scholarly journals Two nuclear localization signals regulate intracellular localization of the Duck enteritis virus UL13 protein

2020 ◽  
Author(s):  
Linjiang Yang ◽  
Xixia Hu ◽  
Anchun Cheng ◽  
Mingshu Wang ◽  
Renyong Jia ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Intracellular Localization ◽  
Duck Enteritis Virus ◽  
Significant Information ◽  
Nuclear Localization Signals ◽  
Dependent Process ◽  
Importin Α ◽  
Localization Signal ◽  
Enteritis Virus

Abstract Background UL13 multifunctional tegument protein duck enteritis virus (DEV) is predicted as conserved herpesvirus protein kinase (CHPK); however, little is known about its subcellular localization signal. Results In this study, by transfection with two predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein (EGFP), two bipartite nuclear localization signals (NLS) were identified. We found that the NLSs block its nuclear import using ivermectin and proved that nuclear localization signal of DEV UL13 is a classical importin α/β-dependent process. And we constructed the DEV UL13 mutant strain, with the NLSs of DEV UL13 deleted, to explore whether it can affect the virus replication Conclusions The DEV pUL13 amino acids 4 to 7 and 90 to 96 was predicted, and proved that this nuclear import occurs via the classical importin α/β-dependent process. We also found NLSs of pUL13 have no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results would provide significant information for the biological function of pUL13 during DEV infection.

scholarly journals Two nuclear localization signals regulate intracellular localization of the Duck enteritis virus UL13 protein

2020 ◽  
Author(s):  
Linjiang Yang ◽  
Xixia Hu ◽  
Anchun Cheng ◽  
Mingshu Wang ◽  
Renyong Jia ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Duck Enteritis Virus ◽  
Significant Information ◽  
Nuclear Localization Signals ◽  
Dependent Process ◽  
Importin Α ◽  
Localization Signal ◽  
Enteritis Virus

Abstract Background UL13 multifunctional tegument protein of duck enteritis virus (DEV) is predicted as protein kinase (CHPK); however, little is known concerning its subcellular localization signal. Results In this study, by transfection with two predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein (EGFP), two bipartite nuclear localization signal (NLS) was identified. We identified the nuclear localization signals (NLSs) that control its nuclear importing using fluorescence assay and proved that nuclear localization of DEV UL13 is a classical importin α/β-dependent process. And we constructed the mutant DEV, with the NLSs of UL13 deleted, to explore whether it will affect the replication of virus particles. Conclusions DEV UL13 protein is directed by amino acids 4 to 7 and 90 to 96, and proved that this nuclear import occurs via the classical importin α/β-dependent process. And Entry nucleus of UL13 protein has no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results would provide significant information for the stud y of the biological function of UL13 during DEV infection.

scholarly journals Two nuclear localization signals regulate intracellular localization of the duck enteritis virus UL13 protein

2021 ◽  
Vol 100 (1) ◽  
pp. 26-38
Author(s):  
Linjiang Yang ◽  
Xixia Hu ◽  
Anchun Cheng ◽  
Mingshu Wang ◽  
Renyong Jia ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Intracellular Localization ◽  
Duck Enteritis Virus ◽  
Nuclear Localization Signals ◽  
Enteritis Virus

scholarly journals Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1

2013 ◽  
Vol 94 (6) ◽  
pp. 1335-1342 ◽  
Author(s):  
Qian Li ◽  
Zhenfeng Zhang ◽  
Zhenhua Zheng ◽  
Xianliang Ke ◽  
Huanle Luo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Human Bocavirus ◽  
Structural Protein ◽  
Nuclear Localization Signals ◽  
Importin Α ◽  
Localization Signal ◽  
Bovine Parvovirus

Human bocavirus (HBoV), closely related to canine minute virus (MVC) and bovine parvovirus (BPV), is a new member of the Bocavirus genus within the Parvoviridae family. The non-structural protein NP1 of HBoV is a nuclear localized protein and plays an important role in DNA replication as well as in the evasion of host innate immunity. In the current study, we provide the first evidence that NP1 possesses a non-classical nuclear localization signal (ncNLS) (amino acids 7–50). Embedded within this ncNLS is a classical bipartite nuclear localization signal (cNLS) (amino acids 14–28), capable of transporting a heterologous cytoplasmic protein β-galactosidase fusion protein (β-gal-EGFP) to the nucleus via the classical importin α/β1-mediated pathway. Amino acids 7–50 containing the cNLS and the ncNLS of NP1 or full-length NP1 interact with importin α1, importin β1 and importin β1Δ, which lacks the importin α binding domain, indicating that the nuclear import of NP1 is through both conventional importin α/β1 heterodimer- and non-classical importinß1-mediated pathways. Given that the arrangement of a cNLS embedded within an ncNLS is unusual in viral proteins, our data together reveal a novel molecular mechanism underlying the nuclear import of HBoV NP1, providing a basis for further understanding its biological function.

scholarly journals Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Aris Haryanto
Keyword(s):  
Fusion Protein ◽  
Nuclear Import ◽  
Recombinant Dna ◽  
Core Protein ◽  
Intracellular Localization ◽  
Full Length ◽  
Nuclear Pore ◽  
E Coli ◽  
Importin Α ◽  
Localization Signal

Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

scholarly journals TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Breanna L. Rice ◽  
Matthew S. Stake ◽  
Leslie J. Parent
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Rous Sarcoma Virus ◽  
Binding Domain ◽  
Nuclear Entry ◽  
Gag Protein ◽  
Nuclear Localization Signals ◽  
Rous Sarcoma ◽  
Importin Α ◽  
Sarcoma Virus

ABSTRACT Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLSs) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-β to mediate nuclear entry. The second NLS (TNPO3), which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), which are known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functions of importin-α/importin-β and importin-11 have been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 directly binds to Gag and mediates its nuclear entry. To our surprise, this interaction did not require the cargo-binding domain (CBD) of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3, including SR domain-containing splicing factors and tRNAs that reenter the nucleus. These results suggest that RSV hijacks this host nuclear import pathway using a unique mechanism, potentially allowing other cargo to simultaneously bind TNPO3. IMPORTANCE RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here, we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the CBD of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

scholarly journals Identification of the Nuclear Localization Signal Region of Duck Enteritis Virus UL14 and Its Interaction with VP16

Intervirology ◽  
2016 ◽  
Vol 59 (4) ◽  
pp. 187-196
Author(s):  
FangJie Li ◽  
YuWei Zhang ◽  
Shun Chen ◽  
MingShu Wang ◽  
RenYong Jia ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Duck Enteritis Virus ◽  
Signal Region ◽  
Localization Signal ◽  
Enteritis Virus

scholarly journals The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals

1999 ◽  
Vol 19 (2) ◽  
pp. 1210-1217 ◽  
Author(s):  
Ray Truant ◽  
Bryan R. Cullen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signals ◽  
Immunodeficiency Virus ◽  
Importin Α

ABSTRACT Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin α (Imp α) NLS receptor. Imp α is in turn bound by importin β (Imp β), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp α and instead bind directly to Imp β. Using in vitro nuclear import assays, we demonstrate that Imp α is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp β proved both sufficient and necessary, in that other β-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp β for Imp α, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp β is also similar to Imp α binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp β and that therefore function independently of Imp α.

scholarly journals Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights

2013 ◽  
Vol 69 (12) ◽  
pp. 2495-2505 ◽  
Author(s):  
Gergely Róna ◽  
Mary Marfori ◽  
Máté Borsos ◽  
Ildikó Scheer ◽  
Enikő Takács ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nucleocytoplasmic Transport ◽  
Wild Type ◽  
Post Translational Modification ◽  
Nuclear Localization Signals ◽  
M Phase ◽  
Importin Α ◽  
Localization Signal

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α–wild-type and the importin-α–hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.

scholarly journals Nuclear Localization Signal and Protein Context both Mediate Importin α Specificity of Nuclear Import Substrates

2006 ◽  
Vol 26 (23) ◽  
pp. 8697-8709 ◽  
Author(s):  
Beate Friedrich ◽  
Christina Quensel ◽  
Thomas Sommer ◽  
Enno Hartmann ◽  
Matthias Köhler
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Import ◽  
Binding Studies ◽  
Vitro Binding ◽  
Importin Α ◽  
Localization Signal ◽  
Experimental Approaches

ABSTRACT The “classical” nuclear protein import pathway depends on importin α and importin β. Importin α binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin β-dependent import pathway. In humans, only one importin β is known to interact with importin α, while six α importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular α importins. Whether the NLS is sufficient to mediate importin α specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin α3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin α binding and transport.

scholarly journals Human mRNA Cap Methyltransferase: Alternative Nuclear Localization Signal Motifs Ensure Nuclear Localization Required for Viability

2005 ◽  
Vol 25 (7) ◽  
pp. 2644-2649 ◽  
Author(s):  
Beth Shafer ◽  
Chun Chu ◽  
Aaron J. Shatkin
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Localization Signals ◽  
Truncation Mutant ◽  
Capping Enzyme ◽  
Importin Α ◽  
Localization Signal ◽  
Targeting Signals ◽  
Sirna Knockdown ◽  
Interfering Rna

ABSTRACT A characteristic feature of gene expression in eukaryotes is the addition of a 5′-terminal 7-methylguanine cap (m7GpppN) to nascent pre-mRNAs in the nucleus catalyzed by capping enzyme and cap methyltransferase. Small interfering RNA (siRNA) knockdown of cap methyltransferase in HeLa cells resulted in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP-tetramethylrhodamine nick end labeling assay, demonstrating the importance of mRNA 5′-end methylation for mammalian cell viability. Nuclear localization of cap methyltransferase is mediated by interaction with importin-α, which facilitates its transport and selective binding to transcripts containing 5′-terminal GpppN. The methyltransferase 96-144 region has been shown to be necessary for importin binding, and N-terminal fusion of this sequence to nonnuclear proteins proved sufficient for nuclear localization. The targeting sequence was narrowed to amino acids 120 to 129, including a required 126KRK. Although full-length methyltransferase (positions 1 to 476) contains the predicted nuclear localization signals 57RKRK, 80KKRK, 103KKRKR, and 194KKKR, mutagenesis studies confirmed functional motifs only at positions 80, 103, and the previously unrecognized 126KRK. All three motifs can act as alternative nu clear targeting signals. Expression of N-truncated cap methyltransferase (120 to 476) restored viability of methyltransferase siRNA knocked-down cells. However, an enzymatically active 144-476 truncation mutant missing the three nuclear localization signals was mostly cytoplasmic and ineffective in preventing siRNA-induced loss of viability.

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