scholarly journals The Nuclear Pore Complex: A Target for NS3 Protease of Dengue and Zika Viruses

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 583 ◽  
Author(s):  
Luis Adrián De Jesús-González ◽  
Margot Cervantes-Salazar ◽  
José Manuel Reyes-Ruiz ◽  
Juan Fidel Osuna-Ramos ◽  
Carlos Noe Farfán-Morales ◽  
...  
Keyword(s):  
Serine Protease ◽  
Nuclear Pore Complex ◽  
Denv Infection ◽  
Nuclear Pore ◽  
Cytoplasmic Transport ◽  
Ns3 Protein ◽  
Nuclear Ring ◽  
Main Regulator ◽  
Cellular Components ◽  
Ns3 Protease

During flavivirus infection, some viral proteins move to the nucleus and cellular components are relocated from the nucleus to the cytoplasm. Thus, the integrity of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC), was evaluated during infection with dengue virus (DENV) and Zika virus (ZIKV). We found that while during DENV infection the integrity and distribution of at least three nucleoporins (Nup), Nup153, Nup98, and Nup62 were altered, during ZIKV infection, the integrity of TPR, Nup153, and Nup98 were modified. In this work, several lines of evidence indicate that the viral serine protease NS2B3 is involved in Nups cleavage. First, the serine protease inhibitors, TLCK and Leupeptin, prevented Nup98 and Nup62 cleavage. Second, the transfection of DENV and ZIKV NS2B3 protease was sufficient to inhibit the nuclear ring recognition detected in mock-infected cells with the Mab414 antibody. Third, the mutant but not the active (WT) protease was unable to cleave Nups in transfected cells. Thus, here we describe for the first time that the NS3 protein from flavivirus plays novel functions hijacking the nuclear pore complex, the main controller of the nuclear-cytoplasmic transport.

scholarly journals The Nonstructural Proteins 3 and 5 from Flavivirus Modulate Nuclear-Cytoplasmic Transport and Innate Immune Response Targeting Nuclear Proteins

2018 ◽  
Author(s):  
Margot Cervantes-Salazar ◽  
Ana L. Gutiérrez-Escolano ◽  
José M. Reyes-Ruiz ◽  
Rosa M. del Angel
Keyword(s):  
Immune Response ◽  
Nuclear Pore Complex ◽  
Innate Immune ◽  
Nuclear Proteins ◽  
Nuclear Pore ◽  
Cytoplasmic Transport ◽  
Infected Cells ◽  
Main Regulator ◽  
Mature Mrnas ◽  
Replicative Cycle

ABSTRACTViruses hijack cellular proteins and components to be replicated in the host cell and to evade the immune response. Although flaviviruses have a cytoplasmic replicative cycle, some viral proteins such as the capsid (C) and the RNA dependent RNA polymerase, NS5, can reach the nucleus of the infected cells. Considering the important roles of NS5 in viral replication and in the control of the immune response, and its striking presence in the nucleus, the possible functions of this protein in some mechanisms orchestrated by the nucleus was analyzed. We isolated and identified nuclear proteins that interact with NS5; one of them, the DEAD-box RNA helicase DDX5 is relocated to the cytoplasm and degraded during infection with DENV, which correlates with its function in IFN dependent response. Since DDX5 and many other proteins are relocated from the nucleus to the cytoplasm during flavivirus infection, the integrity and function of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC) was evaluated. We found that during DENV and ZIKV infection nucleoporins (NUPs) such as TPR, Nup153, Nup98, and Nup62 were cleavaged/degraded. The protease NS2B-NS3 induces NUPs degradation and it causes a dramatic inhibition of mature mRNAs export to the cytoplasm but not the export of DDX5 protein, which is dependent on NS5. Here we describe for the first time that the NS3 and NS5 proteins from flavivirus play novel functions hijacking the NPC and some nuclear proteins relevant in triggering immune response pathways, inducing a favorable environment for viral replication.IMPORTANCEViruses, as intracellular obligate parasites, hijack cellular components to enter and replicate in infected cells. Remarkably, in many cases, viruses hijack molecules with crucial functions for the cells. Here it is described how RNA viruses such as DENV and ZIKV, with a cytoplasmic replicative cycle, use NS3 and NS5, two of their unique non-structural proteins with enzymatic activity, to modulate nuclear-cytoplasmic transport. We found that NS3 disrupts the nuclear pore complex, the main regulator in nuclear-cytoplasmic transport, causing a strong reduction in the amount of mature mRNAs in the cytoplasm and an inhibition in innate immune response. Additionally, NS5 induces the relocation of nuclear proteins to the cytoplasm such as DDX5, involved in immune response, which is later degraded by NS3. These findings allow the understanding of crucial mechanisms that viruses use to deal with the control of the immune response to grant the production of new viral particles.

