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Results for “Nuclear Localization Signals”

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PubMed1993

A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells.

Permissiveness of the host cell to productive infection by oncoretroviruses is cell-cycle dependent, and nuclear localization of viral nucleoprotein preintegration complexes will occur only after cells have passed through mitosis. In contrast, establishment of an integrated provirus after infection by the lentivirus HIV-1 is independent of host cell proliferation. The ability of HIV-1 to replicate in non-dividing cells is partly accounted for by the karyophilic properties of the viral preintegration complex which, after virus infection, is actively transported to the host cell nucleus. Here we report that the gag matrix protein of HIV-1 contains a nuclear localization sequence which, when conjugated to a heterologous protein, directs its nuclear import. In addition, HIV-1 mutants containing amino-acid substitutions in this nuclear localization signal integrate and replicate within dividing but not growth-arrested cells, and thus display a phenotype more representative of an oncoretrovirus.

Amino Acid Sequence↗

PubMed2002

Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus.

In dengue virus (DEN) particles, the core protein is a structural protein of the nucleocapsid. The core protein is known to be present in the nucleus of DEN-infected cells but there have been conflicting reports as to whether it is also present in the nucleolus. To clarify this, the intracellular location of the core protein was examined using a monoclonal antibody, 15B11, which was produced in this study. Immunofluorescence staining with this antibody demonstrated that the core protein first appeared in the cytoplasm and then in the nuclei and nucleoli of infected cells. Nuclear localization of the core protein was determined to be independent of other DEN proteins, since recombinant core proteins still entered the nuclei and nucleoli of cells transfected with only the core protein gene. Three putative nuclear localization signal motifs have been predicted to be present on the core protein. Deletion of the first one (KKAR), located at aa 6-9, and mutation of the second one (KKSK), located at aa 73-76, did not eliminate the nuclear localization property of the core protein. The third motif with a bipartite structure, RKeigrmlnilnRRRR, located at aa 85-100, was determined to be responsible for the nuclear localization of the core protein, since the core protein without this motif was located exclusively in the cytoplasm of DEN-infected cells and that this motif mediated nuclear localization of a normally cytoplasmic protein.

Amino Acid Sequence↗

PubMed1995

Cytoplasmic localization of a mutant M(r) 160,000 topoisomerase II alpha is associated with the loss of putative bipartite nuclear localization signals in a drug-resistant human lung cancer cell line.

Many clinically important antineoplastic agents exert their cytotoxicity through interaction with the M(r) 170,000 topoisomerase II alpha, an essential nuclear enzyme. Resistance to these agents has been associated frequently with either a decrease in the levels of topoisomerase II alpha or a qualitative change that alters the interaction of this enzyme with a drug or DNA. Using a VP-16-selected lung cancer cell line, H209/V6, we have identified a third resistance mechanism which involves an aberrant subcellular location of the topoisomerase II alpha isoenzyme. We have shown previously that H209/V6 cells express two topoisomerase II alpha mRNAs (6.1 and 4.8 kilobases) but only a single catalytically active protein which has a M(r) of 160,000 and is located primarily in the cytoplasm (Mirski et al., Cancer Res., 53: 4866-4873, 1993; Feldhoff et al., Cancer Res., 54: 756-762, 1994). In the present study we have determined that this mutant M(r) 160,000 topoisomerase II alpha is encoded by the shorter 4.8-kilobase mRNA. The sequencing of reverse transcriptase-PCR products from H209/V6 cells and subsequent Northern blot analyses showed that a sequence of 988 nucleotides from the 3'-coding and 3'-noncoding region of the normal topoisomerase II alpha is absent from the 4.8-kilobase mRNA. This shorter mRNA is predicted to encode a topoisomerase II alpha protein that no longer contains the 109 COOH-terminal amino acids of the normal enzyme but instead contains 34 new amino acids encoded by a sequence that was previously in the 3'-noncoding region of the mRNA. Confirmation that the COOH terminus of topoisomerase II alpha is no longer present in the M(r) 160,000 protein in H209/V6 cells was obtained by immunoblot analysis. Sequence analyses indicate that 3 putative bipartite nuclear localization signals in the M(r) 160,000 protein are disrupted or lost. Our results suggest that sequences within the COOH-proximal domain of human topoisomerase II alpha serve an important nuclear localization function.

