Mutational Analysis of Bovine Leukemia Virus Rex: Identification of a Dominant-Negative Inhibitor

ABSTRACT The Rex proteins of the delta-retroviruses act to facilitate the export of intron-containing viral RNAs. The Rex of bovine leukemia virus (BLV) is poorly characterized. To gain a better understanding of BLV Rex, we generated a reporter assay to measure BLV Rex function and used it to screen a series of point and deletion mutations. Using this approach, we were able to identify the nuclear export signal of BLV Rex. Further, we identified a dominant-negative form of BLV Rex. Protein localization analysis revealed that wild-type BLV Rex had a punctate nuclear localization and was associated with nuclear pores. In contrast, the dominant-negative BLV Rex mutation had a diffuse nuclear localization and no nuclear pore association. Overexpression of the dominant-negative BLV Rex altered the localization of the wild-type protein. This dominant-negative derivative of BLV Rex could be a useful tool to test the concept of intracellular immunization against viral infection in a large animal model.

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SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

The Journal of Cell Biology ◽

10.1083/jcb.140.3.499 ◽

1998 ◽

Vol 140 (3) ◽

pp. 499-509 ◽

Cited By ~ 316

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.

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Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.02209-08 ◽

2008 ◽

Vol 83 (6) ◽

pp. 2531-2539 ◽

Cited By ~ 22

Author(s):

Xiaojuan Li ◽

Fanxiu Zhu

Keyword(s):

Subcellular Localization ◽

Nuclear Localization ◽

Nuclear Export ◽

Kaposi's Sarcoma ◽

Nuclear Export Signal ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Wild Type ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.

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A Nuclear Export Signal in Kap95p Is Required for Both Recycling the Import Factor and Interaction with the Nucleoporin GLFG Repeat Regions of Nup116p and Nup100p

The Journal of Cell Biology ◽

10.1083/jcb.137.4.797 ◽

1997 ◽

Vol 137 (4) ◽

pp. 797-811 ◽

Cited By ~ 68

Author(s):

M. Kathryn Iovine ◽

Susan R. Wente

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Protein A ◽

Nuclear Export Signal ◽

Nuclear Pore ◽

Primary Role ◽

Localization Signal ◽

Nuclear Localization Signal Receptor ◽

Export Signal

During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A–tagged Nup116p or protein A–tagged Nup100p complexes. The protein A–tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.

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CDK Phosphorylation of a Novel NLS-NES Module Distributed between Two Subunits of the Mcm2-7 Complex Prevents Chromosomal Rereplication

Molecular Biology of the Cell ◽

10.1091/mbc.e05-05-0412 ◽

2005 ◽

Vol 16 (10) ◽

pp. 5026-5039 ◽

Cited By ~ 72

Author(s):

Muluye E. Liku ◽

Van Q. Nguyen ◽

Audrey W. Rosales ◽

Kaoru Irie ◽

Joachim J. Li

Keyword(s):

Nuclear Localization ◽

Nuclear Export ◽

Protein Localization ◽

Nuclear Export Signal ◽

Replication Initiation ◽

Nuclear Localization Signals ◽

Cdk Phosphorylation ◽

In Cis ◽

Net Export ◽

Cdk Regulation

Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (pre-RCs) at replication origins to prevent inappropriate rereplication. In Saccharomyces cerevisiae, one of these mechanisms promotes the net nuclear export of a pre-RC component, the Mcm2-7 complex, during S, G2, and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raises the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.

