TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

Protein kinase Cα regulates the nucleocytoplasmic shuttling of KRIT1.

KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell-cell and cell-matrix adhesion, and redox homeostasis and signaling. However, rather little is known about how KRIT1 is itself regulated. KRIT1 is found in both the cytoplasm and the nucleus, yet the upstream signaling proteins and mechanisms that regulate KRIT1 nucleocytoplasmic shuttling are not well understood. Here, we identify a key role for protein kinase C (PKC) in this process. In particular, we found that PKC activation promotes the redox-dependent cytoplasmic localization of KRIT1, whereas inhibition of PKC or treatment with the antioxidant N-acetylcysteine leads to KRIT1 nuclear accumulation. Moreover, we demonstrated that the N-terminal region of KRIT1 is crucial for the ability of PKC to regulate KRIT1 nucleocytoplasmic shuttling, and may be a target for PKC-dependent regulatory phosphorylation events. Finally, we found that silencing of PKCα, but not PKCδ, inhibits phorbol 12-myristate 13-acetate (PMA)-induced cytoplasmic enrichment of KRIT1, suggesting a major role for PKCα in regulating KRIT1 nucleocytoplasmic shuttling. Overall, our findings identify PKCα as a novel regulator of KRIT1 subcellular compartmentalization, thus shedding new light on the physiopathological functions of this protein.

Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/ß Nuclear Import Pathway.

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/ß-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/ß-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/ß nuclear import pathway.

Scaffold protein RACK1 regulates BR signaling by modulating the nuclear localization of BZR1.

Brassinosteroids (BRs) are a group of plant-specific steroid hormones, which induces the rapid nuclear localization of the positive transcriptional factors BRASSINAZOLE RESISTANT1/2 (BZR1/2). However, the mechanisms underlying the regulation of nucleocytoplasmic shuttling of BZR1 remain to be fully elucidated. In this study, we show that the scaffold protein Receptor for Activated C Kinase 1 (RACK1) from Arabidopsis is involved in BR signaling cascades through mediating the nuclear localization of BZR1, which is tightly retained in the cytosol by the conserved scaffold protein 14-3-3s. RACK1 can interact with BZR1 and competitively decrease the 14-3-3 interaction with BZR1 in cytosol, which efficiently enhances the nuclear localization of BZR1. 14-3-3 also retains RACK1 in cytosol through their interaction. Conversely, BR treatment enhances the nuclear localization of BZR1 by disrupting the 14-3-3 interaction with RACK1 and BZR1. Our study uncovers a new mechanism that integrates two kinds of conserved scaffold proteins (RACK1 and 14-3-3) coordinating BR signaling event.

NUCLEOCYTOPLASMIC shuttling of ETHYLENE RESPONSE FACTOR 5 mediated by nitric oxide suppresses ethylene biosynthesis in apple fruit.

Nitric oxide (NO) is known to modulate the action of several phytohormones. This includes the gaseous hormone ethylene, but the molecular mechanisms underlying the effect of NO on ethylene biosynthesis are unclear. Here, we observed a decrease in endogenous NO abundance during apple (Malus domestica) fruit development and exogenous treatment of apple fruit with a NO donor suppressed ethylene production, suggesting that NO is a ripening suppressor. Expression of the transcription factor MdERF5 was activated by NO donor treatment. NO induced the nucleocytoplasmic shuttling of MdERF5 by modulating its interaction with the protein phosphatase, MdPP2C57. MdPP2C57-induced dephosphorylation of MdERF5 at Ser260 is sufficient to promote nuclear export of MdERF5. As a consequence of this export, MdERF5 proteins in the cytoplasm interacted with and suppressed the activity of MdACO1, an enzyme that converts 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The NO-activated MdERF5 was observed to increase in abundance in the nucleus and bind to the promoter of the ACC synthase gene MdACS1 and directly suppress its transcription. Together, these results suggest that NO-activated nucleocytoplasmic MdERF5 suppresses the action of ethylene biosynthetic genes, thereby suppressing ethylene biosynthesis and limiting fruit ripening.

Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites.

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2.

Serine phosphorylation of the small phosphoprotein ICAP1 inhibits its nuclear accumulation.