Base Sequence↗

PubMed2001

Characterization of mouse metastasis-associated gene 2: genomic structure, nuclear localization signal, and alternative potentials as transcriptional activator and repressor.

We characterized the mouse metastasis-associated gene 2 product (mmta2), which is a homolog of the metastasis-associated gene 1 product (MTA1). We revealed that the mmta2 gene spanned approximately 10 kb and was separated into 18 exons. The transcription start site of mmta2 was located 377 bp upstream from the putative initiation codon. The subcellular location of the mmta2 protein was the nucleus, and nuclear localization signals were identified in the region between amino acids 456 and 497. To obtain data on the transcription-regulating potential of mmta2, various constructs containing different portions were fused to the GAL4 DNA-binding domain. The entire mmta2 protein repressed the transcription of the reporter genes, whereas treatment with a histone deacetylase inhibitor, trichostatin A (TSA), led to recovery from the repression and to transcriptional activation. However, the N terminus of mmta2 activated transcriptional activity in the absence of TSA. These results suggest that mmta2 has the potential to both repress and activate gene transcription and that its transcription repression activity might be related to histone deacetylation.

3T3 Cells↗

PubMed2001

Simian virus 40 Vp1 DNA-binding domain is functionally separable from the overlapping nuclear localization signal and is required for effective virion formation and full viability.

A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1DeltaC58 pentamer bound DNA with a K(d) of 1.8 x 10(-9) M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1DeltaC17 had comparable apparent K(d)s of 5.3 x 10(-9) to 7.3 x 10(-9) M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1DeltaC58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1. 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1DeltaC58 level for NLSm1. 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209-8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlapping SV40 (NO-SV40) DNA NLSm1, NLSm2, and NLSm1. 2 replicated normally following transfection into host cells and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1. 2 were nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.

Animals↗

PubMed2006

Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions.

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two approximately 2.6 kb DNAs or a monopartite genome of approximately 3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3-20 of TYLCV CP (CP-NLS) or aa 84-106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA-NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin alpha, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin alpha.

Active Transport, Cell Nucleus↗

PubMed2005

Identification of a novel nuclear localization signal in Tbx1 that is deleted in DiGeorge syndrome patients harboring the 1223delC mutation.

DiGeorge syndrome (DGS) is the most common human chromosomal deletion syndrome and is frequently associated with deletions on chromosome 22q11. Approximately 17% of patients with the phenotypic features of this syndrome have no detectable genomic deletion. Animal studies using mouse models have implicated Tbx1 as a critical gene within the commonly deleted region, and several mutations in TBX1 have been identified recently in non-deleted patients, including missense and frameshift mutations. The mechanisms by which these mutations cause disease have remained unclear. We have identified a previously unrecognized and novel nuclear localization signal (NLS) at the C-terminus of Tbx1 that is deleted by the 1223delC mutation, thus explaining the mechanism of disease in these patients. This NLS is conserved across species, among a subfamily of T-box proteins including Brachyury and Tbx10, and among additional nuclear proteins. By providing functional data to indicate loss-of-function produced by the 1223delC TBX1 mutation, our results provide strong support for the conclusion that TBX1 mutations can cause DGS in humans.

Amino Acid Motifs↗

PubMed2000

Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal.

Peptide nucleic acids (PNA) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. In this study, a PNA construct was employed as an anti-gene agent in intact cells in culture. The cell lines studied were derived from Burkitt's lymphomas (BL) that presented a translocated and hyperexpressed c-myc oncogene. A 17-mer anti-myc PNA, complementary to a unique sequence located at the beginning of the second exon of the oncogene, and was covalently linked at its N terminus to a nuclear localization signal (NLS) (PNA-myc(wt)-NLS). When BL cells were exposed to PNA-myc(wt)-NLS, the anti-gene construct was localized predominantly in the cell nuclei and a rapid consequent downregulation of c-myc expression occurred. Under these conditions, both completion of a productive cell cycle and apoptosis were inhibited.

