Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.

Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex.

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro , and in the Syrian golden hamster model in vivo . In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARY: SARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

Nuclear speckle integrity and function require TAO2 kinase.

Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.

Mechanism of karyopherin-ß2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-ß2 (Kapß2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapß2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone.

We report the crystal structure of nuclear import receptor Importin-9 bound to its cargo, the histones H2A-H2B. Importin-9 wraps around the core, globular region of H2A-H2B to form an extensive interface. The nature of this interface coupled with quantitative analysis of deletion mutants of H2A-H2B suggests that the NLS-like sequences in the H2A-H2B tails play a minor role in import. Importin-9•H2A-H2B is reminiscent of interactions between histones and histone chaperones in that it precludes H2A-H2B interactions with DNA and H3-H4 as seen in the nucleosome. Like many histone chaperones, which prevent inappropriate non-nucleosomal interactions, Importin-9 also sequesters H2A-H2B from DNA. Importin-9 appears to act as a storage chaperone for H2A-H2B while escorting it to the nucleus. Surprisingly, RanGTP does not dissociate Importin-9•H2A-H2B but assembles into a RanGTP•Importin-9•H2A-H2B complex. The presence of Ran in the complex, however, modulates Imp9-H2A-H2B interactions to facilitate its dissociation by DNA and assembly into a nucleosome.

Correlation of CRM1-NES affinity with nuclear export activity.

CRM1 (Exportin1/XPO1) exports hundreds of broadly functioning protein cargoes out of the cell nucleus by binding to their classical nuclear export signals (NESs). The 8- to 15-amino-acid-long NESs contain four to five hydrophobic residues and are highly diverse in both sequence and CRM1-bound structure. Here we examine the relationship between nuclear export activities of 24 different NES peptides in cells and their CRM1-NES affinities. We found that binding affinity and nuclear export activity are linearly correlated for NESs with dissociation constants ( Kds) between tens of nanomolar to tens of micromolar. NESs with Kds outside this range have significantly reduced nuclear export activities. These include two unusually tight-binding peptides, one from the nonstructural protein 2 of murine minute virus (MVM NS2) and the other a mutant of the protein kinase A inhibitor (PKI) NES. The crystal structure of CRM1-bound MVM NS2NES suggests that extraordinarily tight CRM1 binding arises from intramolecular contacts within the NES that likely stabilizes the CRM1-bound conformation in free peptides. This mechanistic understanding led to the design of two novel peptide inhibitors that bind CRM1 with picomolar affinity.

Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization.

ALS (Amyotrophic Lateral Sclerosis) is a neurodegenerative disease characterized by the redistribution of the RNA binding protein TDP-43 in affected neurons: from predominantly nuclear to aggregated in the cytosol. However, the determinants of TDP-43 localization and the cellular insults that promote redistribution are incompletely understood. Here, we show that the putative Nuclear Export Signal (NES) is not required for nuclear egress of TDP-43. Moreover, when the TDP-43 domain which contains the putative NES is fused to a reporter protein, YFP, the presence of the NES is not sufficient to mediate nuclear exclusion of the fusion protein. We find that the previously studied "∆NES" mutant, in which conserved hydrophobic residues are mutated to alanines, disrupts both solubility and splicing function. We further show that nuclear export of TDP-43 is independent of the exportin XPO1. Finally, we provide evidence that nuclear egress of TDP-43 is size dependent; nuclear export of dTomato TDP-43 is significantly impaired compared to Flag TDP-43. Together, these results suggest nuclear export of TDP-43 is predominantly driven by passive diffusion.

Karyopherins in cancer.

Malfunction of nuclear-cytoplasmic transport contributes to many diseases including cancer. Defective nuclear transport leads to changes in both the physiological levels and temporal-spatial location of tumor suppressors, proto-oncogenes and other macromolecules that in turn affect the tumorigenesis process and drug sensitivity of cancer cells. In addition to their nuclear transport functions in interphase, Karyopherin nuclear transport receptors also have important roles in mitosis and chromosomal integrity. Therefore, alterations in the expressions or regular functions of Karyopherins may have substantial effects on the course and outcome of diseases.

Recognition Elements in the Histone H3 and H4 Tails for Seven Different Importins.

