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1.
Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ((211)KKYK(214)) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to beta-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products. 相似文献
2.
目的: 分析腺病毒E1A相关蛋白BS69不同亚型的DNA序列,寻找新的核输出信号序列,并在Cos7细胞中表达确定其亚细胞定位。方法: 分析数据库中不同BS69亚型DNA序列,与传统和输出信号序列比对,寻找可能的新的核输出信号序列。采用DNA重组技术把BS69不同亚型片段的cDNA插入到真核表达载体pcDNA3.1上,转染Cos7细胞,用免疫荧光染色方法确定其亚细胞定位,用Western blotting方法验证不同亚型BS69在细胞中的功能。结果: 在BS69亚型2上面发现1段富含亮氨酸的基因序列,与核输出信号序列极为相似。免疫荧光染色方法显示BS69亚型2定位于细胞浆中,而BS69亚型1和2个亚型共同序列编码的蛋白质则定位于细胞核中。BS69亚型2参与了EB病毒潜伏膜蛋白1(Epstein-Barr virus latent membrane protein 1,LMP1)介导的信号转导途径。结论: BS69不同亚型具有不同的亚细胞定位,因此具有不同的生物学功能,其中核蛋白是转录调控因子,细胞浆蛋白则可能参与鼻咽癌发生发展的调控。 相似文献
3.
Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 ((51)RRPR(54)) or pat7 ((48)PRVRRPR(54)) NLS2 abrogated nuclear accumulation, whereas deletion of (48)PRV(50) did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 ((11)RRPRR(15)), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids (485)LSAYLTLFVAL(495). This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm. 相似文献
4.
出核因子CRM1及p27在胶质瘤中的表达 总被引:1,自引:0,他引:1
目的 探讨出核因子CRM1、p27 10位丝氨酸(Ser10)磷酸化及p27蛋白在胶质瘤中的表达、相互关系及意义.方法 免疫组织化学SP法检测70例胶质瘤和10例非肿瘤对照脑组织标本中CRM1、p27Ser10磷酸化形式及p27蛋白的表达,Western blot检测6例新鲜胶质瘤标本中相应蛋白的表达.结果 CRM1及p27Ser10磷酸化形式在对照脑组织中表达不明显,在低级别胶质瘤中表达较少,在高级别胶质瘤中表达较多,两两比较差异均有统计学意义(P<0.01).p27在对照脑组织中表达明显,其表达水平随肿瘤级别增高而降低,差异有统计学意义(P<0.01).Western blot结果显示CRM1、p27Ser10磷酸化形式在胶质瘤中的表达水平与肿瘤细胞的恶性程度相关.相关分析显示:胶质瘤中CRM1蛋白表达与p27蛋白表达呈负相关(r=0.727,P<0.01),与p27Ser10磷酸化形式呈正相关(rs=0.954,P<0.01),与增殖指标Ki-67表达呈正相关(rs=0.799,P<0.01);p27Ser10磷酸化形式与p27蛋白表达呈负相关(rs=-0.744,P<0.01),与Ki-67表达呈正相关(rs=0.785,P<0.01).结论 在胶质瘤中高表达的CRM1可能通过识别并结合高表达的p27Ser10磷酸化形式,促进p27的出核降解,使p27表达降低,失去对细胞周期的调控,从而促进胶质瘤的恶性进展和增殖. 相似文献
5.
目的探讨JC病毒(JCV)主要外壳蛋白VP1所含核定位信号(NLS)在JCV病毒样颗粒(VLP)入核转运中的作用。方法应用位点诱变法将JCV野生型VP1(wtVP1)氨基末端3个氨基酸:第5位赖氨酸、第6位精氨酸、第7位赖氨酸分别置换为丙氨酸、甘氨酸、丙氨酸,制备成NLS变异的VP1(ΔNLS-VP1)。编码ΔNLS-VP1的基因克隆到原核表达质粒pET-15b中,体外表达、重组VP1 NLS变异的病毒样颗粒(ΔNLS-VLP),异硫氰基荧光素(FITC)标记后感染地高辛渗透或未渗透的SVG细胞,荧光显微镜观察VLP入核转运。结果wtVP1组成的病毒样颗粒(wtVLP)可以进入地高辛渗透或未渗透的SVG细胞核,而ΔNLS-VLP只能进入细胞浆,不能进入细胞核;体外转染后,wtVP1主要在SVG细胞核表达,而ΔNLS-VP1主要在细胞浆表达;包裹外源性DNA的VLP感染SVG细胞后,则wtVLP包裹的DNA在细胞浆和细胞核内均可检测到,而ΔNLS-VLP包裹的DNA只能在细胞浆检测到,不能在细胞核内检测到。VLP体外结合分析发现,wtVLP与胞浆转运因子(importin)α、β均可结合,而ΔNLS-VLP与importinα、β均不能结合。结论VP1 NLS对于VLP入核转运是必需的,VIP的入核转运是由VP1 NLS与importin相互作用介导的。 相似文献
6.
