Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.     

Application of bioinformatics-coupled experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of Influenza A virus strain

Background  

Two nuclear localization sequences (NLS) in influenza A virus nucleoprotein (NP) have been demonstrated to be critical for nuclear import of NP and viral ribonucleoprotein complexes. However, a deletion mutant lacking these two signals was still able to localize to the nucleus suggesting the presence of yet another (a third) potential NLS in the NP protein. In order to identify the nature of this potential NLS signal in the NP of a WS/33L influenza virus A strain, we utilized the tools of bioinformatics coupled with functional experimental analyses in the present study.     

Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha

BACKGROUND: Karyopherin alpha (importin alpha) is an adaptor molecule that recognizes proteins containing nuclear localization signals (NLSs). The prototypical NLS that is able to bind to karyopherin alpha is that of the SV40 T antigen, and consists of a short positively charged sequence motif. Distinct classes of NLSs (monopartite and bipartite) have been identified that are only partly conserved with respect to one another but are nevertheless recognized by the same receptor. RESULTS: We report the crystal structures of two peptide complexes of yeast karyopherin alpha (Kapalpha): one with a human c-myc NLS peptide, determined at 2.1 A resolution, and one with a Xenopus nucleoplasmin NLS peptide, determined at 2.4 A resolution. Analysis of these structures reveals the determinants of specificity for the binding of a relatively hydrophobic monopartite NLS and of a bipartite NLS peptide. The peptides bind Kapalpha in its extended surface groove, which presents a modular array of tandem binding pockets for amino acid residues. CONCLUSIONS: Monopartite and bipartite NLSs bind to a different number of amino acid binding pockets and make different interactions within them. The relatively hydrophobic monopartite c-myc NLS binds extensively at a few binding pockets in a similar manner to that of the SV40 T antigen NLS. In contrast, the bipartite nucleoplasmin NLS engages the whole array of pockets with individually more limited but overall more abundant interactions, which include the NLS two basic clusters and the backbone of its non-conserved linker region. Versatility in the specific recognition of NLSs relies on the modular.     

Multiple nuclear localization sequences allow modulation of 5-lipoxygenase nuclear import

The nuclear import of proteins typically requires the presence of a nuclear localization sequence (NLS). Some proteins have more than one NLS, but the significance of having multiple NLSs is unclear. The enzyme 5-lipoxygenase (5-LO) has three NLSs that, unlike the tight cluster of basic residues of the classical SV40 large T antigen NLS, contain dispersed basic residues. When attached to green fluorescent protein (GFP), individual 5-LO NLSs caused quantitatively and statistically less import than the SV40 NLS. Combined 5-LO NLSs produced nuclear import that was comparable to that of the SV40 NLS. As expected, GFP/NLS proteins displayed relatively uniform import in all cells. However, a fusion protein of GFP plus the 5-LO protein, modified to contain only one functional NLS, produced some cells with import and some cells without import. A GFP/5-LO fusion protein containing two functional NLSs produced four identifiable levels of nuclear import. Quantitative and visual analysis of a population of cells expressing the intact GFP/5-LO protein, with three intact NLSs, indicated five levels of nuclear import. This suggested that the subcellular distribution of 5-LO may vary widely in normal cells of the body. Consistent with this, immunohistochemical staining of lung sections found that individual macrophages, in situ, displayed cell-specific levels of import of 5-LO. Since nuclear accumulation is known to affect 5-LO activity, multiple NLSs may allow graded regulation of activity via controlled import. Multiple NLSs on other proteins may likewise allow fine control of protein action through modulation of the level of import.     

Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2.

Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin alpha/beta. This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2. m- and y-IMPalpha recognized both NLSs, with y-IMPalpha exhibiting higher affinity. m-IMPbeta greatly enhanced the binding of m-IMPalpha to the T-ag and N1N2 NLSs, but y-IMPbeta did not significantly affect the affinity of y-IMPalpha for the T-ag NLS. In contrast, y-IMPbeta enhanced y-IMPalpha binding to the NLS of N1N2, but to a lesser extent than the enhancement of m-IMPalpha binding by m-IMPbeta. NLS-dependent nuclear import was reconstituted in vitro using the different importin subunits together with the transport factors Ran and NTF2. Whereas T-ag NLS-mediated nuclear import did not exhibit an absolute requirement for NTF2, N1N2 NLS-mediated transport strictly required NTF2. High levels of NTF2 inhibited nuclear accumulation conferred by both NLSs. We conclude that different NLSs possess distinct nuclear import properties due to differences in recognition by importin and requirements for NTF2.     

Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75

Human lens epithelium-derived growth factor (LEDGF)/p75 protein forms a specific nuclear complex with human immunodeficiency virus type 1 (HIV-1) integrase and is essential for nuclear localization and chromosomal association of the viral protein. We now studied nuclear import of LEDGF/p75 in live and semipermeabilized cells. We show that nuclear import of LEDGF/p75 is GTP-, Ran-, importin-alpha/beta-, and energy-dependent and that the protein competes with the canonical SV40 large T antigen nuclear localization signal (NLS) for nuclear import receptors. We identified the NLS of LEDGF/p75 through deletion analysis and site-directed mutagenesis. The LEDGF/p75 NLS, 148GRKRKAEKQ156, belongs to the canonical SV40-like family. Fusion of this short peptide to the amino terminus of Escherichia coli beta-galactosidase rendered the fusion protein nuclear, confirming that the LEDGF/p75 NLS is transferable. Moreover, a single amino acid change in the NLS was sufficient to exclude the mutant LEDGF/p75 protein from the nucleus and abolish nuclear import of HIV-1 integrase.     

A synthetic peptide bearing the HIV-1 integrase 161-173 amino acid residues mediates active nuclear import and binding to importin alpha: characterization of a functional nuclear localization signal

In spite of recent efforts to elucidate the nuclear import pathway of the human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), its exact route as well as the domains that mediate its import are still unknown. Here, we show that a synthetic peptide bearing the amino acid residues 161-173 of the HIV-1 IN is able to mediate active import of covalently attached bovine serum albumin molecules into nuclei of permeabilized cells and therefore was designated as nuclear localization signal-IN (NLS(IN)). A peptide bearing residues 161-173 in the reversed order showed low karyophilic properties. Active nuclear import was demonstrated by using fluorescence microscopy and a quantitative ELISA-based assay system. Nuclear import was blocked by addition of the NLS(IN) peptide, as well as by a peptide bearing the NLS of the simian virus 40 T-antigen (NLS-SV40). The NLS(IN) peptide partially inhibited nuclear import mediated by the full-length recombinant HIV-1 IN protein, indicating that the sequence of the NLS(IN) is involved in mediating nuclear import of the IN protein. The NLS(IN) as well as the full-length IN protein interacted specifically with importin alpha, binding of which was blocked by the NLS(IN) peptide itself as well as by the NLS-SV40.     

Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex

Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) alpha and beta. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.     

Intramolecular masking of nuclear localization signals: analysis of importin binding using a novel AlphaScreen-based method

Active nuclear import of proteins requires the recognition of a nuclear localization sequence (NLS) by members of the importin (IMP) family of proteins. We have developed a modified AlphaScreen-based assay able to estimate the solution binding affinities of such interactions using biotinylated IMPs and His6-tagged NLS-containing proteins. We describe this assay in detail as well as its application in documenting the phenomenon of intramolecular masking of NLSs using recombinant green fluorescent protein (GFP) fusion proteins containing sequences from the SV40 large tumor T antigen (T-ag). We also use it to examine, for the first time, IMP binding to the cancer cell-specific proapoptotic factor viral protein 3 (VP3) from the chicken anemia virus (CAV). High-affinity binding of the IMPalpha/beta heterodimer to the T-ag NLS was observed when the GFP tag was fused to its N terminus but not to its C terminus. Effects of flanking residues were also observed in GFP-T-ag fusion derivatives containing the Thr128 NLS-inactivating mutation, whereby the absence of flanking sequences N terminal to the T-ag NLS appeared to decrease the specificity of the mutation in terms of oblating IMPalpha/beta binding. IMPbeta, but not IMPalpha or the IMPalpha/beta heterodimer, was found to bind to CAV VP3 with high affinity. Interestingly, GFP-VP3(74-121) bound to IMPbeta with threefold higher affinity than the full-length protein, GFP-VP3(1-121), implying that the NLS is masked to a significant extent in the context of full-length protein. This may represent a regulatory mechanism to control nuclear import in a tumor cell-specific fashion.     