Structure, assembly and interactions of the nuclear lamina and the nuclear pore complex

1992 ◽  
Vol 50 (1) ◽  
pp. 490-491
Author(s):  
N. Panté ◽  
M. Jarnik ◽  
E. Heitlinger ◽  
U. Aebi
Keyword(s):  
Nuclear Pore Complex ◽  
Divalent Cations ◽  
Nuclear Lamina ◽  
Dynamic Structure ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Cytoplasmic Filaments ◽  
Radial Spokes ◽  
Nuclear Ring ◽  
Central Plug

The nuclear pore complex (NPC) is a ∼120 MD supramolecular machine implicated in nucleocytoplasmic transport, that is embedded in the double-membraned nuclear envelope (NE). The basic framework of the ∼120 nm diameter NPC consists of a 32 MD cytoplasmic ring, a 66 MD ‘plug-spoke’ assembly, and a 21 MD nuclear ring. The ‘central plug’ seen in en face views of the NPC reveals a rather variable appearance indicating that it is a dynamic structure. Projecting from the cytoplasmic ring are 8 short, twisted filaments (Fig. 1a), whereas the nuclear ring is topped with a ‘fishtrap’ made of 8 thin filaments that join distally to form a fragile, 30-50 nm distal diameter ring centered above the NPC proper (Fig. 1b). While the cytoplasmic filaments are sensitive to proteases, they as well as the nuclear fishtraps are resistant to RNase treatment. Removal of divalent cations destabilizes the distal rings and thereby opens the fishtraps, addition causes them to reform. Protruding from the tips of the radial spokes into perinuclear space are ‘knobs’ that might represent the large lumenal domain of gp210, a membrane-spanning glycoprotein (Fig. 1c) which, in turn, may play a topogenic role in membrane folding and/or act as a membrane-anchoring site for the NPC. The lectin wheat germ agglutinin (WGA) which is known to recognize the ‘nucleoporins’, a family of glycoproteins having O-linked N-acetyl-glucosamine, is found in two locations on the NPC (Fig. 1. d-f): (i) whereas the cytoplasmic filaments appear unlabelled (Fig. 1d&e), WGA-gold labels sites between the central plug and the cytoplasmic ring (Fig. le; i.e., at a radius of 25-35 nm), and (ii) it decorates the distal ring of the nuclear fishtraps (Fig. 1, d&f; arrowheads).

scholarly journals Structural interaction between the nuclear pore complex and a specific translocating RNP particle.

1995 ◽  
Vol 129 (5) ◽  
pp. 1205-1216 ◽  
Author(s):  
H Mehlin ◽  
B Daneholt ◽  
U Skoglund
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Initial Contact ◽  
Chironomus Tentans ◽  
Central Channel ◽  
Balbiani Ring ◽  
Salivary Gland Cells ◽  
Larval Salivary Gland ◽  
Nuclear Ring ◽  
Precise Area

The transport of Balbiani ring (BR) premessenger RNP particles in the larval salivary gland cells of the dipteran Chironomus tentans can be followed using electron microscopy. A BR RNP particle consists of an RNP ribbon bent into a ringlike structure. Upon translocation through the nuclear pore complex (NPC), the ribbon is straightened and enters the central channel of the NPC with the 5' end of the transcript in the lead. The translocating ribbon is likely to interact with the central channel but, in addition, the remaining portion of the ribbon ring makes contact with the periphery of the NPC. To determine the nature of this latter interaction, we have now studied the connections between the RNP particle and the border of the NPC during different stages of translocation using electron microscope tomography. It was observed that the 3' terminal domain of the ribbon always touches the nuclear ring of the NPC, but the precise area of contact is variable. Sometimes also a region on the opposite side of the ribbon ring reaches the nuclear ring. The pattern of contacts could be correlated to the stage of translocation, and it was concluded that the particle-nuclear ring interactions reflect a rotation of the ribbon ring in front of the central channel, the rotation being secondary to the successive translocation of the ribbon through the channel. The particle's mode of interaction with the NPC suggests that the initial contact between the 5' end domain of the ribbon and the entrance to the central channel is probably crucial to accomplish the ordered translocation of the premessenger RNP particle through the NPC.

scholarly journals Proteasomes tether to two distinct sites at the nuclear pore complex

2017 ◽  
Vol 114 (52) ◽  
pp. 13726-13731 ◽  
Author(s):  
Sahradha Albert ◽  
Miroslava Schaffer ◽  
Florian Beck ◽  
Shyamal Mosalaganti ◽  
Shoh Asano ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Electron Tomography ◽  
Nuclear Pore ◽  
Cellular Environment ◽  
Subtomogram Averaging ◽  
Substrate Processing ◽  
Cellular Components ◽  
Transport Of Macromolecules

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.

