scholarly journals SignalsThat Dictate Nuclear Localization of Human Papillomavirus Type 16Oncoprotein E6 in LivingCells

2003 ◽  
Vol 77 (24) ◽  
pp. 13232-13247 ◽  
Author(s):  
Mingfang Tao ◽  
Michael Kruhlak ◽  
Shuhua Xia ◽  
Elliot Androphy ◽  
Zhi-Ming Zheng
Keyword(s):  
Human Papillomavirus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Protein ◽  
Fluorescent Protein ◽  
Present Report ◽  
Cell Cycle Control ◽  
Point Mutations ◽  
Coding Region ◽  
Nuclear Localization Signals

ABSTRACT Human papillomavirus (HPV) type 16 E6 (16E6) is an oncogenic, multifunctional nuclear protein that induces p53 degradation and perturbs normal cell cycle control, leading to immortalization and transformation of infected keratinocytes and epithelial cells. Although it is unclear how 16E6 disrupts the epigenetic profile of host genes, its presence in the nucleus is a key feature. The present report describes intrinsic properties of 16E6 that influence its nuclear import in living cells. When the coding region of full-length 16E6 was inserted in frame into the C terminus of green fluorescent protein (GFP), it effectively prevented the 16E6 pre-mRNA from being spliced and led to the expression of a GFP-E6 fusion which localized predominantly to the nucleus. Further studies identified three novel nuclear localization signals (NLSs) in 16E6 that drive the protein to accumulate in the nucleus. We found that all three NLS sequences are rich in positively charged basic residues and that point mutations in these key residues could abolish the retention of 16E6 in the nucleus as well as the p53 degradation and cell immortalization activities of the protein. When inserted into corresponding regions of low-risk HPV type 6 E6, the three NLS sequences described for 16E6 functioned actively in converting the normally cytoplasmic HPV type 6 E6 into a nuclear protein. The separate NLS sequences, however, appear to play different roles in nuclear import and retention of HPV E6. The discovery of three unique NLS sequences in 16E6 provides new insights into the nuclear association of 16E6 which may reveal other novel activities of this important oncogenic protein.

scholarly journals Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin α

2005 ◽  
Vol 86 (6) ◽  
pp. 1815-1826 ◽  
Author(s):  
O. Guerra-Peraza ◽  
D. Kirk ◽  
V. Seltzer ◽  
K. Veluthambi ◽  
A. C. Schmit ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Fluorescent Protein ◽  
Yellow Mosaic Virus ◽  
Rice Tungro Bacilliform Virus ◽  
Coat Proteins ◽  
Nuclear Localization Signals ◽  
Mungbean Yellow Mosaic Virus

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant pararetro- and geminiviruses. In both these virus types, the multifunctional coat protein (CP) is thought to be involved in this process. Here, a green fluorescent protein tagging approach was used to demonstrate nuclear import of the CPs of Rice tungro bacilliform virus (RTBV) and Mungbean yellow mosaic virus - Vigna (MYMV) in Nicotiana plumbaginifolia protoplasts. In both cases, at least two nuclear localization signals (NLSs) were identified and characterized. The NLSs of RTBV CP are located within both N- and C-terminal regions (residues 479KRPK/497KRK and 744KRK/758RRK), and those of MYMV CP within the N-terminal part (residues 3KR and 41KRRR). The MYMV and RTBV CP NLSs resemble classic mono- and bipartite NLSs, respectively. However, the N-terminal MYMV CP NLS and both RTBV CP NLSs show peculiarities in the number and position of basic residues. In vitro pull-down assays revealed interaction of RTBV and MYMV CPs with the nuclear import factor importin α, suggesting that both CPs are imported into the nucleus via an importin α-dependent pathway. The possibility that this pathway could serve for docking of virions to the nucleus is discussed.

scholarly journals Hsp40/70/110 chaperones adapt nuclear protein quality control to serve cytosolic clients

2018 ◽  
Vol 217 (6) ◽  
pp. 2019-2032 ◽  
Author(s):  
Rupali Prasad ◽  
Chengchao Xu ◽  
Davis T.W. Ng
Keyword(s):  
Quality Control ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Protein ◽  
Protein Quality ◽  
Ubiquitin Proteasome System ◽  
Cytosolic Proteins ◽  
E3 Ligases ◽  
Nuclear Localization Signals ◽  
Ubiquitin Proteasome

