Beatriz M. A. Fontoura

Influenza virus targets the mRNA export machinery and the nuclear pore complex

The NS1 protein of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 functions to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression, but its mechanisms of action remain to be fully elucidated. We show here that NS1 forms an inhibitory complex with NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5, which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex. Increased levels of NXF1, p15, or Rae1 revert the mRNA export blockage induced by NS1. Furthermore, influenza virus down-regulates Nup98, a nucleoporin that is a docking site for mRNA export factors. Reduced expression of these mRNA export factors renders cells highly permissive to influenza virus replication, demonstrating that proper levels of key constituents of the mRNA export machinery protect against influenza virus replication. Because Nup98 and Rae1 are induced by interferons, down-regulation of this pathway is likely a viral strategy to promote viral replication. These findings demonstrate previously undescribed influenza-mediated viral–host interactions and provide insights into potential molecular therapies that may interfere with influenza infection.

Plasmodium Circumsporozoite Protein Promotes the Development of the Liver Stages of the Parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.

The Nup107‑160 complex and γ‑TuRC regulate microtubule polymerization at kinetochores

The metazoan nuclear pore complex (NPC) disassembles during mitosis, and many of its constituents distribute onto spindles and kinetochores, including the Nup107-160 sub-complex. We have found that Nup107-160 interacts with the γ-tubulin ring complex (γ-TuRC), an essential and conserved microtubule nucleator, and recruits γ-TuRC to unattached kinetochores. The unattached kinetochores nucleate microtubules in a manner that is regulated by Ran GTPase; such microtubules contribute to the formation of kinetochore fibres (k-fibres), microtubule bundles connecting kinetochores to spindle poles. Our data indicate that Nup107-160 and γ-TuRC act cooperatively to promote spindle assembly through microtubule nucleation at kinetochores: HeLa cells lacking Nup107-160 or γ-TuRC were profoundly deficient in kinetochore-associated microtubule nucleation. Moreover, co-precipitated Nup107-160– γ-TuRC complexes nucleated microtubule formation in assays using purified tubulin. Although Ran did not regulate microtubule nucleation by γ-TuRC alone, Nup107-160–γ-TuRC complexes required Ran–GTP for microtubule nucleation. Collectively, our observations show that Nup107-160 promotes spindle assembly through Ran–GTP-regulated nucleation of microtubules by γ-TuRC at kinetochores, and reveal a relationship between nucleoporins and the microtubule cytoskeleton.

Structure-based design of a pathway-specific nuclear import inhibitor

Kapβ2 (also called transportin) recognizes PY nuclear localization signal (NLS), a new class of NLS with a R/H/Kx(2–5)PY motif. Here we show that Kapβ2 complexes containing hydrophobic and basic PY-NLSs, as classified by the composition of an additional N-terminal motif, converge in structure only at consensus motifs, which explains ligand diversity. On the basis of these data and complementary biochemical analyses, we designed a Kapβ2-specific nuclear import inhibitor, M9M.

The Plasmodium eukaryotic initiation factor-2α kinase IK2 controls the latency of sporozoites in the mosquito salivary glands

Sporozoites, the invasive form of malaria parasites transmitted by mosquitoes, are quiescent while in the insect salivary glands. Sporozoites only differentiate inside of the hepatocytes of the mammalian host. We show that sporozoite latency is an active process controlled by a eukaryotic initiation factor-2α (eIF2α) kinase (IK2) and a phosphatase. IK2 activity is dominant in salivary gland sporozoites, leading to an inhibition of translation and accumulation of stalled mRNAs into granules. When sporozoites are injected into the mammalian host, an eIF2α phosphatase removes the PO4 from eIF2α-P, and the repression of translation is alleviated to permit their transformation into liver stages. In IK2 knockout sporozoites, eIF2α is not phosphorylated and the parasites transform prematurely into liver stages and lose their infectivity. Thus, to complete their life cycle, Plasmodium sporozoites exploit the mechanism that regulates stress responses in eukaryotic cells.

The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR

The Nup98 gene codes for several alternatively spliced protein precursors. Two in vitro translated and autoproteolytically cleaved precursors yielded heterodimers of Nup98-6kDa peptide and Nup98-Nup96. TPR (translocated promoter region) is a protein that forms filamentous structures extending from nuclear pore complexes (NPCs) to intranuclear sites. We found that in vitro translated TPR bound to in vitro translated Nup98 and, via Nup98, to Nup96. Double-immunofluorescence microscopy with antibodies to TPR and Nup98 showed colocalization. In confocal sections the nucleolus itself was only weakly stained but there was intensive perinucleolar staining. Striking spike-like structures emanated from this perinucleolar ring and attenuated into thinner structures as they extended to the nuclear periphery. This characteristic staining pattern of the TPR network was considerably enhanced when a myc-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cells. Double-immunoelectron microscopy of these cells using anti-myc and anti-TPR antibodies and secondary gold-coupled antibodies yielded row-like arrangements of gold particles. Taken together, the immunolocalization data support previous electron microscopical data, suggesting that TPR forms filaments that extend from the NPC to the nucleolus. We discuss the possible implications of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.