Nuclear accumulation of the small phosphoprotein integrin cytoplasmic domain-associated protein-1 (ICAP1) results in recruitment of its binding partner, Krev/Rap1 interaction trapped-1 (KRIT1), to the nucleus. KRIT1 loss is the most common cause of cerebral cavernous malformation, a neurovascular dysplasia resulting in dilated, thin-walled vessels that tend to rupture, increasing the risk for hemorrhagic stroke. KRIT1's nuclear roles are unknown, but it is known to function as a scaffolding or adaptor protein at cell-cell junctions and in the cytosol, supporting normal blood vessel integrity and development. As ICAP1 controls KRIT1 subcellular localization, presumably influencing KRIT1 function, in this work, we investigated the signals that regulate ICAP1 and, hence, KRIT1 nuclear localization. ICAP1 contains a nuclear localization signal within an unstructured, N-terminal region that is rich in serine and threonine residues, several of which are reportedly phosphorylated. Using quantitative microscopy, we revealed that phosphorylation-mimicking substitutions at Ser-10, or to a lesser extent at Ser-25, within this N-terminal region inhibit ICAP1 nuclear accumulation. Conversely, phosphorylation-blocking substitutions at these sites enhanced ICAP1 nuclear accumulation. We further demonstrate that p21-activated kinase 4 (PAK4) can phosphorylate ICAP1 at Ser-10 both in vitro and in cultured cells and that active PAK4 inhibits ICAP1 nuclear accumulation in a Ser-10-dependent manner. Finally, we show that ICAP1 phosphorylation controls nuclear localization of the ICAP1-KRIT1 complex. We conclude that serine phosphorylation within the ICAP1 N-terminal region can prevent nuclear ICAP1 accumulation, providing a mechanism that regulates KRIT1 localization and signaling, potentially influencing vascular development.

A C-terminal fragment of Arabidopsis OXIDATIVE STRESS 2 can play a positive role in salt tolerance.

The zinc finger transcription factor OXIDATIVE STRESS 2 (OXS2) was previously reported to be involved in oxidative stress tolerance and stress escape. Here we report that an Arabidopsis oxs2-1 mutant is also more sensitive to salt stress. Conversely, the overproduction of a C-terminal fragment of OXS2, the 'AT3' fragment, can enhance salt tolerance in Arabidopsis by upregulating the transcription of at least six salt-induced genes: COR15A, COR47, RD29B, KIN1, ACS2 and ACS6. Mutant analysis showed that the AT3-mediated salt tolerance requires MPK3, MPK6 and 14-3-3Ω. AT3 was shown to interact with MPK3 in planta, with 14-3-3Ω as a likely linker protein. AT3 can be phosphorylated by MPK3 during salt stress, upon which it relocates from the cytoplasm to the nucleus. It appears that the phosphorylation-induced nuclear localization of OXS2 contributes a positive role to the salt stress response.

PSPC1 is a new contextual determinant of aberrant subcellular translocation of oncogenes in tumor progression.

Dysregulation of nucleocytoplasmic shuttling is commonly observed in cancers and emerging as a cancer hallmark for the development of anticancer therapeutic strategies. Despite its severe adverse effects, selinexor, a selective first-in-class inhibitor of the common nuclear export receptor XPO1, was developed to target nucleocytoplasmic protein shuttling and received accelerated FDA approval in 2019 in combination with dexamethasone as a fifth-line therapeutic option for adults with relapsed refractory multiple myeloma (RRMM). To explore innovative targets in nucleocytoplasmic shuttling, we propose that the aberrant contextual determinants of nucleocytoplasmic shuttling, such as PSPC1 (Paraspeckle component 1), TGIF1 (TGF-ß Induced Factor Homeobox 1), NPM1 (Nucleophosmin), Mortalin and EBP50, that modulate shuttling (or cargo) proteins with opposite tumorigenic functions in different subcellular locations could be theranostic targets for developing anticancer strategies. For instance, PSPC1 was recently shown to be the contextual determinant of the TGF-ß prometastatic switch and PTK6/ß-catenin reciprocal oncogenic nucleocytoplasmic shuttling during hepatocellular carcinoma (HCC) progression. The innovative nucleocytoplasmic shuttling inhibitor PSPC1 C-terminal 131 polypeptide (PSPC1-CT131), which was developed to target both the shuttling determinant PSPC1 and the shuttling protein PTK6, maintained their tumor-suppressive characteristics and exhibited synergistic effects on tumor suppression in HCC cells and mouse models. In summary, targeting the contextual determinants of nucleocytoplasmic shuttling with cargo proteins having opposite tumorigenic functions in different subcellular locations could be an innovative strategy for developing new therapeutic biomarkers and agents to improve cancer therapy.