Misfolded cytosolic proteins are degraded by the ubiquitin proteasome system through quality control (QC) pathways defined by E3 ubiquitin ligases and associated chaperones. Although they work together as a comprehensive system to monitor cytosolic protein folding, their respective contributions remain unclear. To bridge existing gaps, the pathways mediated by the San1 and Ubr1 E3 ligases were studied coordinately. We show that pathways share the same complement of chaperones needed for substrate trafficking, ubiquitination, and degradation. The significance became clear when Ubr1, like San1, was localized primarily to the nucleus. Appending nuclear localization signals to cytosolic substrates revealed that Ydj1 and Sse1 are needed for substrate nuclear import, whereas Ssa1/Ssa2 is needed both outside and inside the nucleus. Sis1 is required to process all substrates inside the nucleus, but its role in trafficking is substrate specific. Together, these data show that using chaperones to traffic misfolded cytosolic proteins into the nucleus extends the nuclear protein QC pathway to include cytosolic clients.

scholarly journals The L2 Minor Capsid Protein of Low-Risk Human Papillomavirus Type 11 Interacts with Host Nuclear Import Receptors and Viral DNA

2006 ◽  
Vol 80 (16) ◽  
pp. 8259-8262 ◽  
Author(s):  
J. Bordeaux ◽  
S. Forte ◽  
E. Harding ◽  
M. S. Darshan ◽  
K. Klucevsek ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Low Risk ◽  
Classical Pathway ◽  
Viral Dna ◽  
Nuclear Localization Signals ◽  
C Terminus ◽  
Import Receptors

ABSTRACT Analysis of the interactions of low-risk human papillomavirus type 11 (HPV11) L2 with karyopherin β (Kap β) nuclear import receptors revealed that L2 interacted with Kap β1, Kap β2, and Kap β3 and formed a complex with the Kap α2β1 heterodimer. HPV11 L2 contains two nuclear localization signals (NLSs)—in the N terminus and the C terminus—that could mediate its nuclear import via a classical pathway. Each NLS was functional in vivo, and deletion of both of them abolished L2 nuclear localization. Both NLSs interacted with the viral DNA. Thus, HPV11 L2 can interact with several karyopherins and the viral DNA and may enter the nucleus via multiple pathways.

scholarly journals Nuclear localization of the Epstein–Barr virus EBNA3B protein

2006 ◽  
Vol 87 (4) ◽  
pp. 789-793 ◽  
Author(s):  
Anita Burgess ◽  
Marion Buck ◽  
Kenia Krauer ◽  
Tom Sculley
Keyword(s):  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Nuclear Antigen ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Computer Analyses ◽  
Epstein Barr ◽  
Green Fluorescent

The Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

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 PDTM-29. STRUCTURE FUNCTION AND CELL TYPE DETERMINANTS OF C11orf95-RELA DRIVEN TUMORS IN MICE

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi193-vi193
Author(s):  
Jesse Dunnack ◽  
Ericka Randazzo ◽  
Jahmique Caines ◽  
Jame He ◽  
Joseph LoTurco
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Point Mutations ◽  
Cell Types ◽  
Tumor Formation ◽  
Wild Type ◽  
Nuclear Localization Signals ◽  
Transcriptional Activation Domain ◽  
Neuronal Progenitor ◽  
Glial Progenitor

Abstract We used a new mouse model to better understand the cellular and molecular determinants of tumors driven by the C11orf95-RELA fusion. Our approach makes use of in utero electroporation and a binary transposase system to introduce human C11orf95-RELA sequence, wild type and mutant, into neural progenitors, and drive expression of the fusion in different glial and neuronal progenitor cell types. Our results indicate that truncations or point mutations in C11orf95 sequence which interfere with nuclear localization result in a complete loss of tumor-inducing activity. The mutations include truncations of the first 60 amino acids, internal truncations that delete possible mono and bipartite nuclear localization signals, and point mutations of two cysteines and histidines that make up a possible zinc finger domain in C11orf95. Interestingly, all of the mutations that block tumorigenesis also block signal independent nuclear localization of the wild type fusion, without blocking induction of NFKB response genes. We further found that over-expression of the NFKB1 subunit P50 which lacks a transcriptional activation domain significantly inhibits tumor formation by the fusion. In addition, we find that driving expression of the wild type fusion in glial progenitor types using promoters for either astrocytes or oligodendrocytes results in the formation of tumors with transcriptomes displaying significant similarities to human supratentorial ependymoma (ST-EPN), but with distinct patterns depending upon the glial progenitor promoter utilized. In contrast, promoters driving expression selectively in neuron restricted progenitors do not result in the formation of ST-EPN. Together our results reveal three new features of C11orf95-RELA driven tumorigenesis: i) multiple sequences within the C11orf95 domain are required for oncogenic driver activity of the fusion, ii) the P50 subunit of NFKB1 can inhibit fusion induced tumorigenesis, and iii) neuron-restricted precursors are less competent than glia-restricted precursors to form tumors induced by C11orf95-RELA.

