Morbillivirus nucleoprotein possesses a novel nuclear localization signal and a CRM1-independent nuclear export signal

Morbilliviruses, which belong to the Mononegavirales, replicate its RNA genome in the cytoplasm of the host cell. However, they also form characteristic intranuclear inclusion bodies, consisting of nucleoprotein (N), in infected cells. To analyze the mechanisms of nucleocytoplasmic transport of N protein, we characterized the nuclear localization (NLS) and nuclear export (NES) signals of canine distemper virus (CDV) N protein by deletion mutation and alanine substitution of the protein. The NLS has a novel leucine/isoleucine-rich motif (TGILISIL) at positions 70-77, whereas the NES is composed of a leucine-rich motif (LLRSLTLF) at positions 4-11. The NLS and NES of the N proteins of other morbilliviruses, that is, measles virus (MV) and rinderpest virus (RPV), were also analyzed. The NLS of CDV-N protein is conserved at the same position in MV-N protein, whereas the NES of MV-N protein is located in the C-terminal region. The NES of RPV-N protein is also located at the same position as CDV-N protein, whereas the NLS motif is present not only at the same locus as CDV-N protein but also at other sites. Interestingly, the nuclear export of all these N proteins appears to proceed via a CRM1-independent pathway.     

Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1

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.     

The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

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.     

BS69的亚细胞定位及其核输出信号序列的分析鉴定

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。     

核定位信号在JC病毒样颗粒入核转运中的作用

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。     

Identification of a nuclear export signal sequence for bovine papillomavirus E1 protein

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.     

Searching for nuclear export elements in hepatitis D virus RNA

AIM: To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export.METHODS: We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export.RESULTS: Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway.CONCLUSION: A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.     

出核因子CRM1及p27在胶质瘤中的表达

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.     

P27^kip1与出核因子CRM1在人淋巴瘤细胞系中的表达及相互关系

目的研究P27kip1与出核因子CRM1在淋巴瘤细胞中的表达及相互关系。方法运用血清饥饿同步化处理淋巴瘤细胞株Jurkat和Raji,10%血清刺激细胞增殖。Western blot分别检测生长阻滞及增殖的淋巴瘤细胞中P27kip1与CRM1的表达。用核浆分离方法检测P27kip1与CRM1分别在胞核、胞质的定位。免疫沉淀检测P27kip1与CRM1在淋巴瘤细胞中的结合。结果在不同状态的淋巴瘤细胞中,P27kip1与CRM1蛋白水平的表达均表现为负相关,并且P27kip1的核内外分布与CRM1的核内外分布一致。免疫沉淀结果验证在淋巴瘤细胞中P27kip1与CRM1相互结合。结论出核因子CRM1可能通过与P27kip1结合而影响P27kip1的核内外分布及表达水平,从而参与调控淋巴瘤细胞的生长。     

Characterization of the nuclear localization signal of the hepatitis delta virus antigen

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.     

A novel virus-encoded nucleocytoplasmic shuttling protein: the UL3 protein of herpes simplex virus type 1

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.     

狂犬病毒糖蛋白及核蛋白在非复制型痘苗病毒天坛株中的共同表达

目的　提高重组痘苗病毒狂犬疫苗的有效性和安全性。方法　利用TK区表达狂犬病毒糖蛋白 (RG)的重组痘苗病毒作为亲本株 ,通过两步同源重组 ,删除痘苗病毒基因组CK片段间与毒力和宿主范围相关的核酸片段 ,同时将狂犬病毒核蛋白 (RN)基因插入C K片段之间 ,获得了含有RG基因和RN基因的非复制型重组痘苗病毒VTKRGΔCKLacZRN。结果　经PCR鉴定 ,RN基因已插入CK区 ;Westernblot结果显示 ,该株病毒能同时表达RG和RN ,相对分子质量 (Mr)分别为 6 5× 10 3 和 5 0 5× 10 3。VTKRGΔCKLacZRN在人源细胞如TK 143细胞中不能正常繁殖 ,在鸡胚成纤维细胞 (CEF)中能正常复制。与非复制型重组病毒VTKRGΔCK相比 ,VTKRGΔCKLacZRN免疫小鼠后能更快地诱生较高滴度的中和抗体 ,效果相当于复制型重组病毒VTKRG免疫组 ;它们均能保护小鼠针对致死剂量狂犬病毒国际标准攻击毒株 (CVS)的攻击。结论　VTKRGΔCKLacZRN具有良好的免疫效果和安全性     

Amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus

The molecular mechanism involved in cytopathogenicity of rabies virus has not been fully elucidated yet. A fixed rabies virus Nishigahara strain does not induce clear cytopathic effect (CPE) in mouse neuroblastoma (NA) cells, whereas Ni-CE strain, which was established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells, induces CPE that is characterized by rounding, shrinkage and detachment of the cells. In this study, to identify which viral gene is associated with the CPE of Ni-CE strain, we analyzed chimeric viruses between Nishigahara and Ni-CE strains generated by reverse genetics systems of both strains. We showed that the matrix gene of Ni-CE strain is responsible for the CPE in NA cells. It was also demonstrated by infection of Nishigahara and Ni-CE mutants with a single amino acid substitution in the matrix protein (M) that an amino acid at position 95 of M is a cytopathic determinant of the virus. We also demonstrated that the CPE is, at least partly, due to apoptosis. This is the first report of identification of an amino acid residue in a rabies virus protein that is important for the cytopathogenicity of the virus.     

