Nuclear Exportin 1 (XPO1) Binds to the Nuclear Localization/Export Signal of the Turnip Mosaic Virus NIb to Promote Viral Infection

The nuclear localization signal (NLS) and nuclear export signal (NES) are key signatures of proteins for controlling nuclear import and export. The NIb protein of turnip mosaic virus (TuMV) is an RNA-dependent RNA polymerase (RdRP) that is absolutely required for viral genome replication. Previous studies have shown that NIb is a nucleocytoplasmic shuttling protein and contains four putative NES and four putative NLS motifs. Here, we analyzed the function of these NESs and NLSs, and identified two functional NESs and one functional NLS. Mutation of the identified functional NESs or NLS inhibited viral RNA accumulation and systemic infection. Exportin 1 (XPO1) is a nuclear export receptor that binds directly to cargo proteins harboring a leucine-rich NES and translocates them to the cytoplasm. We found that XPO1 contains two NIb-binding domains, which recognize the NLS and NES of NIb, respectively, to mediate the nucleocytoplasmic transport of NIb and promote viral infection. Taken together, these data suggest that the nucleocytoplasmic transport of NIb is modulated by XPO1 through its interactions with the functional NLS and NES of NIb to promote viral infection.

Regulation of KHNYN antiviral activity by the extended di-KH domain and nucleo-cytoplasmic trafficking

The zinc finger antiviral protein (ZAP) restricts a broad range of viruses by binding CpG dinucleotides in viral RNA to target it for degradation and inhibit its translation. KHNYN was recently identified as an antiviral protein required for ZAP to inhibit retroviral replication, though little is known about its functional determinants. KHNYN contains an N-terminal extended di-KH-like domain, a PIN endoribonuclease domain and a C-terminal CUBAN domain that binds NEDD8 and ubiquitin. We show that deletion of the extended di-KH domain reduces its antiviral activity. However, despite its similarity to RNA binding KH domains, the extended di-KH domain in KHNYN does not appear to bind RNA. Mutation of residues in the CUBAN domain that bind NEDD8 increase KHNYN abundance but do not alter its antiviral activity, suggesting that this interaction regulates KHNYN homeostatic turnover. In contrast, a CRM1-dependent nuclear export signal (NES) at the C-terminus of the CUBAN domain is required for antiviral activity. Deletion of this signal retains KHNYN in the nucleus and inhibits its interaction with ZAP. Interestingly, this NES appeared in the KHNYN lineage at a similar time as when ZAP evolved in tetrapods, indicating that these proteins may have co-evolved to restrict viral replication.

The Membrane-Anchoring Region of the AcMNPV P74 Protein Is Expendable or Interchangeable with Homologs from Other Species

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.

Inhibition of XPO-1 Mediated Nuclear Export through the Michael-Acceptor Character of Chalcones

The nuclear export receptor exportin-1 (XPO1, CRM1) mediates the nuclear export of proteins that contain a leucine-rich nuclear export signal (NES) towards the cytoplasm. XPO1 is considered a relevant target in different human diseases, particularly in hematological malignancies, tumor resistance, inflammation, neurodegeneration and viral infections. Thus, its pharmacological inhibition is of significant therapeutic interest. The best inhibitors described so far (leptomycin B and SINE compounds) interact with XPO1 through a covalent interaction with Cys528 located in the NES-binding cleft of XPO1. Based on the well-established feature of chalcone derivatives to react with thiol groups via hetero-Michael addition reactions, we have synthesized two series of chalcones. Their capacity to react with thiol groups was tested by incubation with GSH to afford the hetero-Michael adducts that evolved backwards to the initial chalcone through a retro-Michael reaction, supporting that the covalent interaction with thiols could be reversible. The chalcone derivatives were evaluated in antiproliferative assays against a panel of cancer cell lines and as XPO1 inhibitors, and a good correlation was observed with the results obtained in both assays. Moreover, no inhibition of the cargo export was observed when the two prototype chalcones 9 and 10 were tested against a XPO1-mutated Jurkat cell line (XPO1C528S), highlighting the importance of the Cys at the NES-binding cleft for inhibition. Finally, their interaction at the molecular level at the NES-binding cleft was studied by applying the computational tool CovDock.

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.

Abstract P426: Inactivating Grk5 Impairs Basal Cardiac Function And Survival Via P53 Modulation

Introduction: G protein-coupled receptor (GPCR) kinase 5 (GRK5) is a multifunctional protein and depending on its localization within the cell, it has been shown to elicit either protective or deleterious effects. For instance in the heart, when anchored to the plasma membrane, this kinase can regulate specific GPCRs via canonical phosphorylation that can confer cardioprotection. However, when it accumulates in the nucleus its non-canonical activity can drive pathological hypertrophic gene transcription. Interestingly, the latter effects may not be kinase-dependent. Hypothesis: The role played by GRK5’s catalytic activity in the heart has not been fully elucidated and for that reason we sought to assess the in vivo consequences of inactivating the catalytic site of GRK5 with an initial focus at examining the basal cardiac phenotype and response to stress. Methods: We used CRISPR/Cas9 technology to generate a novel knock-in mouse model, with the ATP binding lysine (K) 215 in the catalytic cleft replaced by arginine (R) (GRK5-K215R) resulting in mice devoid of any GRK5 catalytic activity. We studies baseline cardiac function in these mutant mice compared to wild-type (WT) littermates and then stressed them via transverse aortic constriction (TAC). In vitro, we used H9c2 cardiomyocytes and various GRK5 mutants for mechanistic studies. Results: Compared to age-matched WT littermates, GRK5-K215R mice revealed marked and early (9 weeks) deterioration of cardiac function, with augmented apoptosis and fibrosis basally. Importantly, mutant knock-in mice displayed increased p53 gene expression (both at mRNA and protein levels). Moreover, TAC induced increased dysfunction and fibrosis in GRK5-K215R mice compared to WT. Mechanistically, we transduced H9c2 cells with adenoviruses (Ad), encoding for WT GRK5 (Ad-GRK5) or a mutant GRK5 lacking its nuclear localization signal (Ad-NLS) and when GRK5 was localized only outside the nucleus, there was a significant protection against apoptosis, with reduced p53 protein and mRNA levels. Conversely, when we overexpressed a mutant GRK5 without nuclear export signal (GRK5-ΔNES) to trap GRK5 within the nucleus, we found a significant increase in apoptosis, with high p53 protein expression levels. Conclusions: Inactivating GRK5’s catalytic activity impairs its nuclear regulation of p53. This can result in higher levels of p53 mRNA and protein resulting in higher rates of apoptosis in the heart leading to significant cardiac dysfunction and an intolerance to stress.

Caspase-mediated nuclear pore complex trimming in cell differentiation and endoplasmic reticulum stress

Introductory ParagraphDuring programmed cell death, caspases degrade 7 out of ∼30 nucleoporins (Nups) to irreversibly demolish the nuclear pore complex (NPC)1. However, for poorly understood reasons, caspases are also activated in differentiating cells in a non-apoptotic manner2,3. Here, we describe reversible, caspase-mediated NPC “trimming” during early myogenesis. We find that sublethal levels of caspases selectively proteolyze 4 peripheral Nups, Nup358, Nup214, Nup153, and Tpr, resulting in the transient block of nuclear export pathways. Several nuclear export signal (NES)-containing focal adhesion proteins concomitantly accumulate in the nucleus where they function as transcription cofactors4. We show that one such protein, FAK (focal adhesion kinase), drives a global reconfiguration of MBD2 (methyl CpG binding domain protein 2)-mediated genome regulation. We also observe caspase-mediated NPC trimming during neurogenesis and endoplasmic reticulum (ER) stress. Our results illustrate that the NPC can be proteolytically regulated in response to non-apoptotic cues, and call for a reassessment of the death-centric view of caspases.

NOVEL NPM1 EXON 5 MUTATIONS AND GENE FUSIONS LEADING TO ABERRANT CYTOPLASMIC NUCLEOPHOSMIN IN AML

Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but sporadically also exon 9 and 11, all causing changes at protein C-terminal end (loss of tryptophans and creation of a nuclear export signal-NES motif) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 AML patients, we found non-exon 12 NPM1 mutations in 5/387 (1.3%) NPM1c+ cases. Besides mutations in exon 9 (n=1) and exon 11 (n=1), novel mutations in exon 5 were discovered (n=3). One more exon 5 mutation was identified in additional 141 AML patients selected for wild-type NPM1 exon 12. Furthermore, 3 NPM1 rearrangements (i.e. NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13,979 AML samples screened by cytogenetic/FISH and RNA sequencing. Functional studies demonstrated that in AML cases the new NPM1 proteins harboured an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting any AML-associated NPM1 genetic lesions. Also, this study highlights the need for developing new specific assays for molecular diagnosis and monitoring of NPM1-mutated AML.