Kinetic properties of nuclear transport conferred by the retinoblastoma (Rb) NLS

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein central to control of cellular proliferation. We have previously shown that human RB possesses an evolutionarily conserved bipartite nuclear localization sequence (NLS) (KRSAEGSNPPKPLKKLR877) resembling that of nucleoplasmin. Here we analyze the kinetic properties of the RB NLS in detail with respect to recognition by cellular nuclear import factors, the importins (IMPs), and nuclear transport properties, comparing results to those for the NLSs from SV40 large tumor antigen (T-ag) and the Xenopus laevis phosphoprotein N1N2. Binding affinities of different IMPalpha subunits for the Rb NLS, in the absence or presence of IMPbeta subunits were determined, and NLS-dependent nuclear import reconstituted in vitro for the first time using purified IMPalpha/beta subunits together with recombinant human RanGDP and nuclear transport factor 2 (NTF2). RB NLS-mediated transport had a strict requirement for all components, with high NTF2 concentrations inhibiting transport. As in the case of transport mediated by the T-ag- and N1N2-NLSs, nuclear import of an RB-NLS containing beta-Gal fusion protein was reduced or abolished when anti-IMPalpha or beta antibody was added to cytosolic extract, respectively, confirming that RB NLS-mediated nuclear import occurs through action of IMPalpha/beta. We conclude that although mediated by IMPalpha/beta, and similar in most respects to transport mediated by the similarly bipartite N1N2 NLS, nuclear import conferred by the RB NLS has distinct properties, in part due to the affinity of its interaction with IMPalpha.     

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.     

Simian immunodeficiency virus Vpx is imported into the nucleus via importin alpha-dependent and -independent pathways

Vpx protein of human immunodeficiency virus type 2/simian immunodeficiency virus (SIV) has been implicated in the transport of the viral genome into the nuclei of nondividing cells. The mechanism by which Vpx enters the nucleus remains unknown. Here we have identified two distinct noncanonical nuclear localization signals (NLSs) in Vpx of SIV(smPbj1.9) and defined the pathways for its nuclear import. Although nuclear targeting signals identified here are distinct from known nuclear import signals, translocation of Vpx into the nucleus involves the interaction of its N-terminal NLS (amino acids 20 to 40) or C-terminal NLS (amino acids 65 to 75) with importin alpha and, in the latter case, also with importin beta. Collectively, these results suggest that importins interact with Vpx and ensure the effective import of Vpx into the nucleus to support virus replication in nondividing cells.     

A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import [published erratum appears in J Cell Biol 1992 Jul;118(1):215]

We have purified proteins of 70 kD from Drosophila, HeLa cells, and Z. mays that specifically bind nuclear localization sequences (NLSs). These proteins are recognized by antibodies raised against a previously identified NLS-binding protein (NBP) from the yeast S. cerevisiae. All NBPs are associated with nuclei and also present in the cytosol. NBPs are phosphorylated and phosphatase treatment abolished NLS binding. The requirement for NBPs in nuclear protein uptake is demonstrated in semipermeabilized Drosophila melanogaster tissue culture cells. Proper import of a fluorescent protein containing the large T antigen NLS requires cytosol and ATP. In the absence of cytosol and/or ATP, NLS-containing proteins are bound to cytosolic structures and the nuclear envelope. Addition of cytosol and ATP results in movement of this bound intermediate into the nucleus. Anti-NBP antibodies specifically inhibited the binding part of this import reaction. These results indicate that a phosphoprotein common to several eukaryotes acts as a receptor that recognizes NLSs before their uptake into the nucleus.     

Modulation of Nuclear Localization of the Influenza Virus Nucleoprotein through Interaction with Actin Filaments

The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind actin. In cells, exogenously expressed NP when expressed at a high level partitioned to the cytoplasm, where it associated with F-actin stress fibers. In contrast, mutants unable to bind F-actin efficiently were imported into the nucleus even under conditions of high-level expression. Similarly, nuclear import of NLS-deficient NP molecules was restored by concomitant disruption of F-actin binding. We propose that the interaction of NP with F-actin causes the cytoplasmic retention of influenza virus ribonucleoproteins.     

Nuclear localization signal and protein context both mediate importin alpha specificity of nuclear import substrates

The "classical" nuclear protein import pathway depends on importin alpha and importin beta. Importin alpha binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin beta-dependent import pathway. In humans, only one importin beta is known to interact with importin alpha, while six alpha importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular alpha importins. Whether the NLS is sufficient to mediate importin alpha specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin alpha3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin alpha binding and transport.     

核定位信号肽用于基因转移的研究进展

核定位信号(nuclear localization signal,NLS)是一段富含Arg、Lys的氨基酸序列,它存在于真核细胞核蛋白和病毒蛋白中,并具有引导它们趋向定位核区的功能。近年来发展的利用含核定位信号肽的非病毒载体为基因转移提供了一个崭新的途径。