Antigens, Polyomavirus Transforming↗

PubMed2000

Interferon regulatory factor subcellular localization is determined by a bipartite nuclear localization signal in the DNA-binding domain and interaction with cytoplasmic retention factors.

The transduction of type I interferon signals to the nucleus relies on activation of a protein complex, ISGF3, involving two signal transducers and activators of transcription (STAT) proteins, STAT1 and STAT2, and the interferon (IFN) regulatory factor (IRF) protein, p48/ISGF3gamma. The STAT subunits are cytoplasmically localized in unstimulated cells and rapidly translocate to the nucleus of IFN-stimulated cells, but the p48/ISGF3gamma protein is found in both the nucleus and the cytoplasm, regardless of IFN stimulation. Here, we demonstrate that p48 is efficiently and constitutively targeted to the nucleus. Analysis of the subcellular distribution of green fluorescent protein-p48 fragments indicates that p48 contains a bipartite nuclear retention signal within its amino-terminal DNA-binding domain. This signal is preserved in two other IRF proteins involved in immune responses, ICSBP and IRF4. Mutations to clustered basic residues within amino acids 50-100 of p48 or IRF4 disrupt their nuclear accumulation, and DNA-binding ability is not required for nuclear targeting. This is the only example of a nuclear localization signal for any ISGF3 component and assigns a second function to the IRF DNA-binding domain. We also demonstrate that the nuclear distribution of p48 is dramatically altered by coexpression of the STAT2 protein, indicating that STAT2 forms a cytoplasmic complex with p48, overriding the intrinsic p48 nuclear targeting. Retention by STAT2 may serve to regulate the activity of free p48 and/or guarantee that cytoplasmic pools of preassociated STAT2:p48 are available for rapid activation of the IFN response. These findings suggest that analogous mechanisms may exist for regulating the distribution of other IRF proteins.

3T3 Cells↗

PubMed2004

Identification and characterization of nuclear localization signals of CaMKP-N.

Calmodulin-dependent protein kinase phosphatase (CaMKP) and CaMKP-N dephosphorylate and regulate multifunctional Ca(2+)/calmodulin-dependent protein kinases. The enzymatic properties of CaMKP-N and CaMKP resemble each other, whereas their localizations are different. CaMKP-N is localized in the nucleus, whereas CaMKP is localized in the cytosol. In the present study, the nuclear localization signals (NLSs) of CaMKP-N were identified and characterized. CaMKP-N contains two NLSs, NLS1 and NLS2, at the C-terminus. A cluster of basic residues in the NLSs is important for their function. NLS1 and NLS2 function independently, but mutagenesis analysis suggests that these NLSs interact with each other.

Amino Acid Sequence↗

PubMed2006

A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo.

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a DNA-binding, transcriptional co-activator that participates in HIV-1 integration site targeting. Using complementary approaches, we determined the mechanisms of LEDGF/p75 DNA-binding in vitro and chromatin-association in living cells. The binding of highly-purified, recombinant protein was assayed by surface plasmon resonance (SPR) and electrophoretic mobility gel shift. Neither assay revealed evidence for sequence-specific DNA-binding. Residues 146-197 spanning the nuclear localization signal (NLS) and two AT-hook motifs mediated non-specific DNA-binding, and DNA-binding deficient mutants retained the ability to efficiently stimulate HIV-1 integrase activity in vitro. Chromatin-association was assessed by visualizing the localization of EGFP fusion proteins in interphase and mitotic cells. Although a conserved N-terminal PWWP domain was not required for binding to condensed mitotic chromosomes, its deletion subtly affected the nucleoplasmic distribution of the protein during interphase. A dual AT-hook mutant associated normally with chromatin, yet when the mutations were combined with NLS changes or deletion of the PWWP domain, chromatin-binding function was lost. As the PWWP domain did not readily bind free DNA in vitro, our results indicate that chromatin-association is primarily affected through DNA-binding, with the PWWP domain likely contributing a protein interaction to the overall affinity of LEDGF/p75 for human chromatin.