Nucleocytoplasmic shuttling of the HSV-2 serine/threonine kinase Us3

The alphaherpesvirus serine/threonine kinase Us3 plays diverse roles in virus multiplication and modifies both nuclear and cytoplasmic substrates. We recently reported that treatment of HSV-2 Us3-transfected and HSV-2-infected cells with leptomycin B, an inhibitor of nuclear export mediated by interaction of chromosomal regional maintenance protein (CRM1) with leucine rich nuclear export signals (NESs), resulted in nuclear trapping of Us3. Here, we utilized fluorescence loss in photobleaching to monitor nuclear export of HSV-2 Us3 and confirm that this process proceeds solely via a CRM1-mediated mechanism. Analysis of deletion derivatives of HSV-2 Us3 fused to a nuclear export reporter protein implicated the involvement of NES-like sequences in nuclear export. However, nuclear trapping of HSV-2 Us3 proteins carrying mutations in these potential NESs was not observed, indicating that these sequences are not functional in the context of full-length protein. Our analyses also revealed previously unidentified regions of HSV-2 Us3 that contribute to its kinase activity.     

Identification of multiple nuclear export sequences in Fanconi anemia group A protein that contribute to CRM1-dependent nuclear export

The Fanconi anemia (FA) pathway plays an important role in maintaining genomic stability, and defects in this pathway cause cancer susceptibility. The FA proteins have been found to function primarily in a nuclear complex, although a cytoplasmic localization and function for several FA proteins has also been reported. In this study, we investigated the possibility that FANCA, FANCC and FANCG are subjected to active export out of the nucleus. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, the accumulation of epitope-tagged FANCA in the nucleus increased, whereas FANCC was affected to a lesser extent and FANCG showed no response. CRM1-mediated export of FANCA was further confirmed using CRM1 cotransfection, which led to a dramatic relocalization of FANCA to the cytoplasm. Five functional leucine-rich nuclear export sequences (NESs) distributed throughout the FANCA sequence were identified and characterized using an in vivo export assay. Simultaneous inactivation of three of these NESs resulted in a discrete but reproducible increase of FANCA nuclear accumulation. However, these NES mutations did not affect the ability of FANCA to complement the mitomycin C or cisplatin sensitivity of FA-A lymphoblasts. Surprisingly, mutations in the other two NESs resulted in an almost complete relocation of the protein to cytoplasm, suggesting that these motifs overlap with domains that are crucial for nuclear import. Taken together, these findings indicate that FANCA can be actively exported out of the nucleus by CRM1, revealing a new mechanism to regulate the function of the FA protein complex.     

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

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 3C^pro. 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.     

Nuclear export of influenza viral ribonucleoprotein is temperature-dependently inhibited by dissociation of viral matrix protein

The influenza virus copies its genomic RNA in the nuclei of host cells, but the viral particles are formed at the plasma membrane. Thus, the export of new genome from the nucleus into the cytoplasm is essential for viral production. Several viral proteins, such as nucleoprotein (NP) and RNA polymerases, synthesized in the cytoplasm, are imported into the nucleus, and form viral ribonucleoprotein (vRNP) with new genomic RNA. vRNP is then exported into the cytoplasm from the nucleus to produce new viral particles. M1, a viral matrix protein, is suggested to participate in the nuclear export of vRNP. It was found unexpectedly that the production of influenza virus was suppressed in MDCK cells at 41 degrees C, although viral proteins were synthesized and the cytopathic effect was observed in host cells. Indirect immunofluorescent staining with anti-NP or M1 monoclonal antibody showed that NP and M1 remained in the nuclei of infected cells at 41 degrees C, suggesting that a suppression of viral production was caused by inhibition of the nuclear export of these proteins. The cellular machinery for nuclear export depending on CRM1, which mediates the nuclear export of influenza viral RNP, functioned normally at 41 degrees C. Glycerol-density gradient centrifugation demonstrated that vRNP also formed normally at 41 degrees C. However, an examination of the interaction between vRNP and M1 by immunoprecipitation indicated that M1 did not associate with vRNP at 41 degrees C, suggesting that the association is essential for the nuclear export of vRNP. Furthermore, when infected cells incubated at 41 degrees C were cultured at 37 degrees C, the interaction between vRNP and M1 was no longer detected even at 37 degrees C. The results suggest that M1 synthesized at 41 degrees C is unable to interact with vRNP and the dissociation of M1 from vRNP is one of the reasons that the transfer of vRNP into the cytoplasm from the nucleus is prevented at 41 degrees C.