scholarly journals Characterization of the bipartite nuclear localization signal of protein LANA2 from Kaposi's sarcoma-associated herpesvirus

2003 ◽  
Vol 374 (2) ◽  
pp. 545-550 ◽  
Author(s):  
Cesar MUÑOZ-FONTELA ◽  
Estefanía RODRÍGUEZ ◽  
Cesar NOMBELA ◽  
Javier ARROYO ◽  
Carmen RIVAS
Keyword(s):  
Amino Acid ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Kaposi's Sarcoma ◽  
Molecular Mechanisms ◽  
Kaposi’S Sarcoma ◽  
Bipartite Nuclear Localization Signal ◽  
Localization Signal ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

LANA2 is a nuclear latent protein detected exclusively in Kaposi's sarcoma-associated herpesvirus-infected B cells. The protein inhibits p53-dependent transactivation and apoptosis, suggesting an important role in the transforming activity of the virus. To explore the molecular mechanisms of its nuclear localization, fusion proteins of green fluorescent protein (EGFP) and deletion constructs of LANA2 were expressed in HeLa cells. Only the fragment comprising amino acid residues 355–440 of LANA2 localized in the cell nucleus. This fragment contains two closely located basic domains and forms a putative bipartite nuclear localization signal (NLS). The putative LANA2 NLS was able to target EGFP to the nucleus consistently. Site-directed mutation analyses demonstrated that LANA2 contains a functional bipartite NLS between amino acid positions 367 and 384. In addition, analysis of cells transfected with a cytoplasmic LANA2 mutant revealed that an appropriate subcellular localization may be crucial to regulate p53 activity.

Hairless is translocated to the nucleus via a novel bipartite nuclear localization signal and is associated with the nuclear matrix

2001 ◽  
Vol 114 (2) ◽  
pp. 367-376
Author(s):  
K. Djabali ◽  
V.M. Aita ◽  
A.M. Christiano
Keyword(s):  
Amino Acid ◽  
Hair Follicle ◽  
Nuclear Localization ◽  
Nuclear Matrix ◽  
Nuclear Localization Signal ◽  
Amino Acid Residues ◽  
Bipartite Nuclear Localization Signal ◽  
Hairless Gene ◽  
Fractionation Analysis ◽  
Localization Signal

Hair follicle cycling is an exquisitely regulated and dynamic process consisting of phases of growth, regression and quiescence. The transitions between the phases are governed by a growing number of regulatory proteins, including transcription factors. The hairless (hr) gene encodes a putative transcription factor that is highly expressed in the skin, where it appears to be an essential regulator during the regression of the catagen hair follicle. In hairless mice, as well as humans with congenital atrichia, the absence of hr gene function initiates a premature and abnormal catagen due to a dysregulation of apoptosis and cell adhesion, and defects in the signaling required for hair follicle remodeling. Here, we report structure-function studies of the hairless gene product, in which we identify a novel bipartite nuclear localization signal (NLS) of the form KRA(X13) PKR. Deletion analysis of the mouse hr gene mapped the NLS to amino acid residues 409–427. Indirect immunofluorescence microscopy of cells transiently transfected with hairless-green fluorescent fusion proteins demonstrated that these amino acid residues are necessary and sufficient for nuclear localization. Furthermore, nuclear fractionation analysis revealed that the hr protein is associated with components of the nuclear matrix.

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

2008 ◽  
Vol 83 (6) ◽  
pp. 2531-2539 ◽  
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.

scholarly journals Identification of a Novel Cellular Transcriptional Repressor Interacting with the Latent Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

2003 ◽  
Vol 77 (18) ◽  
pp. 9758-9768 ◽  
Author(s):  
Hong-Yi Pan ◽  
Yan-Jin Zhang ◽  
Xin-Ping Wang ◽  
Jian-Hong Deng ◽  
Fu-Chun Zhou ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Kaposi's Sarcoma ◽  
Promoter Activity ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Northern Blot Hybridization ◽  
Nuclear Localization Signals ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Latent Nuclear Antigen

