scholarly journals The Portal Vertex of KSHV Promotes Docking of Capsids at the Nuclear Pores

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 597
Author(s):  
Daniela Dünn-Kittenplon ◽  
Asaf Ashkenazy-Titelman ◽  
Inna Kalt ◽  
Jean-Paul Lellouche ◽  
Yaron Shav-Tal ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Salt Treatment ◽  
Reading Frame ◽  
Protein Subunits ◽  
Tegument Proteins ◽  
Dead End ◽  
Lower Extent ◽  
Icosahedral Capsid

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection ‘dead end’.

scholarly journals The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.

1995 ◽  
Vol 131 (6) ◽  
pp. 1699-1713 ◽  
Author(s):  
M K Iovine ◽  
J L Watkins ◽  
S R Wente
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Structural Basis ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Repetitive Region ◽  
Amino Terminal ◽  
Section Electron ◽  
Rna Export

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.

scholarly journals Nuclear Export Through Nuclear Envelope Remodeling inSaccharomyces cerevisiae

2017 ◽  
Author(s):  
Baojin Ding ◽  
Anne M. Mirza ◽  
James Ashley ◽  
Vivian Budnik ◽  
Mary Munson
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Mrna Export ◽  
Low Frequency ◽  
Nuclear Pore ◽  
Wild Type ◽  
Export Pathway ◽  
Wild Type Yeast

ABSTRACTIn eukaryotes, subsets of exported mRNAs are organized into large ribonucleoprotein (megaRNP) granules. How megaRNPs exit the nucleus is unclear, as their diameters are much larger than the nuclear pore complex (NPC) central channel. We previously identified a non-canonical nuclear export mechanism inDrosophila(Speese et al.,Cell2012) and mammals (Ding et al., in preparation), in which megaRNPs exit the nucleus by budding across nuclear envelope (NE) membranes. Here, we present evidence for a similar pathway in the nucleus of the budding yeast S.cerevisiae, which contain morphologically similar granules bearing mRNAs. Wild-type yeast displayed these granules at very low frequency, but this frequency was dramatically increased when the non-essential NPC protein Nup116 was deleted. These granules were not artifacts of defective NPCs; a mutation in the exportinXPO1(CRM1), in which NPCs are normal, induced similar megaRNP upregulation. We hypothesize that a non-canonical nuclear export pathway, analogous to those observed inDrosophilaand in mammalian cells, exists in yeast, and that this pathway is upregulated for use when NPCs or nuclear export are impaired.SUMMARYDing et al., describe a non-canonical mRNA export pathway in budding yeast similar to that observed inDrosophila. This pathway appears upregulated when the NPC is impaired, nuclear envelope integrity is disrupted, or the export factor Xpo1 (CRM1) is defective.

Abstract MP145: Potential Functions of the Nuclear Basket Associated With Dilated Cardiomyopathy

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Claudia C Preston ◽  
Ryan D Burdine ◽  
Emily C Storm ◽  
Claudia S Preston
Keyword(s):  
Nuclear Envelope ◽  
Dilated Cardiomyopathy ◽  
Nuclear Export ◽  
Gene List ◽  
Bioinformatic Analysis ◽  
Nuclear Pore ◽  
Envelope Gene ◽  
Decay Kinetics ◽  
Chromatin Dynamics ◽  
Induced Pluripotent

Objectives: NUP153 overexpression is clinically correlated with cardiomyopathies, specifically dilated cardiomyopathy (DCM). In combination with the nuclear pore complex (NPC) proteins TPR and NUP50, all three form the basket structure of the NPC that sits within the nuclear envelope. The nuclear basket regulates chromatin dynamics and nuclear export, yet its potential role in cardiopathological development and progression remains unknown. This study was thus carried out to elucidate possible functional roles for nuclear basket nucleoporins in cardiac biology. Methods: Human induced pluripotent stem cells (hiPSCs) with endogenous mEGFP tagged NUP153 were differentiated into cardiomyocytes (hiPSC-CMs) via direct differentiation. Beating started ~ Day 10 and contractile areas were analyzed for contraction velocity and magnitude. NUP153 mobility in hiPSCs and hiPSC-CMs was determined by iFRAP experiments. Bioinformatic analysis of a clinical dilated cardiomyopathy dataset (GSE29819) returned a focused ‘nuclear envelope’ gene list that was uploaded to Ingenuity Pathways Analysis for network analysis. Results: Within hiPSC-CM beating foci (A), NUP153 mobility decreased and went from a biphasic to a monophasic decay curve (B). Bioinformatic analysis of a DCM dataset (C) revealed expression changes in TPR and NUP50 , with no significant changes in NUP153 (D). Conclusions: A switch to monophasic decay kinetics of NUP153 in hiPSC-CMs suggests prioritization of stable slow pools that may reflect changes in global transcriptome regulation. In DCM, differential nucleoporin gene expression changes implicate discrete functional disruptions within the nuclear basket.

Nuclear Envelope Dynamics During Mitosis

1988 ◽  
Vol 46 ◽  
pp. 224-225
Author(s):  
Brian Burke
Keyword(s):  
Nuclear Envelope ◽  
Membrane Vesicles ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Animal Cells ◽  
Interphase Chromosome ◽  
Lamin B ◽  
Chromosome Architecture ◽  
Complex Membrane ◽  
Pore Complexes

The nuclear envelope is a complex membrane structure that forms the boundary of the nuclear compartment in eukaryotes. It regulates the passage of macromolecules between the two compartments and may be important for organizing interphase chromosome architecture. In interphase animal cells it forms a remarkably stable structure consisting of a double membrane ouerlying a protein meshwork or lamina and penetrated by nuclear pore complexes. The latter form the channels for nucleocytoplasmic exchange of macromolecules, At the onset of mitosis, however, it rapidly disassembles, the membranes fragment to yield small vesicles and the lamina, which is composed of predominantly three polypeptides, lamins R, B and C (MW approx. 74, 68 and 65 kDa respectiuely), breaks down. Lamins B and C are dispersed as monomers throughout the mitotic cytoplasm, while lamin B remains associated with the nuclear membrane vesicles.

scholarly journals Mex67p of Schizosaccharomyces pombeInteracts with Rae1p in Mediating mRNA Export

2000 ◽  
Vol 20 (23) ◽  
pp. 8767-8782 ◽  
Author(s):  
Jin Ho Yoon ◽  
Dona C. Love ◽  
Anjan Guhathakurta ◽  
John A. Hanover ◽  
Ravi Dhar
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Synthetic Lethality ◽  
Sequence Similarity ◽  
Mrna Export ◽  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Multicopy Suppressor ◽  
Accessory Factor

ABSTRACT We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 tsmutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast toscMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Δmex67) is synthetically lethal with therae1-167 mutation and accumulates poly(A)+ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of therae1-167 Δmex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149–505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149–505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149–505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

scholarly journals Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rebecca J. Kaddis Maldonado ◽  
Breanna Rice ◽  
Eunice C. Chen ◽  
Kevin M. Tuffy ◽  
Estelle F. Chiari ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Rous Sarcoma Virus ◽  
Nuclear Export ◽  
Live Cell ◽  
Viral Rna ◽  
Wild Type ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome. IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

scholarly journals Regulation and coordination of nuclear envelope and nuclear pore complex assembly

Nucleus ◽  
2013 ◽  
Vol 4 (2) ◽  
pp. 105-114 ◽  
Author(s):  
Michaela Clever ◽  
Yasuhiro Mimura ◽  
Tomoko Funakoshi ◽  
Naoko Imamoto
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Complex Assembly

scholarly journals SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

1998 ◽  
Vol 140 (3) ◽  
pp. 499-509 ◽  
Author(s):  
Michael J. Matunis ◽  
Jian Wu ◽  
Günter Blobel
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Gtpase Activating Protein ◽  
Terminal Domain ◽  
Localization Signal

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

scholarly journals Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

1990 ◽  
Vol 110 (4) ◽  
pp. 883-894 ◽  
Author(s):  
R Reichelt ◽  
A Holzenburg ◽  
E L Buhle ◽  
M Jarnik ◽  
A Engel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Nuclear Envelope ◽  
Three Dimensional ◽  
Structural Features ◽  
Nuclear Pore ◽  
Xenopus Laevis Oocyte ◽  
Three Dimensional Model ◽  
Transmission Electron ◽  
Freeze Dried

Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings," "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 +/- 11 MD; intact NPC without plug, 112 +/- 11 MD; heavy ring, 32 +/- 5 MD; light ring, 21 +/- 4 MD; plug-spoke complex, 66 +/- 8 MD; and spoke complex, 52 +/- 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.

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