SARS coronavirus protein nsp1 disrupts localization of Nup93 from the nuclear pore complex

2019 ◽  
Vol 97 (6) ◽  
pp. 758-766 ◽  
Author(s):  
Garret N. Gomez ◽  
Fareeha Abrar ◽  
Maya P. Dodhia ◽  
Fabiola G. Gonzalez ◽  
Anita Nag
Keyword(s):  
Gene Expression ◽  
Nuclear Pore Complex ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Nuclear Lamina ◽  
Host Gene ◽  
Nuclear Pore ◽  
Host Gene Expression ◽  
Nonstructural Protein 1 ◽  
Cytoplasmic Transport

Severe acute respiratory syndrome coronavirus nonstructural protein 1 (nsp1) is a key factor in virus-induced down-regulation of host gene expression. In infected cells, nsp1 engages in a multipronged mechanism to inhibit host gene expression by binding to the 40S ribosome to block the assembly of translationally competent ribosome, and then inducing endonucleolytic cleavage and the degradation of host mRNAs. Here, we report a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts the nuclear–cytoplasmic transport of biomolecules. We identified members of the nuclear pore complex from the nsp1-associated protein assembly and found that the expression of nsp1 in HEK cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation, while the nuclear lamina remains unperturbed. Consistent with its role in host shutoff, nsp1 alters the nuclear–cytoplasmic distribution of an RNA binding protein, nucleolin. Our results suggest that nsp1, alone, can regulate multiple steps of gene expression including nuclear–cytoplasmic transport.

scholarly journals The Nuclear Pore Complex is a Key Target of Viral Proteases to Promote Viral Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 706
Author(s):  
Luis Adrián De Jesús-González ◽  
Selvin Palacios-Rápalo ◽  
José Manuel Reyes-Ruiz ◽  
Juan Fidel Osuna-Ramos ◽  
Carlos Daniel Cordero-Rivera ◽  
...  
Keyword(s):  
Viral Replication ◽  
Nuclear Pore Complex ◽  
Viral Proteins ◽  
Critical Groups ◽  
Nuclear Pore ◽  
Viral Mrnas ◽  
Viral Proteases ◽  
Cytoplasmic Transport ◽  
Nuclear Factors ◽  
Mrna Maturation

Various viruses alter nuclear pore complex (NPC) integrity to access the nuclear content favoring their replication. Alteration of the nuclear pore complex has been observed not only in viruses that replicate in the nucleus but also in viruses with a cytoplasmic replicative cycle. In this last case, the alteration of the NPC can reduce the transport of transcription factors involved in the immune response or mRNA maturation, or inhibit the transport of mRNA from the nucleus to the cytoplasm, favoring the translation of viral mRNAs or allowing access to nuclear factors necessary for viral replication. In most cases, the alteration of the NPC is mediated by viral proteins, being the viral proteases, one of the most critical groups of viral proteins that regulate these nucleus–cytoplasmic transport changes. This review focuses on the description and discussion of the role of viral proteases in the modification of nucleus–cytoplasmic transport in viruses with cytoplasmic replicative cycles and its repercussions in viral replication.

scholarly journals 8 Å structure of the nuclear ring of the Xenopus laevis nuclear pore complex solved by cryo-EM and AI

2021 ◽  
Author(s):  
He Ren ◽  
Linhua Tai ◽  
Yun Zhu ◽  
Xiaojun Huang ◽  
Fei Sun ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Pore Complex ◽  
Protein Complexes ◽  
Complex Structure ◽  
Nucleocytoplasmic Transport ◽  
Structural Features ◽  
Nuclear Pore ◽  
Electron Microscopic ◽  
Nuclear Ring ◽  
Great Advance

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the nuclear ring (NR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the NR, including the Y complexes and flanking components. In this most comprehensive and accurate model to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. Each NR asymmetric subunit contains two copies of Y complexes, one copy of Nup205 that connects the Y complexes to the neighbouring complex, one copy of ELYS that stabilizes the long arm region of the inner Y complex, and one copy of newly identified Nup93 that forms a bridge across the stems of Y complexes. These in-depth structural features represent a great advance in understanding the assembly of NPCs.

scholarly journals A Drosophila Tpr protein homolog is localized both in the extrachromosomal channel network and to nuclear pore complexes