MRTF: Basic Biology and Role in Kidney Disease.

A lesser known but crucially important downstream effect of Rho family GTPases is the regulation of gene expression. This major role is mediated via the cytoskeleton, the organization of which dictates the nucleocytoplasmic shuttling of a set of transcription factors. Central among these is myocardin-related transcription factor (MRTF), which upon actin polymerization translocates to the nucleus and binds to its cognate partner, serum response factor (SRF). The MRTF/SRF complex then drives a large cohort of genes involved in cytoskeleton remodeling, contractility, extracellular matrix organization and many other processes. Accordingly, MRTF, activated by a variety of mechanical and chemical stimuli, affects a plethora of functions with physiological and pathological relevance. These include cell motility, development, metabolism and thus metastasis formation, inflammatory responses and-predominantly-organ fibrosis. The aim of this review is twofold: to provide an up-to-date summary about the basic biology and regulation of this versatile transcriptional coactivator; and to highlight its principal involvement in the pathobiology of kidney disease. Acting through both direct transcriptional and epigenetic mechanisms, MRTF plays a key (yet not fully appreciated) role in the induction of a profibrotic epithelial phenotype (PEP) as well as in fibroblast-myofibroblast transition, prime pathomechanisms in chronic kidney disease and renal fibrosis.

RGS14 Regulation of Post-Synaptic Signaling and Spine Plasticity in Brain.

The regulator of G-protein signaling 14 (RGS14) is a multifunctional signaling protein that regulates post synaptic plasticity in neurons. RGS14 is expressed in the brain regions essential for learning, memory, emotion, and stimulus-induced behaviors, including the basal ganglia, limbic system, and cortex. Behaviorally, RGS14 regulates spatial and object memory, female-specific responses to cued fear conditioning, and environmental- and psychostimulant-induced locomotion. At the cellular level, RGS14 acts as a scaffolding protein that integrates G protein, Ras/ERK, and calcium/calmodulin signaling pathways essential for spine plasticity and cell signaling, allowing RGS14 to naturally suppress long-term potentiation (LTP) and structural plasticity in hippocampal area CA2 pyramidal cells. Recent proteomics findings indicate that RGS14 also engages the actomyosin system in the brain, perhaps to impact spine morphogenesis. Of note, RGS14 is also a nucleocytoplasmic shuttling protein, where its role in the nucleus remains uncertain. Balanced nuclear import/export and dendritic spine localization are likely essential for RGS14 neuronal functions as a regulator of synaptic plasticity. Supporting this idea, human genetic variants disrupting RGS14 localization also disrupt RGS14's effects on plasticity. This review will focus on the known and unexplored roles of RGS14 in cell signaling, physiology, disease and behavior.

Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17.

Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19LSLRELAI26 of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein.IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein.

Novel nuclear translocation of inositol polyphosphate 4-phosphatase is associated with cell cycle, proliferation and survival.

Inositol polyphosphate 4 phosphatase type I enzyme (INPP4A) has a well-documented function in the cytoplasm where it terminates the phosphatidylinositol 3-kinase (PI 3-K) pathway by acting as a negative regulator. In this study, we demonstrate for the first time that INPP4A shuttles between the cytoplasm and the nucleus. Nuclear INPP4A is enzymatically active and in dynamic equilibrium between the nucleus and cytoplasm depending on the cell cycle stage, with highest amounts detected in the nucleus during the G0/G1 phase. Moreover, nuclear INPP4A is found to have direct proliferation suppressive activity. Cells constitutively overexpressing nuclear INPP4A exhibit massive apoptosis. In human tissues as well as cell lines, lower nuclear localization of INPP4A correlate with cancerous growth. Together, our findings suggest that nuclear compartmentalization of INPP4A may be a mechanism to regulate cell cycle progression, proliferation and apoptosis. Our results imply a role for nuclear-localized INPP4A in tumor suppression in humans.

