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

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.

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The L2 Minor Capsid Protein of Human Papillomavirus Type 16 Interacts with a Network of Nuclear Import Receptors

Journal of Virology ◽

10.1128/jvi.78.22.12179-12188.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12179-12188 ◽

Cited By ~ 55

Author(s):

Medha S. Darshan ◽

John Lucchi ◽

Emily Harding ◽

Junona Moroianu

Keyword(s):

Human Papillomavirus ◽

Nuclear Import ◽

Capsid Proteins ◽

Permeabilized Cells ◽

Nuclear Localization Signals ◽

Human Papillomavirus Type 16 ◽

C Terminus ◽

Binding Data ◽

Import Receptors ◽

Productive Phase

ABSTRACT The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapβ2 and Kapβ3. Moreover, binding of Ran-GTP to either Kapβ2 or Kapβ3 inhibits its interaction with L2, suggesting that the Kapβ/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapα2β1 heterodimer via interaction with the Kapα2 adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapα2β1 heterodimers, Kapβ2, or Kapβ3. Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapα2β1 heterodimers, Kapβ2, and Kapβ3.

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

The Journal of Cell Biology ◽

10.1083/jcb.200608141 ◽

2006 ◽

Vol 175 (4) ◽

pp. 579-593 ◽

Cited By ~ 100

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.

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

Journal of Virology ◽

10.1128/jvi.77.24.13232-13247.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13232-13247 ◽

Cited By ~ 36

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.

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The N-terminus of fission yeast DNA polymerase alpha contains a basic pentapeptide that acts in vivo as a nuclear localization signal.

Molecular Biology of the Cell ◽

10.1091/mbc.6.12.1697 ◽

1995 ◽

Vol 6 (12) ◽

pp. 1697-1705 ◽

Cited By ~ 6

Author(s):

D Bouvier ◽

G Baldacci

Keyword(s):

Fission Yeast ◽

Dna Polymerase ◽

Nuclear Localization ◽

Nuclear Import ◽

Terminal Sequence ◽

Nuclear Localization Signals ◽

Dna Polymerase Alpha ◽

Localization Signal ◽

Necessary And Sufficient

The N-terminal sequence of the catalytic subunit of fission yeast DNA polymerase alpha (pol alpha) contains two putative nuclear localization signals (NLS). To check the functionality of these signals in vivo, the N-terminal sequence was experimentally divided into three amino acid blocks, two of which contain a distinct presumptive NLS. Each block was deleted, either individually or in combination with one of the two others. The deleted gene products were expressed in fission yeast, and assayed by indirect immunofluorescence for their aptitude to localize to the cell nucleus. Block II, which contains the putative NLS pentapeptide 97RKRKK, was both necessary and sufficient to promote nuclear import of pol alpha, as well as of a pyruvate kinase fusion protein. Precise excision of the NLS pentapeptide from block II inhibited the nuclear import of pol alpha, thus confirming the role of this sequence as the functional NLS of the fission yeast enzyme.

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Importin β Can Mediate the Nuclear Import of an Arginine-Rich Nuclear Localization Signal in the Absence of Importin α

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1218 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1218-1225 ◽

Cited By ~ 158

Author(s):

Diana Palmeri ◽

Michael H. Malim

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Protein Import ◽

T Antigen ◽

Classical Pathway ◽

Permeabilized Cell ◽

Cell Leukemia Virus Type ◽

Nuclear Localization Signals ◽

Human T Cell ◽

Importin Α

ABSTRACTThe import of proteins into the nucleus is dependent oncis-acting targeting sequences, nuclear localization signals (NLSs), and members of the nuclear transport receptor (importin-β-like) superfamily. The most extensively characterized import pathway, often termed the classical pathway, is utilized by many basic-type (lysine-rich) NLSs and requires an additional component, importin α, to serve as a bridge between the NLS and the import receptor importin β. More recently, it has become clear that a variety of proteins enter the nucleus via alternative import receptors and that their NLSs bind directly to those receptors. By using the digitonin-permeabilized cell system for protein import in vitro, we have defined the import pathway for the Rex protein of human T-cell leukemia virus type 1. Interestingly, the arginine-rich NLS of Rex uses importin β for import but does so by a mechanism that is importin α independent. Based on the ability of the Rex NLS to inhibit the import of the lysine-rich NLS of T antigen and of both NLSs to be inhibited by the domain of importin α that binds importin β (the IBB domain), we infer that the Rex NLS interacts with importin β directly. In addition, and in keeping with other receptor-mediated nuclear import pathways, Rex import is dependent on the integrity of the Ran GTPase cycle. Based on these results, we suggest that importin β can mediate the nuclear import of arginine-rich NLSs directly, or lysine-rich NLSs through the action of importin α.

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The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors

Virology ◽

10.1016/j.virol.2010.07.045 ◽

2010 ◽

Vol 407 (1) ◽

pp. 100-109 ◽

Cited By ~ 7

Author(s):

Zachary Piccioli ◽

Courtney H. McKee ◽

Anna Leszczynski ◽

Zeynep Onder ◽

Erin C. Hannah ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Binding Domain ◽

Low Risk ◽

Zinc Binding ◽

Import Receptors ◽

Zinc Binding Domain

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Nuclear Import of the Stem–Loop Binding Protein and Localization during the Cell Cycle

Molecular Biology of the Cell ◽

10.1091/mbc.e04-11-1023 ◽

2005 ◽

Vol 16 (6) ◽

pp. 2960-2971 ◽

Cited By ~ 24

Author(s):

Judith A. Erkmann ◽

Eric J. Wagner ◽

Jian Dong ◽

Yanping Zhang ◽

Ulrike Kutay ◽

...

Keyword(s):

Cell Cycle ◽

Nuclear Localization ◽

Nuclear Import ◽

Binding Protein ◽

Histone Mrna ◽

Stem Loop ◽

Histone Mrnas ◽

Import Receptors ◽

Mrna Binding

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem–loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impα/Impβ and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impα/Impβ binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impα/Impβ pathway contributes to SLBP nuclear import in HeLa cells.

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Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

Molecular Endocrinology ◽

10.1210/me.2004-0334 ◽

2005 ◽

Vol 19 (7) ◽

pp. 1884-1892 ◽

Cited By ~ 39

Author(s):

Helena Sim ◽

Kieran Rimmer ◽

Sabine Kelly ◽

Louisa M. Ludbrook ◽

Andrew H. A. Clayton ◽

...

Keyword(s):

Y Chromosome ◽

Nuclear Import ◽

High Mobility ◽

Sex Reversal ◽

High Mobility Group ◽

Missense Mutations ◽

Nuclear Localization Signals ◽

Xy Sex Reversal

Abstract The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-β binding to the C-terminal NLS (c-NLS), whereas in others, importin-β recognition is normal, suggesting the existence of an importin-β-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

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