1997 ◽  
Vol 110 (8) ◽  
pp. 927-944 ◽  
Author(s):  
G. Zimowska ◽  
J.P. Aris ◽  
M.R. Paddy
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Periphery ◽  
Coiled Coil ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Channel Network ◽  
Nuclear Interior ◽  
Cytoplasmic Transport ◽  
Complex Protein ◽  
Pore Complexes

Here we report structural, molecular, and biochemical characterizations of Bx34, a Drosophila melanogaster nuclear coiled-coil protein which is localized to extrachromosomal and extranucleolar spaces in the nuclear interior and which is homologous to the mammalian nuclear pore complex protein Tpr. In the nuclear interior, Bx34 is excluded from chromosomes and the nucleolus and generally localizes to regions between these structures and the nuclear periphery. This distribution matches the ‘extrachromosomal channel network’ described previously. In the nuclear periphery, Bx34 localizes on or near nuclear pore complexes. Biochemically, Bx34 isolates exclusively with the nuclear matrix fraction. The Bx34 cDNA sequence predicts a large protein (262 kDa) with two distinct structural domains. The Bx34 N-terminal 70% (180 kDa) is predicted to form an extended region of coiled-coil, while the C-terminal 30% (82 kDa) is predicted to be unstructured and acidic. Bx34 shows moderate sequence identity over its entire length to the mammalian nuclear pore complex protein ‘Tpr’ (28% amino acid identity and 50% similarity). Furthermore, several of the sequence motifs and biochemical similarities between Bx34 and Tpr are sufficiently striking that it is likely that Bx34 and Tpr are functionally related. The Bx34 gene exists in a single copy in region 48C of chromosome 2R. The localization of coiled-coil Bx34 to both the nuclear interior and nuclear pore complexes and its sequence similarity to a known nuclear pore complex protein leads to speculations about a role for Bx34 in nucleo-cytoplasmic transport which we can test using molecular genetic approaches.

40 KNOCKOUT OF GOAT NUCLEOPORIN 155 (NUP155) GENE USING CRISPR/Cas9 SYSTEMS

2014 ◽  
Vol 26 (1) ◽  
pp. 134 ◽  
Author(s):  
S. Hu ◽  
Z. Wang ◽  
I. Polejaeva
Keyword(s):  
Single Cell ◽  
Nuclear Pore Complex ◽  
Gene Knockout ◽  
Strand Break ◽  
Transgenic Animal ◽  
Nuclear Pore ◽  
Cytoplasmic Transport ◽  
Research Initiative ◽  
Pcr Products ◽  
Transgenic Animal Models

Nucleoporin 155 (NUP155) is a major component of the nuclear pore complex involved in the cellular nucleo-cytoplasmic transport. NUP155 knockout in mice leads to atrial fibrillation and early sudden cardiac death. Due to the small size of the heart, functional electrophysiology is problematic in transgenic mice. Large transgenic animal models have the potential to offer insights into the role of nuclear pore complex in cardiovascular disease. The goal of this study was to generate NUP155 knockout goats by using the CRISPR/Cas9 systems. Two CRISPR target sites were chosen in the first exon of the goat NUP155 gene and CRISPR knockout vectors were constructed. Surveyor assay showed that the 2 CRISPR vectors efficiently induced a double-strand break with cleavage efficiencies of 30 to 50% in transfected goat fibroblast cells. Single cell-derived clones were obtained by limiting dilution of the transfected cells in 48-well plates. In total, 23 single cell clones were isolated and subjected to Surveyor assay followed by sequencing the PCR products spanning the target site. Five of 23 single cell colonies had heterozygous mutations and one carried biallelic modifications of NUP155. The heterozygous cells will be used to produce NUP155± goats by somatic cell nuclear transfer. In conclusion, we demonstrated that CRISPR/Cas9 systems are highly efficient for gene knockout applications in goats. This work was supported by the Utah Science Technology and Research Initiative, Utah Multidisciplinary Arrhythmia Consortium and Utah Agricultural Experiment Station project #1100.

scholarly journals Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process.

1990 ◽  
Vol 111 (4) ◽  
pp. 1571-1582 ◽  
Author(s):  
N Bataillé ◽  
T Helser ◽  
H M Fried
Keyword(s):  
Nuclear Pore Complex ◽  
Wheat Germ Agglutinin ◽  
Xenopus Oocyte ◽  
Wheat Germ ◽  
Ribosomal Subunit ◽  
Oocyte Nucleus ◽  
Nuclear Pore ◽  
Xenopus Laevis Oocyte ◽  
Ribosomal Subunits ◽  
Cytoplasmic Transport

To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures.

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