Nucleocytoplasmic shuttling of SAMHD1 is important for LINE-1 suppression.

Currently, SAMHD1 is the only known dNTPase in human cells. It also suppresses the replication of both retroviruses and retroelements. SAMHD1 contains a classic nuclear localization sequence (NLS) and resides in the nucleus in live or fixed cells. It has been reported that alteration or removal of NLS does not affect the dNTPase or the antiviral activity of SAMHD1. However, it was unclear whether the nuclear localization was involved in SAMHD1-mediated suppression against retroelements such as long interspersed element type 1 (LINE-1 or L1). In this study, we reported that SAMHD1 is a nucleocytoplasmic shuttling protein. Digitonin-based cytoplasm/nucleus fractionation tests suggested that SAMHD1 is capable of being exported from the nucleus, which was confirmed by introducing exogenous exportin Xpo1 in live cells. Interestingly, altering the protein's subcellular localization by mutating or removing NLS significantly enhances SAMHD1's potency in L1 suppression. Further tests with SAMHD1 mutants indicated that nucleocytoplasmic shuttling is important for SAMHD1-mediated L1 suppression. Finally, we demonstrated that the cytoplasmic distribution of SAMHD1 leads to an enhanced depletion of L1 ORF2p. Taken together, our data have revealed SAMHD1 as a nucleocytoplasmic shuttling protein, and associated such a new feature of SAMHD1 with its potency against L1 retrotransposition, which provides more insights to the understanding of SAMHD1 and its role in L1 suppression.

Intersectin goes nuclear: secret life of an endocytic protein.

Intersectin 1-short (ITSN1-s) is a 1220 amino acid ubiquitously expressed scaffold protein presenting a multidomain structure that allows to spatiotemporally regulate the functional interaction of a plethora of proteins. Besides its well-established role in endocytosis, ITSN1-s is involved in the regulation of cell signaling and is implicated in tumorigenesis processes, although the signaling pathways involved are still poorly understood. Here, we identify ITSN1-s as a nucleocytoplasmic trafficking protein. We show that, by binding to importin (IMP)α, a small fraction of ITSN1-s localizes in the cell nucleus at the steady state, where it preferentially associates with the nuclear envelope and interacts with lamin A/C. However, upon pharmacological ablation of chromosome region maintenance 1 (CRM-1)-dependent nuclear export pathway, the protein accumulates into the nucleus, thus revealing its moonlighting nature. Analysis of deletion mutants revealed that the coiled coil (CC) and Src homology (SH3) regions play the major role in its nucleocytoplasmic shuttling. While no evidence of nuclear localization signal (NLS) was detected in the CC region, a functional bipartite NLS was identified within the SH3D region of ITSN1-s (RKKNPGGWWEGELQARGKKRQIGW-1127), capable of conferring energy-dependent nuclear accumulation to reporter proteins and whose mutational ablation affects nuclear import of the whole SH3 region. Thus, ITSN1-s is an endocytic protein, which shuttles between the nucleus and the cytoplasm in a CRM-1- and IMPα-dependent fashion.

Subcellular Localization Signals of bHLH-PAS Proteins: Their Significance, Current State of Knowledge and Future Perspectives.

The bHLH-PAS (basic helix-loop-helix/ Period-ARNT-Single minded) proteins are a family of transcriptional regulators commonly occurring in living organisms. bHLH-PAS members act as intracellular and extracellular "signals" sensors, initiating response to endo- and exogenous signals, including toxins, redox potential, and light. The activity of these proteins as transcription factors depends on nucleocytoplasmic shuttling: the signal received in the cytoplasm has to be transduced, via translocation, to the nucleus. It leads to the activation of transcription of particular genes and determines the cell response to different stimuli. In this review, we aim to present the current state of knowledge concerning signals that affect shuttling of bHLH-PAS transcription factors. We summarize experimentally verified and published nuclear localization signals/nuclear export signals (NLSs/NESs) in the context of performed in silico predictions. We have used most of the available NLS/NES predictors. Importantly, all our results confirm the existence of a complex system responsible for protein localization regulation that involves many localization signals, which activity has to be precisely controlled. We conclude that the current stage of knowledge in this area is still not complete and for most of bHLH-PAS proteins an experimental verification of the activity of further NLS/NES is needed.

Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism.

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin-α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN-NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian-origin cells (DF-1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference.

Nucleocytoplasmic shuttling: The ins and outs of quantitative imaging.

Nucleocytoplasmic protein shuttling is integral to the transmission of signals between the nucleus and the cytoplasm. The nuclear/cytoplasmic distribution of proteins of interest can be determined via fluorescence microscopy, following labelling of the target protein with fluorophore-conjugated antibodies (immunofluorescence) or by tagging the target protein with an autofluorescent protein, such as green fluorescent protein (GFP). The latter enables live cell imaging, a powerful approach that precludes many of the artefacts associated with indirect immunofluorescence in fixed cells. In this review, we discuss important considerations for the design and implementation of fluorescence microscopy experiments to quantify the nuclear/cytoplasmic distribution of a protein of interest. We summarise the pros and cons of detecting endogenous proteins in fixed cells by immunofluorescence and ectopically-expressed fluorescent fusion proteins in living cells. We discuss the suitability of widefield fluorescence microscopy and of 2D, 3D and 4D imaging by confocal microscopy for different applications, and describe two different methods for quantifying the nuclear/cytoplasmic distribution of a protein of interest from the fluorescent signal. Finally, we discuss the importance of eliminating sources of bias and subjectivity during image acquisition and post-imaging analyses. This is critical for the accurate and reliable quantification of nucleocytoplasmic shuttling.

Dual function of the nuclear export signal of the Borna disease virus nucleoprotein in nuclear export activity and binding to viral phosphoprotein.

BACKGROUND: Borna disease virus (BoDV), which has a negative-sense, single-stranded RNA genome, causes persistent infection in the cell nucleus. The nuclear export signal (NES) of the viral nucleoprotein (N) consisting of leucine at positions 128 and 131 and isoleucine at positions 133 and 136 overlaps with one of two predicted binding sites for the viral phosphoprotein (P). A previous study demonstrated that higher expression of BoDV-P inhibits nuclear export of N; however, the function of N NES in the interaction with P remains unclear. We examined the subcellular localization, viral polymerase activity, and P-binding ability of BoDV-N NES mutants. We also characterized a recombinant BoDV (rBoDV) harboring an NES mutation of N. RESULTS: BoDV-N with four alanine-substitutions in the leucine and isoleucine residues of the NES impaired its cytoplasmic localization and abolished polymerase activity and P-binding ability. Although an alanine-substitution at position 131 markedly enhanced viral polymerase activity as determined by a minigenome assay, rBoDV harboring this mutation showed expression of viral RNAs and proteins relative to that of wild-type rBoDV. CONCLUSIONS: Our results demonstrate that BoDV-N NES has a dual function in BoDV replication, i.e., nuclear export of N and an interaction with P, affecting viral polymerase activity in the nucleus.

A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1.

The human RNA-editing enzyme adenosine deaminase acting on RNA (ADAR1) carries a unique nuclear localization signal (NLS) that overlaps one of its double-stranded RNA-binding domains (dsRBDs). This dsRBD-NLS is recognized by the nuclear import receptor transportin 1 (Trn1; also called karyopherin-ß2) in an RNA-sensitive manner. Most Trn1 cargos bear a well-characterized proline-tyrosine-NLS, which is missing from the dsRBD-NLS. Here, we report the structure of the dsRBD-NLS, which reveals an unusual dsRBD fold extended by an additional N-terminal α-helix that brings the N- and C-terminal flanking regions in close proximity. We demonstrate experimentally that the atypical ADAR1-NLS is bimodular and is formed by the combination of the two flexible fragments flanking the folded domain. The intervening dsRBD acts only as an RNA-sensing scaffold, allowing the two NLS modules to be properly positioned for interacting with Trn1. We also provide a structural model showing how Trn1 can recognize the dsRBD-NLS and how dsRNA binding can interfere with Trn1 binding.