Ritter W Plank C Lausier J Rudolph C Zink D Reinhardt D Rosenecker J 《Journal of molecular medicine (Berlin, Germany)》2003,81(11):708-717
The transport of exogenous DNA into the nucleus of eukaryotic cells is a prerequisite for successful gene delivery. To favor nuclear transport we synthesized a tetramer of the nuclear localization signal (NLS) of the SV40 large T-antigen as a novel nonviral gene delivery vector. This 4.4-kDa lysine-rich peptide (NLSV404) binds and compacts DNA by electrostatic interaction and forms stable polyplexes. Apart from its sequence-specific potency to mediate nuclear accumulation of conjugated albumin, NLSV404 also displays properties of nuclear transport for plasmid DNA as confirmed by fluorescence in situ hybridization. Further, NLSV404 polyplexes are shown to efficiently transfect various cell lines such as 16HBE14o–, HeLa S6, and Cos7 cells. NLSV404 polyplexes displayed at least 20-fold higher transfection rates than analogous polyplexes formed by the nuclear transport-deficient mutant sequence cNLS. Using growth-arrested cells, NLSV404 complexes were at least 100-fold more efficient than cNLS complexes. Combination of NLSV404 peptide but not of cNLS peptide with preformed polyethylenimine and dendrimer DNA complexes resulted in a strong increase in transfection efficiency. Incubation of cells prior to transfection with NLSV404 polyplexes with excess free peptide NLSV404 but not with cNLS resulted in a dose-dependent dramatic decrease in the transfection rate, suggesting a sequence-specific competitive inhibition. These results indicate that NLSV404 mediates nuclear accumulation of transfected plasmid DNA and that it can be a highly useful component of nonviral gene vectors.Abbreviations
BSA-BODIPY
Fluorescence-labeled bovine serum albumin
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EGFP
Enhanced green fluorescent protein
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FISH
Fluorescent in situ hybridization
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NLS
Nuclear localization signal
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PEI
Polyethylenimine
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SV40
Simian virus 40 相似文献
7.
The delta antigen (HDAg) is the only protein encoded by the hepatitis delta virus (HDV) RNA genome. The HDAg contains an RNA binding domain, a dimerization domain, and a nuclear localization signal (NLS). The nuclear import of HDV RNPs is thought to be one of the first tasks of the HDAg during the HDV replication cycle. Using c-myc-PK fusions with several regions of the HDAg in transfection assays in Huh7 cells, we found that the HDAg NLS consists of a single stretch of 10 amino acids, EGAPPAKRAR, located in positions 66-75. Deletion and mutation analysis of this region showed that both the acidic glutamic acid residue at position 66 and the basic arginine residue at position 75 are essential for promoting nuclear import. 相似文献
8.
Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state. 相似文献
9.
J.Y. Chen L.X. Yang Z.F. Huang 《Brazilian journal of medical and biological research》2013,46(12):1021-1027
Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation. 相似文献
10.
The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway. 相似文献
11.
Hong Guo Qiong Ding Fusen Lin Weiwei Pan Jianyin Lin Alan C. Zheng 《Virus research》2009,145(2):312-320
Bovine herpesvirus-1 infected cell protein 27 (BICP27) was detected predominantly in the nucleolus. The open reading frame of BICP27 was fused with the enhanced yellow fluorescent protein (EYFP) gene to investigate its subcellular localization in live cells and BICP27 was able to direct monomeric, dimeric or trimeric EYFP exclusively to the nucleolus. By constructing a series of deletion mutants, the putative nuclear localization signal (NLS) and nucleolar localization signal (NoLS) were mapped to 81RRAR84 and 86RPRRPRRRPRRR97 respectively. Specific deletion of the putative NLS, NoLS or both abrogated nuclear localization, nucleolar localization or both respectively. Furthermore, NLS was able to direct trimeric EYFP predominantly to the nucleus but excluded from the nucleolus, whereas NoLS targeted trimeric EYFP primarily to the nucleus, and enriched in the nucleolus with faint staining in the cytoplasm. NLS + NoLS directed trimeric EYFP predominantly to the nucleolus with faint staining in the nucleus. Moreover, deletion of NLS + NoLS abolished the transactivating activity of BICP27 on gC promoter, whereas deletion of either NLS or NoLS did not. The study demonstrated that BICP27 is a nucleolar protein, adding BICP27 to the growing list of transactivators which localize to the nucleolus. 相似文献
12.
核蛋白定位信号(nuclear localization signal,NLS)是一段富含精氨酸、赖氨酸等碱性氨基酸的短肽,是介导蛋白通过核孔复合体入核的必要信号序列。NLS存在于真核细胞核蛋白及病毒蛋白中,鱼精蛋白是应用较为广泛的天然NLS类短肽。针对NLS能够介导DNA、蛋白质、纳米粒等进入细胞核的特点,国内外学者对其在生物医学中的应用进行了广泛研究。 相似文献
13.