Sec13 Shuttles between the Nucleus and the Cytoplasm and Stably Interacts with Nup96 at the Nuclear Pore Complex

ABSTRACT Sec13 is a constituent of the endoplasmic reticulum and the nuclear pore complex (NPC). At the endoplasmic reticulum, Sec13 is involved in the biogenesis of COPII-coated vesicles, whereas at the NPC its function is unknown. We show here, by yeast two-hybrid screenings and biochemical assays, that a region at the amino terminus of the human nuclear pore complex protein Nup96 interacts with the WD (Trp-Asp) repeat region of human Sec13. By using immunofluorescence and confocal and immunoelectron microscopy, we found that in interphase, Sec13 and Nup96 are localized at both sides of the NPC in addition to other intracellular sites. In mitosis, Sec13 was found dispersed throughout the cell, whereas a pool of Nup96 colocalized with the spindle apparatus. Photobleaching experiments showed that Sec13 shuttles between intranuclear sites and the cytoplasm, and a fraction of Sec13 is stably associated with NPCs. Cotransfection of Sec13 and the Sec13 binding site of Nup96 decreased the mobile pool of Sec13, demonstrating the interaction of Sec13 and Nup96 in vivo. Targeting studies showed that Sec13 is actively transported into the nucleus and contains a nuclear localization signal. These results indicate that Sec13 stably interacts with Nup96 at the NPC during interphase and that the shuttling of Sec13 between the nucleus and the cytoplasm may couple and regulate functions between these two compartments.

The Nucleoporin Nup98 Is a Site for GDP/GTP Exchange on Ran and Termination of Karyopherin β2-mediated Nuclear Import

Karyopherin β2 (Kapβ2, transportin) binds the M9 sequence of human ribonucleoprotein A1 and mediates its nuclear import. Here we show a role for the nucleoporin Nup98 in the disassembly of Kapβ2 import complexes at the nuclear side of the nuclear pore complex (NPC). Kapβ2 bound to a region at the N terminus of Nup98 that contains an M9-like sequence. The human ribonucleoprotein A1 M9 sequence competed with Nup98 for binding to Kapβ2, indicating that Nup98 can dissociate Kapβ2 from its substrate. Binding of Kapβ2 to Nup98 was inhibited by Ran loaded with guanylyl imidophosphate, suggesting that RanGTP dissociates Kapβ2 from Nup98. RanGTP is produced from RanGDP through nucleotide exchange mediated by RanGEF (RCC1). Immunoelectron microscopy and nucleotide exchange assays revealed functional RanGEF on both sides of the NPC. On the nuclear side, the localization of RanGEF coincided with that of Nup98. RanGEF bound to Nup98 at a region adjacent to the Kapβ2-binding site. These findings suggest a model where 1) import substrate is released from Kapβ2 at the nucleoplasmic side of the NPC by competition with the Nup98 M9-like site, 2) Nup98-bound RanGEF catalyzes the formation of RanGTP, and 3) RanGTP dissociates Kapβ2 from Nup98 allowing repeated cycles of import.

Nucleoporin Levels Regulate Cell Cycle Progression and Phase-Specific Gene Expression

The Nup107-160 complex, the largest subunit of the nuclear pore, is multifunctional. It mediates mRNA export in interphase, and has roles in kinetochore function, spindle assembly, and postmitotic nuclear pore assembly. We report here that the levels of constituents of the Nup107-160 complex are coordinately cell cycle-regulated. At mitosis, however, a member of the complex, Nup96, is preferentially downregulated. This occurs via the ubiquitin-proteasome pathway. When the levels of Nup96 are kept high, a significant delay in G1/S progression occurs. Conversely, in cells of Nup96(+/-) mice, which express low levels of Nup96, cell cycle progression is accelerated. These lowered levels of Nup96 yield specific defects in nuclear export of certain mRNAs and protein expression, among which are key cell cycle regulators. Thus, Nup96 levels regulate differential gene expression in a phase-specific manner, setting the stage for proper cell cycle progression.

Cytoplasmic p53 polypeptide is associated with ribosomes.

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.