo signalling.

The disordered expression of ZNF143 is closely related to the malignant progression of tumours. However, the basic control mechanism of ZNF143 in glioma has not yet been clarified. Therefore, we tried to find a new pathway to illustrate the function of ZNF143 in glioma. To explore the function of KPNA2 in the development of glioma, we used survival analysis by the Kaplan‒Meier method to assess the overall survival (OS) of patients with low and high KPNA2 expression in the TCGA and CGGA cohorts. Western blotting assays and RT‒PCR assays were utilized to determine the expression level of KPNA2 in glioma cells. The interaction between ZNF143 and KPNA2 was confirmed by ChIP assays. Proliferation was assessed by CCK-8 assays, and migration was evaluated by wound healing and Transwell assays. Apoptosis was determined by flow cytometry, and the expression level of YAP/TAZ was visualized using an immunofluorescence assay. The expression levels of LATS1, LATS2, YAP1, and p-YAP1 were determined. Patients with low KPNA2 expression showed a better prognosis than those with high KPNA2 expression. KPNA2 was found to be upregulated in human glioma cells. ZNF143 can bind to the promoter region of KPNA2. Downregulation of ZNF143 and KPNA2 can activate the Hippo signalling pathway and reduce YAP/TAZ expression in human glioma cells, thus inducing apoptosis of human glioma cells and weakening their proliferation, migration and invasion. In conclusion, ZNF143 mediates the Hippo/YAP signalling pathway and inhibits the growth and migration of glioma cells by regulating KPNA2.

Germline predisposition to clonal hematopoiesis.

We now recognize that with aging, hematopoietic stem and progenitor cells (HSPCs) acquire mutations that confer a fitness advantage and clonally expand in a process now termed clonal hematopoiesis (CH). Because CH predisposes to a variety of health problems, including cancers, cardiovascular diseases, and inflammatory conditions, there is intense interest in the inherited alleles associated with the development of CH. DNA variants near TERT, SMC4, KPNA4, IL12A, CD164, and ATM confer the strongest associations. In this review, we discuss our current state of knowledge regarding germline predisposition to CH.

Circular RNA circ_0000376 promotes paclitaxel resistance and tumorigenesis of non-small cell lung cancer via positively modulating KPNA4 by sponging miR-1298-5p.

BACKGROUND: Circ_0000376 could promote non-small cell lung cancer (NSCLC) progression; however, the role of circ_0000376 in paclitaxel (PTX) resistance of NSCLC is still unknown. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were applied for measuring circ_0000376, microRNA-1298-5p (miR-1298-5p), and karyopherin subunit alpha 4 (KPNA4) expression. The half inhibitory concentration (IC50) of PTX was assessed by cell counting kit-8 assay. 5-ethynyl-2'-deoxyuridine assay, wound healing assay, transwell assay, and flow cytometry were performed to measure the proliferation, migration, invasion, and apoptosis of cells. The regulatory mechanisms of circ_0000376, miR-1298-5p, and KPNA4 were validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and rescue experiments. Xenograft assay was used for the functional analysis of circ_0000376 in vivo. RESULTS: Circ_0000376 and KPNA4 expressions were upregulated, while miR-1298-5p expression was downregulated in PTX-resistant tissues and cells. Circ_0000376 depletion promoted PTX sensitivity and apoptosis, while suppressing the proliferation, migration, and invasion of PTX-resistant NSCLC cells. Furthermore, circ_0000376 could modulate KPNA4 expression by sponging miR-1298-5p. Rescue experiments showed that miR-1298-5p inhibition reversed the circ_0000376 depletion-mediated anticancer effects and PTX sensitivity. Moreover, miR-1298-5p inhibited PTX resistance and tumorigenesis of PTX-resistant cells, which were abolished by KPNA4 overexpression. In addition, circ_0000376 knockdown suppressed tumor growth and enhanced PTX sensitivity in vivo. CONCLUSION: Circ_0000376 facilitated PTX resistance and tumorigenesis of NSCLC by miR-1298-5p/KPNA4 axis, suggesting an underlying therapeutic strategy for NSCLC.

Toxoplasma Gondii Importin α Shows Weak Auto-Inhibition.

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin ß-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

Ebolavirus Species-Specific Interferon Antagonism Mediated by VP24.

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-ß), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms.

Multi-omic underpinnings of epigenetic aging and human longevity.

Biological aging is accompanied by increasing morbidity, mortality, and healthcare costs; however, its molecular mechanisms are poorly understood. Here, we use multi-omic methods to integrate genomic, transcriptomic, and metabolomic data and identify biological associations with four measures of epigenetic age acceleration and a human longevity phenotype comprising healthspan, lifespan, and exceptional longevity (multivariate longevity). Using transcriptomic imputation, fine-mapping, and conditional analysis, we identify 22 high confidence associations with epigenetic age acceleration and seven with multivariate longevity. FLOT1, KPNA4, and TMX2 are novel, high confidence genes associated with epigenetic age acceleration. In parallel, cis-instrument Mendelian randomization of the druggable genome associates TPMT and NHLRC1 with epigenetic aging, supporting transcriptomic imputation findings. Metabolomics Mendelian randomization identifies a negative effect of non-high-density lipoprotein cholesterol and associated lipoproteins on multivariate longevity, but not epigenetic age acceleration. Finally, cell-type enrichment analysis implicates immune cells and precursors in epigenetic age acceleration and, more modestly, multivariate longevity. Follow-up Mendelian randomization of immune cell traits suggests lymphocyte subpopulations and lymphocytic surface molecules affect multivariate longevity and epigenetic age acceleration. Our results highlight druggable targets and biological pathways involved in aging and facilitate multi-omic comparisons of epigenetic clocks and human longevity.

Acetylation regulates the nucleocytoplasmic distribution and oncogenic function of karyopherin alpha 2 in lung adenocarcinoma.

Karyopherin subunit alpha 2 (KPNA2, importin α1) is a nucleoplasmic protein responsible for the nuclear import of proteins with classical nuclear localization signals. Aberrant nuclear accumulation of KPNA2 has been observed in numerous cancer tissues. AMP-activated protein kinase (AMPK) is involved in the phosphorylation and acetylation of KPNA2 in enterocytes. However, the impact of these post-translational modifications on modulating the nucleocytoplasmic distribution of KPNA2 and its oncogenic role remain unclear. Unlike nuclear accumulation of wild-type KPNA2, which promoted lung cancer cell migration, KPNA2 Lys22 acetylation-mimicking mutations (K22Q and K22Q/S105A) prevented nuclear localization of KPNA2 and reduced the cell migration ability. Cytosolic KPNA2 K22Q interacted with and restricted the nuclear entry of E2F transcription factor 1 (E2F1), an oncogenic cargo protein of KPNA2, in lung cancer cells. Intriguingly, the AMPK activator EX229 promoted the nuclear export of KPNA2 S105A. However, the CBP/p300 inhibitor CCS-1477 abolished this phenomenon, suggesting that CBP/p300-mediated acetylation of KPNA2 promoted KPNA2 nuclear export in lung cancer cells. Collectively, our findings suggest that the CBP/p300 positively regulates KPNA2 acetylation, which enhances its cytosolic localization and suppresses its oncogenic activity in lung cancer.

Novel roles of karyopherin subunit alpha 2 in hepatocellular carcinoma.

Hepatocellular carcinoma is the most common type of liver cancer and associated with a high fatality rate. This disease poses a major threat to human health worldwide. A considerable number of genetic and epigenetic factors are involved in the development of hepatocellular carcinoma. However, the molecular mechanism underlying the progression of hepatocellular carcinoma remains unclear. Karyopherin subunit alpha 2 (KPNA2), also termed importin α1, is a member of the nuclear transporter family. In recent years, KPNA2 has been gradually linked to the nuclear transport pathway for a variety of tumor-associated proteins. Furthermore, it promotes tumor development by participating in various pathophysiological processes such as cell proliferation, apoptosis, immune response, and viral infection. In hepatocellular carcinoma, it has been found that KPNA2 expression is significantly higher in liver cancer tissues versus paracancerous tissues. Moreover, it has been identified as a marker of poor prognosis and early recurrence in patients with hepatocellular carcinoma. Nevertheless, the role of KPNA2 in the development of hepatocellular carcinoma remains to be determined. This review summarizes the current knowledge on the pathogenesis and role of KPNA2 in hepatocellular carcinoma, and provides new directions and strategies for the diagnosis, treatment, and prediction of prognosis of this disease.

Importin α/ß-dependent nuclear transport of human parvovirus B19 nonstructural protein 1 is essential for viral replication.

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/ß-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/ß 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.