The Disruption of a Nuclear Export Signal in the C-Terminus of the Herpes Simplex Virus 1 Determinant of Pathogenicity UL24 Protein Leads to a Syncytial Plaque Phenotype.

UL24 of herpes simplex virus 1 (HSV-1) has been shown to be a determinant of pathogenesis in mouse models of infection. The N-terminus of UL24 localizes to the nucleus and drives the redistribution of nucleolin and B23. In contrast, when expressed alone, the C-terminal domain of UL24 accumulates in the Golgi apparatus; its importance during infection is unknown. We generated a series of mammalian expression vectors encoding UL24 with nested deletions in the C-terminal domain. Interestingly, enhanced nuclear staining was observed for several UL24-deleted forms in transient transfection assays. The substitution of a threonine phosphorylation site had no effect on UL24 localization or viral titers in cell culture. In contrast, mutations targeting a predicted nuclear export signal (NES) significantly enhanced nuclear localization, indicating that UL24 is able to shuttle between the nucleus and the cytoplasm. Recombinant viruses that encode UL24-harboring substitutions in the NES led to the accumulation of UL24 in the nucleus. Treatment with the CRM-1-specific inhibitor leptomycin B blocked the nuclear export of UL24 in transfected cells but not in the context of infection. Viruses encoding UL24 with NES mutations resulted in a syncytial phenotype, but viral yield was unaffected. These results are consistent with a role for HSV-1 UL24 in late cytoplasmic events in HSV-1 replication.

Herpes Simplex Virus-1 ICP27 Nuclear Export Signal Mutants Exhibit Cell Type-Dependent Deficits in Replication and ICP4 Expression.

Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.

APEX3 - An Optimized Tool for Rapid and Unbiased Proximity Labeling.

Macromolecular interactions regulate all aspects of biology. The identification of interacting partners and complexes is important for understanding cellular processes, host-pathogen conflicts, and organismal development. Multiple methods exist to label and enrich interacting proteins in living cells. Notably, the soybean ascorbate peroxidase, APEX2, rapidly biotinylates adjacent biomolecules in the presence of biotin-phenol and hydrogen peroxide. However, during initial experiments with this system, we found that APEX2 exhibits a cytoplasmic-biased localization and is sensitive to the nuclear export inhibitor leptomycin B (LMB). This led us to identify a putative nuclear export signal (NES) at the carboxy-terminus of APEX2 (NESAPEX2), structurally adjacent to the conserved heme binding site. This putative NES is functional as evidenced by cytoplasmic localization and LMB sensitivity of a mCherry-NESAPEX2 chimeric construct. Single amino acid substitutions of multiple hydrophobic residues within NESAPEX2 eliminate cytoplasm-biased localization of both mCherry-NESAPEX2 as well as full-length APEX2. However, all but one of these NES substitutions also compromises peroxide-dependent labeling. This unique separation-of-function mutant, APEX2-L242A, is termed APEX3. Localization and functionality of APEX3 are confirmed by fusion to the nucleocytoplasmic shuttling transcriptional factor, RELA. APEX3 is therefore an optimized tool for unbiased proximity labeling of cellular proteins and interacting factors..

Parallel T Cell Immunogenic Regions in Influenza B and A Viruses with Distinct Nuclear Export Signal Functions: The Balance between Viral Life Cycle and Immune Escape.

Influenza A viruses (IAVs) and influenza B viruses (IBVs) cause annual epidemics in human populations with seasonal circulation spikes. Peptide AM58-66GL9 located at residues 58-66 of M1 protein of IAVs has been recognized as an immunodominant T cell epitope with HLA-A*0201 restriction and broadly used as a positive reference in influenza immunity. This peptide also almost completely overlaps with a nuclear export signal (NES) 59-68 in IAV M1, which explains the limited escape mutations under the T cell immune pressure in this region. In this study, we investigated the potential immunogenicity and NES in the corresponding region of IBV. The long peptide covering this region can be recognized by specific T cells and induce robust expression of IFN-γ among HLA-B*1501 donors in vivo, but not in HLA-A*0201 donors. Among a series of truncated peptides derived from this region, we identified an immunodominant HLA-B*1501-restricted T cell epitope BM58-66AF9 (ALIGASICF) in the M1 protein of IBV. Furthermore, the structure of the HLA-B*1501/BM58-66AF9 complex shows that BM58-66AF9 performs a flat and featureless conformation that is similar to AM58-66GL9 presented by HLA-A*0201. In contrast with IAV, the sequence around residues 55-70 of IBV M1 does not contain an NES. Our comparative study on IBVs and IAVs provides new insights into the immune and evolution characteristics of IBVs and may shed light on vaccine development for influenza viruses.