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The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants

Journal of Cell Science ◽

10.1242/jcs.114.3.589 ◽

2001 ◽

Vol 114 (3) ◽

pp. 589-597 ◽

Cited By ~ 2

Author(s):

J.K. Hood ◽

W.W. Hwang ◽

P.A. Silver

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Localization ◽

Nuclear Export ◽

Fluorescent Protein ◽

Nuclear Export Signal ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Cell Cycle Regulatory Proteins ◽

Localization Signal ◽

Green Fluorescent

The cyclin-dependent kinase Cdc28p associates with the cyclin Clb2p to induce mitosis in the yeast Saccharomyces cerevisiae. Several cell cycle regulatory proteins have been shown to require specific nuclear transport events to exert their regulatory functions. Therefore, we investigated the subcellular localization of wild-type Clb2p and several mutant versions of the protein using green fluorescent protein (GFP) fusion constructs. Wild-type Clb2p is primarily nuclear at all points of the cell. A point mutation in a potential leucine-rich nuclear export signal (NES) enhances the nuclear localization of the protein, and delta-yrb2 cells exhibit an apparent Clb2p nuclear export defect. Clb2p contains a bipartite nuclear localization signal (NLS), and its nuclear localization requires the alpha and beta importins (Srp1p and Kap95p), as well as the yeast Ran GTPase and its regulators. Deletion of the Clb2p NLS causes increased cytoplasmic localization of the protein, as well as accumulation at the bud neck. These data indicate that Clb2p exists in multiple places in the yeast cell, possibly allowing Cdc28p to locally phosphorylate substrates at distinct subcellular sites.

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Novel REST Truncation Mutations Causing Hereditary Gingival Fibromatosis

Journal of Dental Research ◽

10.1177/0022034521996620 ◽

2021 ◽

pp. 002203452199662

Author(s):

J.T. Chen ◽

C.H. Lin ◽

H.W. Huang ◽

Y.P. Wang ◽

P.C. Kao ◽

...

Keyword(s):

Nuclear Localization ◽

Genetic Disorder ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Wild Type ◽

Negative Effect ◽

Localization Signal ◽

Gingival Fibromatosis

Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

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The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

Journal of Virology ◽

10.1128/jvi.73.2.1293-1301.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1293-1301 ◽

Cited By ~ 21

Author(s):

Kazunori Inabe ◽

Masako Nishizawa ◽

Shigeru Tajima ◽

Kazuyoshi Ikuta ◽

Yoko Aida

Keyword(s):

Viral Entry ◽

Envelope Protein ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Transient Transfection ◽

Viral Envelope ◽

Tyrosine Residue ◽

Wild Type ◽

Viral Envelope Protein

ABSTRACT The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

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OsWRKY62 and OsWRKY76 interact with Importin α1s for Negative Regulation of Defensive Responses in Nucleus

10.21203/rs.3.rs-636974/v1 ◽

2021 ◽

Author(s):

Xiaohui Xu ◽

Han Wang ◽

Jiqin Liu ◽

Shuying Han ◽

Miaomiao Lin ◽

...

Keyword(s):

Amino Acids ◽

Nuclear Localization ◽

Nuclear Export ◽

Nuclear Translocation ◽

Nuclear Export Signal ◽

Defense Responses ◽

Nuclear Localization Signals ◽

Defensive Responses ◽

Export Signal ◽

Positively Charged

Abstract Background: OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation?Results: In this study, we characterized they interacted with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαDIBB1a lacking the auto-inhibitory importin β-binding domain. OsIMαDIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal. Similarly, we found that OsIMαDIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1 with an extra nuclear export signal compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae.Conclusion: These results indicated that OsWRKY62 localization is a consequence of competition binding between rice importins and exportins. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

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Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1571-1582.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1571-1582 ◽

Cited By ~ 49

Author(s):

Shrikesh Sachdev ◽

Sriparna Bagchi ◽

Donna D. Zhang ◽

Angela C. Mings ◽

Mark Hannink

Keyword(s):

Nuclear Import ◽

Nuclear Export ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Nuclear Pore ◽

Transport Pathway ◽

Repeat Domain ◽

Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

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The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.79.21.13463-13472.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13463-13472 ◽

Cited By ~ 69

Author(s):

Danso Ako-Adjei ◽

Marc C. Johnson ◽

Volker M. Vogt

Keyword(s):

Nuclear Export ◽

Leukemia Virus ◽

Particle Diameter ◽

Particle Morphology ◽

Structural Protein ◽

Wild Type ◽

Virus Like Particles ◽

Gag Proteins ◽

Hiv 1

ABSTRACT The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

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