005.Efficiency of nuclear import of the chromatin-remodelling factor SRY is critical for sex determination

2005 ◽  
Vol 17 (9) ◽  
pp. 64
Author(s):  
D. A. Jans ◽  
G. Kaur ◽  
I. K. H. Poon ◽  
A. Delluc-Clavieries ◽  
K. M. Wagstaff
Keyword(s):  
Sex Determination ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Sex Reversal ◽  
Nuclear Accumulation ◽  
Missense Mutations ◽  
Nuclear Localization Signals ◽  
Testicular Cells ◽  
Xy Sex Reversal

15% of cases of human XY sex reversal are due to mutations in SRY (sex determining region on the Y chromosome), many of which map to one of SRY’s two independently acting nuclear localization signals (NLSs) flanking its DNA binding domain. The C-terminal NLS (C-NLS) targets SRY to the nucleus through a ‘conventional’ pathway dependent on the nuclear import receptor importin-β (Imp-β). No importin has been shown to bind the N-terminal NLS (N-NLS), but it is known to interact with the Ca2+-binding protein calmodulin (CaM). We examined seven distinct missense mutations in the SRY NLSs from XY sex-reversed human females for effects on nuclear import and ability to interact with CaM/Imp-β1. All mutations were found to result in reduced nuclear localization in transfected testicular cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce SRY nuclear accumulation, indicating a dependence of SRY nuclear import on CaM. Intriguingly, N-NLS mutants were resistant to CDZ’s effects, implying a loss of interaction with CaM; this was confirmed directly by in vitro binding experiments using recombinantly expressed protein. Either impaired CaM or Imp-β1 binding can thus be the basis of sex-reversal in human patients. Our results implicate a CaM-dependent nuclear import pathway for SRY mediated by the N-NLS that, together with the C-NLS, is required to achieve threshold levels of SRY in the nucleus for male sex determination.

scholarly journals Nonclassical nuclear localization signals mediate nuclear import of CIRBP

2020 ◽  
Vol 117 (15) ◽  
pp. 8503-8514 ◽  
Author(s):  
Benjamin Bourgeois ◽  
Saskia Hutten ◽  
Benjamin Gottschalk ◽  
Mario Hofweber ◽  
Gesa Richter ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Interaction ◽  
Stress Granule ◽  
Rich Region ◽  
Nuclear Localization Signals ◽  
Cargo Proteins

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)–rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

scholarly journals Epstein–Barr virus nuclear antigen 3A contains six nuclear-localization signals

2006 ◽  
Vol 87 (10) ◽  
pp. 2879-2884 ◽  
Author(s):  
Marion Buck ◽  
Anita Burgess ◽  
Roslynn Stirzaker ◽  
Kenia Krauer ◽  
Tom Sculley
Keyword(s):  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Viral Proteins ◽  
Site Directed Mutagenesis ◽  
Nuclear Antigen ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Epstein Barr

The Epstein–Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein–Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix. By using enhanced green fluorescent protein-tagged deletion mutants of EBNA3A in combination with site-directed mutagenesis, an additional five functional nuclear-localization signals have been identified in the EBNA3A protein. Two of these (aa 63–66 and 375–381) were computer-predicted, whilst the remaining three (aa 394–398, 573–578 and 598–603) were defined functionally in this study.

scholarly journals Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo

2006 ◽  
Vol 175 (4) ◽  
pp. 579-593 ◽  
Author(s):  
Benjamin L. Timney ◽  
Jaclyn Tetenbaum-Novatt ◽  
Diana S. Agate ◽  
Rosemary Williams ◽  
Wenzhu Zhang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Yeast Cells ◽  
Limiting Factor ◽  
Nuclear Pore ◽  
Nuclear Localization Signals ◽  
The Poor ◽  
Cytoplasmic Proteins

Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump–leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm.

Sign in / Sign up

Export Citation Format

Share Document