Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.
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PMID:The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. 942 62

Influenza A viruses are important worldwide pathogens in humans and different animal species. The functions of most of the ten different viral proteins of this negative-strand RNA virus have been well elucidated. However, little is known about the virus-induced intracellular signalling events that support viral replication. The Raf/MEK/ERK cascade is the prototype of mitogen-activated protein (MAP) kinase cascades and has an important role in cell growth, differentiation and survival. Investigation of the function of this pathway has been facilitated by the identification of specific inhibitors such as U0126, which blocks the cascade at the level of MAPK/ERK kinase (MEK). Here we show that infection of cells with influenza A virus leads to biphasic activation of the Raf/MEK/ERK cascade. Inhibition of Raf signalling results in nuclear retention of viral ribonucleoprotein complexes (RNPs), impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production. Thus, signalling through the mitogenic cascade seems to be essential for virus production and RNP export from the nucleus during the viral life cycle.
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PMID:Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade. 1123 81

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.
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PMID:Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs. 1131 64

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.
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PMID:Influenza B and C virus NEP (NS2) proteins possess nuclear export activities. 1146 9

The cellular nuclear transport machinery relies on the assembly of specialized transport complexes between soluble transport receptors, transport substrates, and additional accessory proteins. This study focuses on the structural characteristics of influenza virus protein NS2 (NEP), which interacts with the nuclear export machinery during viral replication, and has been proposed to act as an adapter molecule between the nuclear export machinery and the viral ribonucleoprotein complex. For this purpose, we have purified recombinant NS2 under nondenaturing conditions, and have investigated its structure and aggregation state using optical spectroscopy, differential scanning calorimetry, as well as hydrodynamic techniques. Our results indicate that isolated NS2 exists as a monomer in solution, and adopts a compact, but very flexible conformation, which shows characteristics of the molten globule state under near physiological conditions. Proteolytic sensitivity suggests that, despite its overall plasticity, the structure of NS2 is heterogeneous. While the C terminus of the protein adopts a relatively rigid conformation, its N terminus, which is recognized by the nuclear export machinery, exists in a highly mobile and exposed state. It is proposed that the flexibility observed in the nuclear export domain of NS2 is an important element in the recognition of substrate proteins by the nuclear export machinery.
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PMID:Structural plasticity in influenza virus protein NS2 (NEP). 1175 4

During influenza virus infection, viral ribonucleoproteins (vRNPs) are replicated in the nucleus and must be exported to the cytoplasm before assembling into mature viral particles. Nuclear export is mediated by the cellular protein Crm1 and putatively by the viral protein NEP/NS2. Proteolytic cleavage of NEP defines an N-terminal domain which mediates RanGTP-dependent binding to Crm1 and a C-terminal domain which binds to the viral matrix protein M1. The 2.6 A crystal structure of the C-terminal domain reveals an amphipathic helical hairpin which dimerizes as a four-helix bundle. The NEP-M1 interaction involves two critical epitopes: an exposed tryptophan (Trp78) surrounded by a cluster of glutamate residues on NEP, and the basic nuclear localization signal (NLS) of M1. Implications for vRNP export are discussed.
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PMID:Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). 1297 Jan 77

The influenza virus genome replicates and forms a viral ribonucleoprotein complex (vRNP) with nucleoprotein (NP) and RNA polymerases in the nuclei of host cells. vRNP is then exported into the cytoplasm for viral morphogenesis at the cell membrane. Matrix protein 1 (M1) and nonstructural protein 2/nuclear export protein (NS2/NEP) work in the nuclear export of vRNP by associating with it. It was previously reported that influenza virus production was inhibited in Madin-Darby canine kidney (MDCK) cells cultured at 41 degrees C because nuclear export of vRNP was blocked by the dissociation of M1 from vRNP (A. Sakaguchi, E. Hirayama, A. Hiraki, Y. Ishida, and J. Kim, Virology 306:244-253, 2003). Previous data also suggested that a certain protein(s) synthesized only at 41 degrees C inhibited the association of M1 with vRNP. The potential of heat shock protein 70 (HSP70) as a candidate obstructive protein was investigated. Induction of HSP70 by prostaglandin A1 (PGA1) at 37 degrees C caused the suppression of virus production. The nuclear export of viral proteins was inhibited by PGA1, and M1 was not associated with vRNP, indicating that HSP70 prevents M1 from binding to vRNP. An immunoprecipitation assay showed that HSP70 was bound to vRNP, suggesting that the interaction of HSP70 with vRNP is the reason for the dissociation of M1. Moreover, NS2 accumulated in the nucleoli of host cells cultured at 41 degrees C, showing that the export of NS2 was also disturbed at 41 degrees C. However, NS2 was exported normally from the nucleus, irrespective of PGA1 treatment at 37 degrees C, suggesting that HSP70 does not influence NS2.
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PMID:Heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus ribonucleoprotein complex. 1472 81

The NS2 (NEP) protein of influenza A virus contains a highly conserved nuclear export signal (NES) motif in its amino-terminal region (12ILMRMSKMQL21, A/WSN/33), which is thought to be required for nuclear export of viral ribonucleoprotein complexes (vRNPs) mediated by a cellular export factor, CRM1. However, simultaneous replacement of three hydrophobic residues in the NES with alanine does not affect NS2 (NEP) binding to CRM1, although the virus with these mutations is not viable. To determine the extent of sequence conservation required by the NS2 (NEP) NES for its export function during viral replication, we randomly introduced mutations by degenerative mutagenesis into the region of NS cDNA encoding the NS2 (NEP) NES and then attempted to generate mutant viruses containing these alterations by reverse genetics. Sequence analysis of the recovered viruses showed that although some of the mutants possessed amino acids other than those conserved in the NES, hydrophobicity within this motif was maintained. Nuclear export of vRNPs representing all of the mutant viruses was completely inhibited in the presence of a CRM1 inhibitor, leptomycin B, as was the transport of wild-type virus, indicating that the CRM1-mediated pathway is responsible for the nuclear export of both wild-type and mutant vRNPs. The vRNPs of some of the mutant viruses were exported in a delayed manner, resulting in limited viral growth in cell culture and in mice. These results suggest that the NES motif may be an attractive target for the introduction of attenuating mutations in the production of live vaccine viruses.
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PMID:Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence. 1533 47

Influenza virus acquires a lipid raft-containing envelope by budding from the apical surface of epithelial cells. Polarised budding involves specific sorting of the viral membrane proteins, but little is known about trafficking of the internal virion components. We show that during the later stages of virus infection, influenza nucleoprotein (NP) and polymerase (the protein components of genomic ribonucleoproteins) localised to apical but not lateral or basolateral membranes, even in cell types where haemagglutinin was found on all external membranes. Other cytosolic components of the virion either distributed throughout the cytoplasm (NEP/NS2) or did not localise solely to the apical plasma membrane in all cell types (M1). NP localised specifically to the apical surface even when expressed alone, indicating intrinsic targeting. A similar proportion of NP associated with membrane fractions in flotation assays from virus-infected and plasmid-transfected cells. Detergent-resistant flotation at 4 degrees C suggested that these membranes were lipid raft microdomains. Confirming this, cholesterol depletion rendered NP detergent-soluble and furthermore, resulted in its partial redistribution throughout the cell. We conclude that NP is independently targeted to the apical plasma membrane through a mechanism involving lipid rafts and propose that this helps determine the polarity of influenza virus budding.
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PMID:Lipid raft-dependent targeting of the influenza A virus nucleoprotein to the apical plasma membrane. 1552 99

During influenza virus infection, transcription and replication of the viral RNA take place in the cell nucleus. Directly after entry in the nucleus the viral ribonucleoproteins (RNPs, the viral subunits containing vRNA, nucleoprotein and the viral polymerase) are tightly associated with the nuclear matrix. Here, we have analysed the binding of RNPs, M1 and NS2/NEP proteins to purified nucleosomes, reconstituted histone octamers and purified single histones. RNPs and M1 both bind to the chromatin components but at two different sites, RNP to the histone tails and M1 to the globular domain of the histone octamer. NS2/NEP did not bind to nucleosomes at all. The possible consequences of these findings for nuclear release of newly made RNPs and for other processes during the infection cycle are discussed.
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PMID:Interaction of influenza virus proteins with nucleosomes. 1566 Nov 64


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