AT-Hook Motifs↗

PubMed2000

Corf, the Rev/Rex homologue of HTDV/HERV-K, encodes an arginine-rich nuclear localization signal that exerts a trans-dominant phenotype when mutated.

Regulation of nucleo-cytoplasmic export of viral transcripts by a viral protein (Rev/Rex) is a characteristic feature in the replication cycle of complex retroviruses. We recently reported that the endogenous retrovirus family HTDV/HERV-K encodes a protein, Corf, that is a cellular Counterpart of Rev/Rex function and thus a new component of nucleo-cytoplasmic pathways. In HTDV/HERV-K-expressing cells, Corf is localized within the nucleoli. Here we describe the nuclear localization signal (NLS) of the Corf protein. Mutations in the NLS lead to cytoplasmic accumulation of the mutated protein and abrogate Corf function in a trans-dominant way.

Amino Acid Sequence↗

PubMed1994

The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes.

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), are unusual among retroviruses in their ability to infect nondividing cells. The matrix proteins of several lentiviruses contain a short stretch of amino acids reminiscent of known nuclear localization signals. In HIV-1, this motif has been shown to function as a nuclear targeting sequence when conjugated to a heterologous protein, and to permit the active nuclear import of the HIV-1 preintegration complex in growth-arrested cells. In the present work, mutations were introduced in the matrix nuclear localization region of T-cell- and macrophage-tropic HIV-1 clones. The resulting viral mutants replicated with normal or even accelerated kinetics in dividing cells, including activated peripheral blood lymphocytes. However, in sharp contrast with wild-type virus, the mutants could not grow efficiently in terminally differentiated macrophages or establish a stable and inducible infection intermediate in unstimulated peripheral blood lymphocytes. Because macrophages represent a major viral reservoir in vivo, and because at any given time most T cells in the body are quiescent, these results strongly suggest that the karyophilic properties of the matrix protein are critical for the spread of the virus in HIV-infected individuals, and consequently for AIDS pathogenesis.

Cell Division↗

PubMed2002

Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells.

The integrase of the phage PhiC31 recombines an attP site in the phage genome with a chromosomal attB site of its Streptomyces host. We have utilized the integrase-mediated reaction to achieve episomal and genomic deletion of a reporter gene in mammalian cells, and provide the first comparison of its efficiency with other recombinases in a new assay system. This assay demonstrated that the efficiency of PhiC31-integrase is significantly enhanced by the C-terminal, but not the N-terminal, addition of a nuclear localization signal and becomes comparable with that of the widely used Cre/loxP system. Furthermore, we found that the improved FLP recombinase, FLPe, exhibits only 10% recombination activity on chromosomal targets as compared with Cre, whereas the Anabaena derived XisA recombinase is essentially inactive in mammalian cells. These results provide the first demonstration that a nuclear localisation signal and its position within a recombinase can be important for its efficiency in mammalian cells and establish the improved PhiC31-integrase as a new tool for genome engineering.

3T3 Cells↗

PubMed2005

Transcription inhibition of oncogenic KRAS by a mutation-selective peptide nucleic acid conjugated to the PKKKRKV nuclear localization signal peptide.