A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods.

The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity.

Methyl eugenol protects the kidney from oxidative damage in mice by blocking the Nrf2 nuclear export signal through activation of the AMPK/GSK3ß axis.

Disrupted redox homeostasis contributes to renal ischemia-reperfusion (IR) injury. Abundant natural products can activate nuclear factor erythroid-2-related factor 2 (Nrf2), thereby providing therapeutic benefits. Methyl eugenol (ME), an analog of the phenolic compound eugenol, has the ability to induce Nrf2 activity. In this study, we investigated the protective effects of ME against renal oxidative damage in vivo and in vitro. An IR-induced acute kidney injury (AKI) model was established in mice. ME (20 mg·kg-1·d-1, i.p.) was administered to mice on 5 consecutive days before IR surgery. We showed that ME administration significantly attenuated renal destruction, improved the survival rate, reduced excessive oxidative stress and inhibited mitochondrial lesions in AKI mice. We further demonstrated that ME administration significantly enhanced Nrf2 activity and increased the expression of downstream antioxidative molecules. Similar results were observed in vitro in hypoxia/reoxygenation (HR)-exposed proximal tubule epithelial cells following pretreatment with ME (40 µmol·L-1). In both renal oxidative damage models, ME induced Nrf2 nuclear retention in tubular cells. Using specific inhibitors (CC and DIF-3) and molecular docking, we demonstrated that ME bound to the binding pocket of AMPK with high affinity and activated the AMPK/GSK3ß axis, which in turn blocked the Nrf2 nuclear export signal. In addition, ME alleviated the development of renal fibrosis induced by nonfatal IR, which is frequently encountered in the clinic. In conclusion, we demonstrate that ME modulates the AMPK/GSK3ß axis to regulate the cytoplasmic-nuclear translocation of Nrf2, resulting in Nrf2 nuclear retention and thereby enhancing antioxidant target gene transcription that protects the kidney from oxidative damage.

Enhanced mitochondrial DNA editing in mice using nuclear-exported TALE-linked deaminases and nucleases.

We present two methods for enhancing the efficiency of mitochondrial DNA (mtDNA) editing in mice with DddA-derived cytosine base editors (DdCBEs). First, we fused DdCBEs to a nuclear export signal (DdCBE-NES) to avoid off-target C-to-T conversions in the nuclear genome and improve editing efficiency in mtDNA. Second, mtDNA-targeted TALENs (mitoTALENs) are co-injected into mouse embryos to cleave unedited mtDNA. We generated a mouse model with the m.G12918A mutation in the MT-ND5 gene, associated with mitochondrial genetic disorders in humans. The mutant mice show hunched appearances, damaged mitochondria in kidney and brown adipose tissues, and hippocampal atrophy, resulting in premature death.

Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

The germ cell-specific TAP-like protein NXF-2 forms a novel granular structure and is required for tra-2 3'UTR-dependent mRNA export in Caenorhabditis elegans.

TAP is a general mRNA export receptor and is highly conserved among eukaryotes. The nematode Caenorhabditis elegans has another TAP-like protein, NXF-2, but little is known about its function. In this study, we show that NXF-2 is specifically expressed in germ cells and forms a novel granular structure that is different from that of P granules and that NXF-2 granules are anchored to the nuclear periphery in the mitotic region of the hermaphrodite gonad. In contrast, NXF-2 granules are released within the whole cytoplasm in the meiotic region, where the feminization gene tra-2 starts to function. Both inhibition of XPO-1 (an ortholog of the export receptor CRM1) and mutation of the nuclear export signal of NXF-2 caused the release of NXF-2 granules from the nuclear periphery, indicating that anchoring of NXF-2 granules depends on XPO-1 function. Moreover, inhibition of NXF-2 resulted in a substantial nuclear accumulation of the reporter mRNA carrying the tra-2 3'UTR. These results suggest that, together with XPO-1, NXF-2 exports and anchors tra-2 mRNA to the nuclear periphery to avoid precocious translation until the germ cells reach the meiotic region, thereby contributing to the regulation of tra-2 mRNA expression.

RanGTPase links nucleo-cytoplasmic transport to the recruitment of cargoes into small extracellular vesicles.

