scholarly journals Nuclear Localization of the Nipah Virus W Protein Allows for Inhibition of both Virus- and Toll-Like Receptor 3-Triggered Signaling Pathways

2005 ◽  
Vol 79 (10) ◽  
pp. 6078-6088 ◽  
Author(s):  
Megan L. Shaw ◽  
Washington B. Cardenas ◽  
Dmitriy Zamarin ◽  
Peter Palese ◽  
Christopher F. Basler
Keyword(s):  
Nuclear Localization ◽  
Nipah Virus ◽  
Toll Like Receptor ◽  
Promoter Activation ◽  
Double Stranded Rna ◽  
V Protein ◽  
Terminal Domain ◽  
P Gene ◽  
Localization Signal ◽  
Stimulation Of

ABSTRACT The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta interferon (IFN-β) promoter and the IFN regulatory factor 3 (IRF3)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor 3 (TLR3) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 439, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin-α 3 and karyopherin-α 4. Nuclear localization of W protein therefore enables it to target both virus and TLR3 pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKKε. We demonstrate that, when the TLR3 pathway is stimulated, the levels of phosphorylated IRF3 are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF3.

scholarly journals Mechanisms and consequences of Newcastle disease virus W protein subcellular localization in the nucleus or mitochondria

2021 ◽  
Author(s):  
Yanling Yang ◽  
Jia Xue ◽  
Qingyuan Teng ◽  
Xiao Li ◽  
Yawen Bu ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Nuclear Localization ◽  
Newcastle Disease ◽  
Nuclear Export ◽  
Protein Localization ◽  
Dependent Manner ◽  
V Protein ◽  
Terminal Domain ◽  
P Gene

We previously demonstrated that W proteins from different Newcastle disease virus (NDV) strains localize in either the cytoplasm (e.g., NDV strain SG10) or the nucleus (e.g., NDV strain La Sota). To clarify the mechanism behind these cell localization differences, we overexpressed W protein derived from four different NDV strains or W protein associated with different cellular regions in Vero cells. This revealed that the key region for determining W protein localization is 180–227aa. Further experiments found that there is a nuclear export signal (NES) motif in W protein 211–224aa. W protein could be transported into the nucleus via interaction with KPNA1, KPNA2, and KPNA6 in a nuclear localization signal-dependent manner, and W protein containing an NES was transported back to the cytoplasm in a CRM1-independent manner. Interestingly, we observed that the cytoplasm-localized W protein colocalizes with mitochondria. We rescued the NES-deletion W protein NDV strain rSG10-ΔWC/WΔNES using an NDV reverse genetics system and found that the replication ability, virulence, and pathogenicity of an NDV strain were all higher when the W protein cellular localization was in the nucleus rather than the mitochondria. Further experiments revealed that W protein nuclear localization reduced the expression of IFN-β otherwise stimulated by NDV. Our research reveals the mechanism by which NDV W protein becomes localized to different parts of the cell and demonstrates the outcomes of nuclear or cytoplasmic localization both in vitro and in vivo, laying a foundation for subsequent functional studies of the W protein in NDV and other paramyxoviruses. IMPORTANCE In Newcastle disease virus (NDV), the W protein, like the V protein, is a nonstructural protein encoded by the P gene via RNA editing. Compared with V protein, W protein has a common N-terminal domain but a unique C-terminal domain. V protein is known as a key virulence factor and an important interferon antagonist across the family Paramyxoviridae. In contrast, very little is known about the function of NDV W protein, and this limited information is based on studies of the Nipah virus W protein. Here, we investigated the localization mechanism of NDV W protein and its subcellular distribution in mitochondria. We found that W protein localization differences impact IFN-β production, consequently affecting NDV virulence, replication, and pathogenicity. This work provides new insights on the differential localization mechanism of NDV W proteins, along with fundamental knowledge for understanding the functions of W proteins in NDV and other paramyxoviruses.

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 Nuclear Entry Mechanism of Rat PER2 (rPER2): Role of rPER2 in Nuclear Localization of CRY Protein

2001 ◽  
Vol 21 (19) ◽  
pp. 6651-6659 ◽  
Author(s):  
Koyomi Miyazaki ◽  
Miho Mesaki ◽  
Norio Ishida
Keyword(s):  
Nuclear Localization ◽  
Clock Gene ◽  
Clock Genes ◽  
Nuclear Entry ◽  
Cry Protein ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Localization Signal ◽  
Carboxyl Terminal

ABSTRACT Mammalian PERIOD2 protein (PER2) is the product of a clock gene that controls circadian rhythms, because PER2-deficient mice have an arrhythmic phenotype. The nuclear entry regulation of clock gene products is a key step in proper circadian rhythm formation in bothDrosophila and mammals, because the periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutants of rat PER2 (rPER2) to identify the functional nuclear localization signal (NLS) in rPER2. The elimination of putative NLS (residues 778 to 794) from the rPER2 fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (bacterial alkaline phosphatase) translocates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in rPER2. Furthermore, intact rPER2 was preferentially translocated from the cytoplasm to the nucleus when coexpressed with human CRY1 (hCRY1). However, rPER2 mutants lacking a carboxyl-terminal domain could not enter the nucleus even in the presence of hCRY1. In addition, coexpression of the nuclear localization domain (residues 512 to 794) lacking rPER2 and CRY1 changed the subcellular localization of CRY1 from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carboxyl-terminal domain of rPER2 is essential for binding to CRY1. The data suggested that both the rPER2 NLS and carboxyl-terminal CRY binding domain are essential for nuclear entry of the rPER2-CRY1 complex.

scholarly journals Nipah Virus Sequesters Inactive STAT1 in the Nucleus via a P Gene-Encoded Mechanism

2009 ◽  
Vol 83 (16) ◽  
pp. 7828-7841 ◽  
Author(s):  
Michael J. Ciancanelli ◽  
Valentina A. Volchkova ◽  
Megan L. Shaw ◽  
Viktor E. Volchkov ◽  
Christopher F. Basler
Keyword(s):  
Tyrosine Phosphorylation ◽  
Nipah Virus ◽  
Mutant Virus ◽  
Wild Type ◽  
Amino Terminal ◽  
Terminal Domain ◽  
P Gene ◽  
Infected Cells ◽  
Amino Terminal Domain ◽  
In Cells

ABSTRACT The Nipah virus (NiV) phosphoprotein (P) gene encodes the C, P, V, and W proteins. P, V, and W, have in common an amino-terminal domain sufficient to bind STAT1, inhibiting its interferon (IFN)-induced tyrosine phosphorylation. P is also essential for RNA-dependent RNA polymerase function. C is encoded by an alternate open reading frame (ORF) within the common amino-terminal domain. Mutations within residues 81 to 113 of P impaired its polymerase cofactor function, as assessed by a minireplicon assay, but these mutants retained STAT1 inhibitory function. Mutations within the residue 114 to 140 region were identified that abrogated interaction with and inhibition of STAT1 by P, V, and W without disrupting P polymerase cofactor function. Recombinant NiVs were then generated. A G121E mutation, which abrogated inhibition of STAT1, was introduced into a C protein knockout background (Cko) because the mutation would otherwise also alter the overlapping C ORF. In cell culture, relative to the wild-type virus, the Cko mutation proved attenuating but the G121E mutant virus replicated identically to the Cko virus. In cells infected with the wild-type and Cko viruses, STAT1 was nuclear despite the absence of tyrosine phosphorylation. This latter observation mirrors what has been seen in cells expressing NiV W. In the G121E mutant virus-infected cells, STAT1 was not phosphorylated and was cytoplasmic in the absence of IFN stimulation but became tyrosine phosphorylated and nuclear following IFN addition. These data demonstrate that the gene for NiV P encodes functions that sequester inactive STAT1 in the nucleus, preventing its activation and suggest that the W protein is the dominant inhibitor of STAT1 in NiV-infected cells.

scholarly journals Conserved Regions in the Epstein-Barr Virus Leader Protein Define Distinct Domains Required for Nuclear Localization and Transcriptional Cooperation with EBNA2

2000 ◽  
Vol 74 (21) ◽  
pp. 9953-9963 ◽  
Author(s):  
RongSheng Peng ◽  
Jie Tan ◽  
Paul D. Ling
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Epstein Barr Virus ◽  
Intracellular Localization ◽  
Barr Virus ◽  
Conserved Regions ◽  
Localization Signal ◽  
Epstein Barr ◽  
Stimulation Of

ABSTRACT Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood. Recent studies have shown that EBNA-LP may be an important EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introduced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the latency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are located in the repeat domains that are essential for the EBNA2 cooperativity function. In addition, three serine residues are also well conserved in the repeat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA-LP with two repeats, which is the minimal protein able to cooperate with EBNA2. Mutations introduced into CR1a had no effect on EBNA-LP function, while mutations introduced into CR1b resulted in EBNA-LP with slightly decreased activity. Mutations in CR1c and CR2 resulted in proteins that no longer localized exclusively to the nucleus and also had no EBNA2 cooperation activity. Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved serines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP proteins with substantially reduced activity. The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleotides encoding these positively charged amino acid groupings into a cytoplasmic test protein, herpes simplex virus ΔIE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (109 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function independently as a nuclear localization signal. However, a combination of amino acids 29 to 50 resulted in more efficient nuclear localization than with amino acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is essential for EBNA2 cooperativity function. Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explanation for why this isoform has no activity. In addition, two conserved serine residues which are distinct from nuclear import functions are important for EBNA2 cooperativity function.

scholarly journals Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marco Schiavina ◽  
Edoardo Salladini ◽  
Maria Grazia Murrali ◽  
Giancarlo Tria ◽  
Isabella C. Felli ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Chemical Shifts ◽  
Nipah Virus ◽  
Conformational Ensemble ◽  
Genome Replication ◽  
Nmr Studies ◽  
V Protein ◽  
Intrinsically Disordered ◽  
Terminal Domain ◽  
Starting Point

Abstract Using SAXS and NMR spectroscopy, we herein provide a high-resolution description of the intrinsically disordered N-terminal domain (PNT, aa 1–406) shared by the Nipah virus (NiV) phosphoprotein (P) and V protein, two key players in viral genome replication and in evasion of the host innate immune response, respectively. The use of multidimensional NMR spectroscopy allowed us to assign as much as 91% of the residues of this intrinsically disordered domain whose size constitutes a technical challenge for NMR studies. Chemical shifts and nuclear relaxation measurements provide the picture of a highly flexible protein. The combination of SAXS and NMR information enabled the description of the conformational ensemble of the protein in solution. The present results, beyond providing an overall description of the conformational behavior of this intrinsically disordered region, also constitute an asset for obtaining atomistic information in future interaction studies with viral and/or cellular partners. The present study can thus be regarded as the starting point towards the design of inhibitors that by targeting crucial protein–protein interactions involving PNT might be instrumental to combat this deadly virus.

scholarly journals Inhibition of Human Immunodeficiency Virus Type 1 Rev Function by a Dominant-Negative Mutant of Sam68 through Sequestration of Unspliced RNA at Perinuclear Bundles

2001 ◽  
Vol 75 (17) ◽  
pp. 8203-8215 ◽  
Author(s):  
Vanessa B. Soros ◽  
Héctor Valderrama Carvajal ◽  
Stéphane Richard ◽  
Alan W. Cochrane
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Localization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hiv Rna ◽  
Immunodeficiency Virus ◽  
Translation Apparatus ◽  
Localization Signal ◽  
Stimulation Of

ABSTRACT Human immunodeficiency virus (HIV) type 1 encodes an essential protein, Rev, which functions to transport unspliced and singly spliced viral transcripts from the nucleus to the cytoplasm to allow expression of the viral structural proteins. It has previously been reported that Sam68 synergistically stimulates Rev activity (T. Reddy et al., Nat. Med. 5:635–642, 1999). Here we report that the Sam68-like mammalian proteins SLM1 and SLM2 also stimulate Rev activity. Their stimulation ability cannot be attributed to a shuttling property, since Sam68, SLM1, and SLM2 do not display significant shuttling activity alone or in the presence of Rev. In addition, Sam68, SLM1, and SLM2 do not affect the equilibrium between unspliced and completely spliced HIV RNA. The C-terminally truncated Sam68 mutant (Sam68ΔC) previously observed to inhibit the Sam68-mediated stimulation of Rev activity (Reddy et al., 1999) also inhibits SLM1- and SLM2-mediated stimulation of Rev activity. This suggests that the mechanism by which Sam68, SLM1, and SLM2 stimulate Rev activity may be common. Sam68ΔC does not inhibit Rev activity by inhibiting Rev from shuttling between the nucleus and cytoplasm. Inhibition by Sam68ΔC is a consequence of its mislocalization to the cytoplasm, as evidenced by the fact that addition of an exogenous nuclear localization signal to Sam68ΔC restores nuclear localization and stimulation of Rev activity. We demonstrate that Sam68ΔC causes perinuclear accumulation of unspliced HIV env RNA and propose that Sam68ΔC inhibits Rev activity by sequestering Rev-responsive RNA away from the translation apparatus.

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