Mutations in the Kirsten ras (KRAS) gene are present in almost all pancreatic adenocarcinomas, and one common mutation is at codon 12: GGT (Gly) is transformed into GAT (Asp). In this work we have targeted the KRAS coding sequence embracing the GAT mutation with a sense PNA molecule (P14), with the aim of downregulating the expression of the mutant allele. P14 was designed with a 15-base sequence complementary to the antisense strand of KRAS at the GAT (Asp) mutation and conjugated to the nuclear localization signal peptide PKKKRKV. CD spectra as a function of temperature show that P14 (2 microM) binds to the antisense strand of the GAT target in the mutated allele with a T(M) of 78 degrees C and to the antisense strand of the GGT target in the wild-type allele with a T(M) of 69 degrees C, in 50 mM Tris-HCl, pH 7.4, and 1 M NaCl. Moreover, P14 showed a high capacity to enter and accumulate in the nuclei of pancreatic cells (Panc-1 and BxPC3), whereas the nonconjugated analogue did not. Quantitative RT-PCR showed that 1 microM P14 was able to specifically suppress KRAS transcription in Panc-1 cells, which harbor mutant KRAS, but not in BxPC3 cells, which contain only wild-type KRAS. However, P14 inhibited KRAS transcription also in BxPC3 cells when used at concentrations of 5 and 10 microM. Following a single PNA treatment, changes in protein level were evident only in Panc-1 cells. As we found that all three genes of the ras family are expressed in the pancreatic cells, we designed PNA-NLS conjugates (P16 and P17) to target also HRAS and NRAS. The binding of each PNA conjugate to the ras genes was assayed by electrophoresis, and their capacity to inhibit transcription was measured by RT-PCR. All of the data obtained, both in vivo and in vitro, are discussed in terms of sequence specificity in the binding between PNA-NLS molecules and genomic DNA.

Cell Line, Tumor↗

PubMed2002

Cationic cholesterol promotes gene transfection using the nuclear localization signal in protamine.

PURPOSE: The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin). METHOD: Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37 degrees C. The cells then were washed and cultured for another 40 h in the growth medium at 37 degrees C before luciferase assay. RESULTS: The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA. In addition, its transfection efficiency was enhanced greatly by the addition of protamine. Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine. Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine. CONCLUSION: Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine.

3T3 Cells↗

PubMed1998

Two proline-rich nuclear localization signals in the amino- and carboxyl-terminal regions of the Borna disease virus phosphoprotein.

Borna disease virus (BDV) uses a unique strategy of replication and transcription which takes place in the nucleus, unlike other known, nonsegmented, negative-stranded RNA viruses of animal origin. In this process, viral constituents necessary for replication must be transported to the nucleus from the cytoplasm. We report here the evidence that BDV P protein, which may play an important role in viral replication and transcription, is transported into the nucleus in the absence of other viral constituents. This transportation is accomplished by its own nuclear localization signals (NLSs), which are present in both N-terminal (29PRPRKIPR36) and C-terminal (181PPRIYPQLPSAPT193) regions of the protein. These two NLSs can function independently and both have several Pro residues as key amino acids.

Amino Acid Sequence↗

PubMed2000

Modular domain structure of Arabidopsis COP1. Reconstitution of activity by fragment complementation and mutational analysis of a nuclear localization signal in planta.

The Arabidopsis COP1 protein functions as a developmental regulator, in part by repressing photomorphogenesis in darkness. Using complementation of a cop1 loss-of-function allele with transgenes expressing fusions of cop1 mutant proteins and beta-glucuronidase, it was confirmed that COP1 consists of two modules, an amino terminal module conferring a basal function during development and a carboxyl terminal module conferring repression of photomorphogenesis. The amino-terminal zinc-binding domain of COP1 was indispensable for COP1 function. In contrast, the debilitating effects of site-directed mutations in the single nuclear localization signal of COP1 were partially compensated by high-level transgene expression. The carboxyl-terminal module of COP1, though unable to substantially ameliorate a cop1 loss-of-function allele on its own, was sufficient for conferring a light-quality-dependent hyperetiolation phenotype in the presence of wild-type COP1. Moreover, partial COP1 activity could be reconstituted in vivo from two non-covalently linked, complementary polypeptides that represent the two functional modules of COP1. Evidence is presented for efficient association of the two sub-fragments of the split COP1 protein in Arabidopsis and in a yeast two-hybrid assay.

Arabidopsis↗