The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline, M., Merle, E., and Moroianu, J. (2003). The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway. Virology 317(1), 13-23.). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E738-98 from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence (76IRTLEDLLM84) changed the localization of 2xEGFP-E738-98 from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments. 相似文献
14.
15.
Herpes simplex virus type 1 (HSV-1) UL3 protein is a nuclear protein. In this study, the molecular mechanism of the subcellular localization of UL3 was characterized by fluorescence microscopy in living cells. A nuclear localization signal (NLS) and a nuclear export signal (NES) were also identified. UL3 was demonstrated to target to the cytoplasm through the NES via chromosomal region maintenance 1 (CRM-1) dependent pathway, and to the nucleus through RanGTP-dependent mechanism. Heterokaryon assays confirmed that UL3 was capable of shuttling between the nucleus and the cytoplasm. These results demonstrate that the UL3 protein is a novel HSV-1 encoded nucleocytoplasmic shuttling protein. 相似文献
16.
Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3Cpro. Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli. 相似文献
17.
《European journal of medical genetics》2022,65(3):104441
Fragile X syndrome (FXS; MIM 300624) is an X-linked genetic disorder characterized by physical abnormalities associated with intellectual disability and a wide spectrum of neurological and psychiatric impairments. FXS occurs more frequently in males, 1 in 5000 males and 1 in 8000 females accounting for 1–2% of overall intellectual disability (ID). In more than 99% of patients, FXS results from expansions of a CGG triplet repeat (>200 in male) of the FMR1 gene. In the last years an increasing number, albeit still limited, of FXS subjects carrying FMR1 mutations including deletions, splicing errors, missense, and nonsense variants was reported. Nevertheless, the studies concerning the functional consequences of mutations in the FMR1 gene are rare so far and, therefore, we do not have sufficient knowledge regarding the genotype/phenotype correlation. We report a child carrying a hemizygous missense FMR1 (NM_002024.5:c.1325G > A p.Arg442Gln) variant, maternally inherited, associated with facial abnormalities, developmental delay, and social and communication deficits assessed with formal neuropsychological tests. The study contributes to highlighting the clinical differences between the CGG triplet repeat dependent phenotype and FMR1variant dependent phenotype and it also confirms the pathogenicity of the variant being reported for the second time in the literature. 相似文献
18.
Processivity factor of KSHV contains a nuclear localization signal and binding domains for transporting viral DNA polymerase into the nucleus 总被引:3,自引:0,他引:3
Kaposi's sarcoma-associated human herpesvirus (KSHV) encodes a processivity factor (PF-8, ORF59) that forms homodimers and binds to viral DNA polymerase (Pol-8, ORF9). PF-8 is essential for stabilizing Pol-8 on template DNA so that Pol-8 can incorporate nucleotides continuously. Here, the intracellular interaction of these two viral proteins was examined by confocal immunofluorescence microscopy. When individually expressed, PF-8 was observed exclusively in the nucleus, whereas Pol-8 was found only in the cytoplasm. However, when co-expressed, Pol-8 was co-translocated with PF-8 into the nucleus. Mutational analysis revealed that PF-8 contains a nuclear localization signal (NLS) as well as domains located at the N-terminus and the C-proximal regions that are required for Pol-8 binding. This study suggests that the mechanism that enables PF-8 to transport Pol-8 into the nucleus is the first critical step required for Pol-8 and PF-8 to function processively in KSHV DNA synthesis. 相似文献
19.
20.
Endogenous presentation of a nuclear antigen on MHC class II by autophagy in the absence of CRM1-mediated nuclear export 总被引:1,自引:0,他引:1
Riedel A Nimmerjahn F Burdach S Behrends U Bornkamm GW Mautner J 《European journal of immunology》2008,38(8):2090-2095
Accumulating evidence suggests that intracellular antigens are endogenously presented on MHC class II, but it is still unknown whether antigens within different subcellular compartments are presented with similar efficiency, and via the same or different pathways. We have previously shown that endogenous MHC class II presentation of the cytosolic bacterial antigen neomycin phosphotransferase II (NeoR) is mediated by autophagy. Here, we addressed whether secluding NeoR from this cytoplasmic pathway by directing the protein into the cell nucleus (NucNeoR) would affect antigen presentation. Unexpectedly, NucNeoR was presented at least as efficiently as the cytosolic version of the antigen. Furthermore, presentation of NucNeoR was also dependent on autophagocytosis and lysosomal processing, indicating that both antigens were presented via the same pathway. Inhibition of CRM1-mediated nuclear export did not impede antigen presentation, indicating that NucNeoR gained access to this autophagy-dependent MHC class II presentation pathway by a CRM1-independent route. Thus, this endogenous presentation pathway broadens the spectrum of intracellular antigens surveyed by CD4(+) T cells by efficiently sampling cytoplasmic as well as nuclear antigens. 相似文献