Small extracellular vesicle (sEV)-mediated intercellular communication regulates multiple aspects of growth and development in multicellular organisms. However, the mechanism underlying cargo recruitment into sEVs is currently unclear. We show that the key nucleo-cytoplasmic transport (NCT) protein-RanGTPase, in its GTP-bound form (RanGTP), is enriched in sEVs secreted by mammalian cells. This recruitment of RanGTP into sEVs depends on the export receptor CRM1 (also called XPO1). The recruitment of GAPDH, a candidate cargo protein, into sEVs is regulated by the RanGTP-CRM1axis in a nuclear export signal (NES)-dependent manner. Perturbation of NCT through overexpression or depletion of nuclear transport components affected the recruitment of Ran, CRM1 and GAPDH into sEVs. Our studies, thus, suggest a link between NCT, particularly the Ran-CRM1 axis, and recruitment of NES-containing cargoes into the sEVs. Collectively, these findings implicate RanGTPase as a link between NCT and sEV mediated intercellular communication.

Binding Affinity Measurement of Nuclear Export Signal Peptides to Their Exporter CRM1.

CRM1 recognizes hundreds to thousands of protein cargoes by binding to the eight to fifteen residue-long nuclear export signals (NESs) within their polypeptide chains. Various assays to measure the binding affinity of NESs for CRM1 have been developed. CRM1 binds to NESs with a wide range of binding affinities, with dissociation constants that span from low nanomolar to tens of micromolar. An optimized binding affinity assay with improved throughput was recently developed to measure binding affinities of NES peptides for CRM1 in the presence of excess RanGTP. The assay can measure affinities, with multiple replicates, for up to seven different NES peptides per screening plate. Here, we present a protocol for the purification of the necessary proteins and for measuring CRM1-NES binding affinities.

Crystallization of Nuclear Export Signals or Small-Molecule Inhibitors Bound to Nuclear Exporter CRM1.

The Karyopherin protein CRM1 or XPO1 is the major nuclear export receptor that regulates nuclear exit of thousands of macromolecules in the cell. CRM1 recognizes protein cargoes by binding to their 8-15 residue-long nuclear export signals (NESs). A ternary CRM1-Ran-RanBP1 complex engineered to be suitable for crystallization has enabled structure determination by X-ray crystallography of CRM1 bound to many NES peptides and small-molecule inhibitors. Here, we present a protocol for the purification of the individual proteins, formation of the ternary CRM1-Ran-RanBP1 complex and crystallization of this complex for X-ray crystallography.

Recurrent missense variant in the nuclear export signal of FMR1 associated with FXS-like phenotype including intellectual disability, ASD, facial abnormalities.

Fragile X syndrome (FXS; MIM 300624) is an X-linked genetic disorder characterized by physical abnormalities associated with intellectual disability and a wide spectrum of neurological and psychiatric impairments. FXS occurs more frequently in males, 1 in 5000 males and 1 in 8000 females accounting for 1-2% of overall intellectual disability (ID). In more than 99% of patients, FXS results from expansions of a CGG triplet repeat (>200 in male) of the FMR1 gene. In the last years an increasing number, albeit still limited, of FXS subjects carrying FMR1 mutations including deletions, splicing errors, missense, and nonsense variants was reported. Nevertheless, the studies concerning the functional consequences of mutations in the FMR1 gene are rare so far and, therefore, we do not have sufficient knowledge regarding the genotype/phenotype correlation. We report a child carrying a hemizygous missense FMR1 (NM_002024.5:c.1325G > A p.Arg442Gln) variant, maternally inherited, associated with facial abnormalities, developmental delay, and social and communication deficits assessed with formal neuropsychological tests. The study contributes to highlighting the clinical differences between the CGG triplet repeat dependent phenotype and FMR1variant dependent phenotype and it also confirms the pathogenicity of the variant being reported for the second time in the literature.

Ubiquitination on Lysine 247 of Newcastle Disease Virus Matrix Protein Enhances Viral Replication and Virulence by Driving Nuclear-Cytoplasmic Trafficking.

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding, and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M protein nuclear-cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were analyzed. Here, two types of combined NLSs and NESs were identified within the NDV-M protein. The Herts/33-type M protein was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M protein was retained mostly in the nuclei and showed retarded VLP production. Two critical residues, namely, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, of which its modification regulates the nuclear-cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued LaSota (rLaSota) strains rLaSota-R247K, -S263R, and -double mutation (DM) showed about 2-fold higher hemagglutination (HA) titers and 10-fold higher 50% egg infective dose (EID50) titers than wild-type (wt) rLaSota. Furthermore, the mean death time (MDT) and intracerebral pathogenicity index (ICPI) values of those recombinant viruses were slightly higher than those of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV and even those of all other paramyxoviruses. This information is beneficial for the development of vaccines and therapies for paramyxoviruses. IMPORTANCE Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked Newcastle disease (ND) as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and open up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach for improving paramyxovirus vaccines.