ABSTRACT The latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpesvirus (KSHV) has an essential role in viral latent infection. LNA maintains the stability of KSHV episomes and modulates the expression of cellular genes. A novel cellular protein KLIP1 was identified to interact with LNA through yeast two-hybrid screening, and confirmed by a glutathione S-transferase pull down assay. Domain mapping showed that KLIP1 interacted with the N-terminal domain of LNA. Northern blot hybridization with a KLIP1 probe identified a major transcript of 1.8 kb and a minor transcript of 2.8 kb. cDNA library screening and 5′-RACE revealed that the major transcript encoded an open-reading-frame of 1,257 bp and had a 5′-untranslated region of 73 nucleotides. The major KLIP1 transcript was ubiquitously present in different cell types examined. A KLIP1 synthetic peptide antibody detected a doublet of 58-kDa and 63-kDa proteins in a Western blot assay. KLIP1 had two putative nuclear localization signals and showed punctate nuclear localization when expressed as a GFP-fusion protein. KLIP1 interacted with LNA in vivo, as demonstrated by coimmunoprecipitation using KSHV-infected cells and colocalization when they were expressed as GFP- and DsRed-fusion proteins, respectively. Consistent with its interaction with LNA, nuclear localization, and possession of two leucine zipper motifs, KLIP1 behaved like a transcriptional factor and repressed herpes simplex virus thymidine kinase (TK) promoter activity in a mammalian one-hybrid assay. In addition, cotransfection with LNA alleviated the transcriptional repression effect of KLIP1 on TK promoter activity. These results suggest that KLIP1 is a new member of cellular transcriptional repressors, and that LNA is involved in deregulating cellular transcription process.

scholarly journals The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Supports Latent DNA Replication in Dividing Cells

2002 ◽  
Vol 76 (22) ◽  
pp. 11677-11687 ◽  
Author(s):  
Jianhong Hu ◽  
Alexander C. Garber ◽  
Rolf Renne
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Dna Binding ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Terminal Repeat ◽  
Nuclear Antigen ◽  
Dividing Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is a multifunctional protein that is consistently expressed in all KSHV-associated malignancies. LANA interacts with a variety of cellular proteins, including the transcriptional cosuppressor complex mSin3 and the tumor suppressors p53 and Rb, thereby regulating viral and cellular gene expression. In addition, LANA is required for maintenance of the episomal viral DNA during latency in dividing cells. Colocalization studies suggest that LANA tethers the viral genome to chromosomes during mitosis. In support of this model, a specific LANA- binding site has recently been identified within the terminal repeat unit, and a chromatin interaction domain was mapped to a short amino acid stretch within the N-terminal domain of LANA. Epstein-Barr virus nuclear antigen 1 (EBNA-1), a functional homologue of LANA, is also required for genome segregation; in addition, EBNA-1 also supports efficient DNA replication of oriP-containing plasmids. By performing short-term replication assays, we demonstrate here for the first time that de novo synthesis of terminal-repeat (TR)-containing plasmids is highly dependent on the presence of LANA. We map the required cis-acting sequences within the TR to a 79-bp region and demonstrate that the DNA-binding domain of LANA is required for this DNA replication activity. Surprisingly, the 233-amino-acid C domain of LANA by itself partially supports replication. Our data show that LANA is a sequence-specific DNA-binding protein that, like EBNA-1, plays an important role in DNA replication and genome segregation. In addition, we show that all necessary cis elements for the origin of replication (ori) function are located within a single TR, suggesting that the putative ori of KSHV is different from those of other gammaherpesviruses, which all contain ori sequences within the unique long sequence outside of their TR. This notion is further strengthened by the unique modular structure of the KSHV TR element.

scholarly journals Importin α/β and Ran-GTP Regulate XCTK2 Microtubule Binding through a Bipartite Nuclear Localization Signal

2004 ◽  
Vol 15 (1) ◽  
pp. 46-57 ◽  
Author(s):  
Stephanie C. Ems-McClung ◽  
Yixian Zheng ◽  
Claire E. Walczak
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Molecular Mechanisms ◽  
Spindle Assembly ◽  
Small Gtpase ◽  
Microtubule Binding ◽  
Bipartite Nuclear Localization Signal ◽  
Importin Α ◽  
Localization Signal ◽  
Ran Gtp

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin α and/or importin β to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin α and β (α/β) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin α/β-mediated regulation of XCTK2 microtubule binding. Our data show that importin α/β directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.

scholarly journals Identification of a short amino acid sequence essential for efficient nuclear targeting of the Kaposi’s sarcoma-associated herpesvirus/human herpesvirus-8 K8 protein

2001 ◽  
Vol 82 (3) ◽  
pp. 507-512 ◽  
Author(s):  
Stéphanie Portes-Sentis ◽  
Evelyne Manet ◽  
Géraldine Gourru ◽  
Alain Sergeant ◽  
Henri Gruffat
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Nuclear Localization Sequence ◽  
Herpesvirus 8 ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

The K8 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 is a member of the bZIP family of transcription factors, which has homology with the Epstein–Barr virus transcription and replication factor, EB1. In this report, we have analysed the subcellular localization of the K8 protein and characterized a 12 amino acid sequence rich in basic residues which is responsible for targeting the protein to the cell nucleus. Furthermore, we show that a K8 mutant lacking the nuclear localization sequence can be directed to the nucleus by co-expression with an intact K8 protein, suggesting that K8 homodimerizes in the cytoplasm of the cell in vivo.

Sign in / Sign up

Export Citation Format

Share Document