N-terminal tails of histones H3 and H4 are known to bind several different Importins to import the histones into the cell nucleus. However, it is not known what binding elements in the histone tails are recognized by the individual Importins. Biochemical studies of H3 and H4 tails binding to seven Importins, Impß, Kapß2, Imp4, Imp5, Imp7, Imp9, and Impα, show the H3 tail binding more tightly than the H4 tail. The H3 tail binds Kapß2 and Imp5 with KD values of 77 and 57 nm, respectively, and binds the other five Importins more weakly. Mutagenic analysis shows H3 tail residues 11-27 to be the sole binding segment for Impß, Kapß2, and Imp4. However, Imp5, Imp7, Imp9, and Impα bind two separate elements in the H3 tail: the segment at residues 11-27 and an isoleucine-lysine nuclear localization signal (IK-NLS) motif at residues 35-40. The H4 tail also uses either one or two basic segments to bind the same set of Importins with a similar trend of relative affinities as the H3 tail, albeit at least 10-fold weaker. Of the many lysine residues in the H3 and H4 tails, only acetylation of the H3 Lys14 substantially decreased binding to several Importins. Lastly, we show that, in addition to the N-terminal tails, the histone fold domains of H3 and H4 and/or the histone chaperone Asf1b are important for Importin-histone recognition.

LocNES: a computational tool for locating classical NESs in CRM1 cargo proteins.

MOTIVATION: Classical nuclear export signals (NESs) are short cognate peptides that direct proteins out of the nucleus via the CRM1-mediated export pathway. CRM1 regulates the localization of hundreds of macromolecules involved in various cellular functions and diseases. Due to the diverse and complex nature of NESs, reliable prediction of the signal remains a challenge despite several attempts made in the last decade. RESULTS: We present a new NES predictor, LocNES. LocNES scans query proteins for NES consensus-fitting peptides and assigns these peptides probability scores using Support Vector Machine model, whose feature set includes amino acid sequence, disorder propensity, and the rank of position-specific scoring matrix score. LocNES demonstrates both higher sensitivity and precision over existing NES prediction tools upon comparative analysis using experimentally identified NESs. AVAILABILITY AND IMPLEMENTATION: LocNES is freely available at http://prodata.swmed.edu/LocNES CONTACT: yuhmin.chook@utsouthwestern.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Circuit-level input integration in bacterial gene regulation.

Gene regulatory circuits can receive multiple simultaneous inputs, which can enter the system through different locations. It is thus necessary to establish how these genetic circuits integrate multiple inputs as a function of their relative entry points. Here, we use the dynamic circuit regulating competence for DNA uptake in Bacillus subtilis as a model system to investigate this issue. Specifically, we map the response of single cells in vivo to a combination of (i) a chemical signal controlling the constitutive expression of key competence genes, and (ii) a genetic perturbation in the form of copy number variation of one of these genes, which mimics the level of stress signals sensed by the bacteria. Quantitative time-lapse fluorescence microscopy shows that a variety of dynamical behaviors can be reached by the combination of the two inputs. Additionally, the integration depends strongly on the relative locations where the two perturbations enter the circuit. Specifically, when the two inputs act upon different circuit elements, their integration generates novel dynamical behavior, whereas inputs affecting the same element do not. An in silico bidimensional bifurcation analysis of a mathematical model of the circuit offers good quantitative agreement with the experimental observations, and sheds light on the dynamical mechanisms leading to the different integrated responses exhibited by the gene regulatory circuit.

Localized cell death focuses mechanical forces during 3D patterning in a biofilm.

From microbial biofilm communities to multicellular organisms, 3D macroscopic structures develop through poorly understood interplay between cellular processes and mechanical forces. Investigating wrinkled biofilms of Bacillus subtilis, we discovered a pattern of localized cell death that spatially focuses mechanical forces, and thereby initiates wrinkle formation. Deletion of genes implicated in biofilm development, together with mathematical modeling, revealed that ECM production underlies the localization of cell death. Simultaneously with cell death, we quantitatively measured mechanical stiffness and movement in WT and mutant biofilms. Results suggest that localized cell death provides an outlet for lateral compressive forces, thereby promoting vertical mechanical buckling, which subsequently leads to wrinkle formation. Guided by these findings, we were able to generate artificial wrinkle patterns within biofilms. Formation of 3D structures facilitated by cell death may underlie self-organization in other developmental systems, and could enable engineering of macroscopic structures from cell populations.

Temporal competition between differentiation programs determines cell fate choice.

Multipotent differentiation, where cells adopt one of several possible fates, occurs in diverse systems ranging from bacteria to mammals. This decision-making process is driven by multiple differentiation programs that operate simultaneously in the cell. How these programs interact to govern cell fate choice is poorly understood. To investigate this issue, we simultaneously measured activities of the competing sporulation and competence programs in single Bacillus subtilis cells. This approach revealed that these competing differentiation programs progress independently without cross-regulation before the decision point. Cells seem to arrive at a fate choice through differences in the relative timing between the two programs. To test this proposed dynamic mechanism, we altered the relative timing by engineering artificial cross-regulation between the sporulation and competence circuits. Results suggest a simple model that does not require a checkpoint or intricate cross-regulation before cellular decision-making. Rather, cell fate choice appears to be the outcome of a 'molecular race' between differentiation programs that compete in time, providing a simple dynamic mechanism for decision-making.