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.

Phosphorylation in the accessory domain of yeast histone chaperone protein 1 exposes the nuclear export signal sequence.

Histone chaperone proteins assist in the formation of the histone octamers, the scaffold proteins that facilitate the packing of DNA into nucleosomes in the cell nucleus. One such histone chaperone protein is yeast nucleosome assembly protein 1 (yNap1), the crystal structure of which has been determined and found to have a nuclear export signal (NES) sequence within its long α-helix. Experimental evidence obtained from mutagenesis studies of the budding yeast suggests that the NES is necessary for the transport of yNap1 from the cell nucleus to the cytosol. However, the NES sequence is masked by an accessory domain, the exact role of which has not yet been elucidated, especially in nucleocytoplasmic transport. To clarify the role of the accessory domain, we focused on its phosphorylation, because proteomic experiments have identified multiple phosphorylation sites on yNap1. To study this phenomenon computationally, all-atom molecular dynamics simulations of the non-phosphorylated yNap1 (Nap1-nonP) and phosphorylated yNap1 (Nap1-P) systems were performed. Specifically, we addressed how the NES sequence is exposed to the protein surface by measuring its solvent-accessible surface area (SASA). It was found that the median of the SASA distribution of Nap1-P was greater than that of Nap1-nonP, indicating that phosphorylation in the accessory domain exposes the NES, resulting in its increased accessibility. In conclusion, yNap1 might modulate the accessibility of the NES by dislocating the accessory domain through its phosphorylation.

Implication of the nuclear trafficking of rabies virus P3 protein in viral pathogenicity.

Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.

A Curious Novel Combination of Nucleophosmin (NPM1) Gene Mutations Leading to Aberrant Cytoplasmic Dislocation of NPM1 in Acute Myeloid Leukemia (AML).

Nucleophosmin (NPM1) mutations occurring in acute myeloid leukemia (AML) (about 50 so far identified) cluster almost exclusively in exon 12 and lead to common changes at the NPM1 mutants C-terminus, i.e., loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES), at the bases of exportin-1(XPO1)-mediated aberrant cytoplasmic NPM1. Immunohistochemistry (IHC) detects cytoplasmic NPM1 and is predictive of the molecular alteration. Besides IHC and molecular sequencing, Western blotting (WB) with anti-NPM1 mutant specific antibodies is another approach to identify NPM1-mutated AML. Here, we show that among 382 AML cases with NPM1 exon 12 mutations, one was not recognized by WB, and describe the discovery of a novel combination of two mutations involving exon 12. This appeared as a conventional mutation A with the known TCTG nucleotides insertion/duplication accompanied by a second event (i.e., an 8-nucleotide deletion occurring 15 nucleotides downstream of the TCTG insertion), resulting in a new C-terminal protein sequence. Strikingly, the sequence included a functional NES ensuring cytoplasmic relocation of the new mutant supporting the role of cytoplasmic NPM1 as critical in AML leukemogenesis.

Biodegradable Hypocrellin B nanoparticles coated with neutrophil membranes for hepatocellular carcinoma photodynamics therapy effectively via JUNB/ROS signaling.

Hepatocellular carcinoma (HCC) is an inflammation-induced and chemotherapy-resistant common liver cancer, and a major cause of death. Some natural products have been found to be used as photosensitizers in photodynamic therapy of HCC. Due to its specific molecular structure diversities and biological activities, current status of HCC treatment with nature production remains unsatisfactory, owing largely to the toxicity, side effect and inefficiency to drug targeting. Herein, we show a nanoparticle-based broad-spectrum anti-inflammatory strategy that naïve neutrophil membrane-coated PLGA nanoparticles (NM-HB NPs) were constructed for synchronous nearinfrared fluorescence (NIR FL) imaging and photodynamic therapy (PDT) for HCC. Moreover, NM-HB NPs inhibited the expression of JUNB and promoted the ROS production. JUNB depletion enhanced the anti-HCC effect of NM-HB NPs. Importantly, it was shown that NM-HB NPs are well targeted to the tumor site and overcomes the blood circulation and immune elimination in vivo and vitro. In a mouse model of HCC, the neutrophil membrane-coated nanoparticles (NM-HB NPs) show significant therapeutic efficacy by PDT and suppressing tumor tissue increase. All results demonstrated that NM coated HB NPs representing a viable and effective treatment option for HCC.