Mutations in the human naked cuticle homolog NKD1 found in colorectal cancer alter Wnt/Dvl/beta-catenin signaling.

BACKGROUND: Mutation of Wnt signal antagonists Apc or Axin activates beta-catenin signaling in many cancers including the majority of human colorectal adenocarcinomas. The phenotype of apc or axin mutation in the fruit fly Drosophila melanogaster is strikingly similar to that caused by mutation in the segment-polarity gene, naked cuticle (nkd). Nkd inhibits Wnt signaling by binding to the Dishevelled (Dsh/Dvl) family of scaffold proteins that link Wnt receptor activation to beta-catenin accumulation and TCF-dependent transcription, but human NKD genes have yet to be directly implicated in cancer. METHODOLOGY/PRINCIPAL FINDINGS: We identify for the first time mutations in NKD1--one of two human nkd homologs--in a subset of DNA mismatch repair-deficient colorectal tumors that are not known to harbor mutations in other Wnt-pathway genes. The mutant Nkd1 proteins are defective at inhibiting Wnt signaling; in addition, the mutant Nkd1 proteins stabilize beta-catenin and promote cell proliferation, in part due to a reduced ability of each mutant Nkd1 protein to bind and destabilize Dvl proteins. CONCLUSIONS/SIGNIFICANCE: Our data raise the hypothesis that specific NKD1 mutations promote Wnt-dependent tumorigenesis in a subset of DNA mismatch-repair-deficient colorectal adenocarcinomas and possibly other Wnt-signal driven human cancers.

Architecture-dependent noise discriminates functionally analogous differentiation circuits.

Gene regulatory circuits with different architectures (patterns of regulatory interactions) can generate similar dynamics. This raises the question of why a particular circuit architecture is selected to implement a given cellular process. To investigate this problem, we compared the Bacillus subtilis circuit that regulates differentiation into the competence state to an engineered circuit with an alternative architecture (SynEx) in silico and in vivo. Time-lapse microscopy measurements showed that SynEx cells generated competence dynamics similar to native cells and reconstituted the physiology of differentiation. However, architectural differences between the circuits altered the dynamic distribution of stochastic fluctuations (noise) during circuit operation. This distinction in noise causes functional differences between the circuits by selectively controlling the timing of competence episodes and response of the system to various DNA concentrations. These results reveal a tradeoff between temporal precision and physiological response range that is controlled by distinct noise characteristics of alternative circuit architectures.

Cell-autonomous, myristyl-independent activity of the Drosophila Wnt/Wingless antagonist Naked cuticle (Nkd).

Robust animal development, tissue homeostasis, and stem cell renewal requires precise control of the Wnt/beta-catenin signaling axis. In the embryo of the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene attenuates signaling by the Wnt ligand Wingless (Wg) during segmentation. nkd mutants have been reported to exhibit abnormalities in wg transcription, Wg protein distribution and/or transport, and the intracellular response to Wg, but the relationship between each alteration and the molecular mechanism of Nkd action remains unclear. In addition, whether Nkd acts in a cell-autonomous or nonautonomous fashion in the embryo is not known. Mammalian Nkd homologs have N-terminal consensus sequences that direct the post-translational addition of a lipophilic myristoyl moiety, but fly and mosquito Nkd, while sharing N-terminal sequence homology, lack a myristoylation consensus sequence. Here we provide evidence that fly Nkd acts cell-autonomously in the embryo, with its N-terminus able to confer unique functional properties and membrane association that cannot be mimicked in vivo by heterologous myristoylation consensus sequences. In conjunction with our recent observation that Nkd requires nuclear localization for function, our data suggest that Nkd acts at more than one subcellular location within signal-receiving cells to attenuate Wg signaling.

Viable mice with compound mutations in the Wnt/Dvl pathway antagonists nkd1 and nkd2.

Gradients of Wnt/beta-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/beta-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkd(lacZ) allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/beta-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development.

An unconventional nuclear localization motif is crucial for function of the Drosophila Wnt/wingless antagonist Naked cuticle.

Wnt/beta-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, we first must understand how Wnt signals are normally controlled. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/beta-catenin signaling that is crucial for segmentation in the model genetic organism, the fruit fly Drosophila melanogaster. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. Here we show that nkd mutants exhibit elevated levels of the beta-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, we identify a conserved 30-amino-acid motif, separable from Dsh-binding regions, that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. Our studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal.