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。     

JC病毒VLP-Z的制备及其生物活性鉴定

目的 利用先期重组的原核表达质粒pET15b-VP1-Z体外表达重组的蛋白VP1-Z;研究VP1-Z是否组装成VLP-Z,以及VLP-z是否具有与野生型VLP相同的生物学活性.方法 重组质粒pET15b-VP1-Z转染大肠杆菌BL21(DB),IPTG诱导重组蛋白VP1-Z表达;按照野生型VLP的制备方法纯化蛋白;Western blot和考马斯亮蓝染色确定纯化蛋白及其纯度;电镜观察VLP-Z是否形成病毒样颗粒;血凝抑制试验检测VLP-Z是否具有血凝抑制活性;细胞免疫荧光检测VLP-Z能否转运进入细胞.结果 超速离心法得到纯度及浓度较高的重组蛋白VLP-Z,其相对分子质量约50×10~3;电镜观察发现VLP-Z组装成病毒样颗粒,直径约45～50 nm,较野生型VLP直径稍大;血凝抑制试验证实VLP-Z可以抑制人"O"型红细胞聚集;细胞免疫荧光显示,感染后VLP-Z可以转运进入HeLa细胞质及细胞核,与野生型VLP感染后相同.结论 成功制备了VLP-Z,且VLP-Z具有与野生型VLP相同的生物学活性.     

甲型H1N1流感病毒样颗粒疫苗的初步研究

目的对甲型H1N1病毒样颗粒的免疫原性和保护效果进行初步研究,为研发不依赖鸡胚生产工艺的流感疫苗提供新的思路。方法使用昆虫-杆状病毒表达系统表达并纯化甲型H1N1流感病毒A/California/07/2009(H1N1)的病毒样颗粒;通过Western blot、血凝、单向免疫扩散,透射电镜等方法鉴定病毒样颗粒;使用A/California/07/2009(H1N1)裂解疫苗作为对照,腹腔注射免疫Balb/c小鼠,并使用A/Beijing/501/2009(H1N1)进行攻毒,评价病毒样颗粒疫苗的免疫原性及保护效果。结果经鉴定,纯化后的病毒样颗粒血凝滴度为1:512,HA含量为92.9μg/ml,颗粒大小在100 nm左右,二免10 d后IgG抗体效价7.9×103,血凝抑制实验测得血抑(Hemagglutination Inhibition,HI)效价384,对50LD50的A/Beijing/501/2009(H1N1)病毒的保护率达到100%。结论甲型H1N1流感病毒样颗粒的免疫原性显著高于裂解疫苗,为发展不依赖鸡胚生产的流感亚单位疫苗提供了技术保证。     

登革2型病毒ZS01/01株病毒样颗粒免疫原性研究

目的 研究登革2型病毒（Dengue virus type 2,DENV-2）病毒样颗粒（virus-Like particles,VLPs）的免疫原性.方法 利用已构建的DENV-2 ZS01/01株病毒样颗粒的表达质粒转染293T细胞,对分泌型VLPs进行大量培养并通过蔗糖密度梯度离心法对其进行纯化.纯化的VLPs经Western Blot及透射电镜观察等方法鉴定后免疫BALB/c小鼠.利用ELISA及中和试验等方法对体液免疫反应进行检测,ELISPOT法测定细胞免疫水平.结果 登革2型病毒样颗粒表达质粒转染哺乳动物细胞所得上清经蔗糖密度梯度离心后,电镜下可观察到类似于天然登革病毒的大小在45～55nm之间的病毒样颗粒.体液及细胞免疫检测结果显示登革2型VLPs可以刺激小鼠产生较高水平的登革E蛋白特异性抗体及一定水平的中和抗体,免疫小鼠脾淋巴细胞经体外刺激后IFN-γ水平显著升高.结论 登革2型病毒病毒样颗粒免疫BALB/c小鼠后可引起一定水平的细胞免疫及体液免疫反应,该研究结果为四价登革病毒样颗粒疫苗的研制奠定了基础.     

Assembly of JC virus-like particles in COS7 cells

JC virus lacks an appropriate cell line to support virus replication. The establishment of a JC pseudovirus assembly system could play an alternative role for a virus culture system. COS7 cells and a transfer vector, pcDL-SRα296, were used to express JC viral structural genes. VP231-SRα, which encodes VP2/VP3 and VP1, but lacks 137 bp of the 5′-terminus of agnogene, showed both efficient nuclear migration and quantitative expression of the major capsid protein VP1. JC pseudovirus assembly was observed in the nucleus of VP231-SRα transfected cells. Evidence of JC pseudovirus assembly is presented. The further utilization of this system, which includes a study for the viral morphogenesis, serological diagnosis, as well as the potential application for gene transfer vector, is discussed. J. Med. Virol. 51:265–272, 1997. © 1997 Wiley-Liss, Inc.     

以病毒样颗粒为平台的分子载体和疫苗设计

病毒样颗粒（vims-like particles，VIPs）在形态结构上类似于病毒，但不含病毒基因组。依据来源可分为源于病毒VLPs和人工VIPs。前者主要通过基因工程制备，后者由人工化学合成而来。VIPs的表面组分可进行灵活多样的修饰改造以满足不同需求；在适宜条件下VIPs可以包裹核酸或其它小分子物质。作为分子运载工具，VLPs可靶向性地递送基因、药物或其它小分子物质。VLPs具有独特的免疫学特性，不但能通过影响APC发挥佐剂效应，还可以作为其它佐剂、肽疫苗和核酸疫苗的整合平台设计多种形式的疫苗。VLPs在载体和疫苗设计中具有良好的应用前景。     

Secretion of noninfectious dengue virus-like particles and identification of amino acids in the stem region involved in intracellular retention of envelope protein

DNA plasmids that express flavivirus premembrane/membrane (prM/M) and envelope (E) proteins in the form of virus-like particles (VLPs) have an excellent potential as DNA vaccine candidates against virus infection. The plasmid-expressed VLPs are also useful as safe, noninfectious antigens in serodiagnostic assays. We have constructed plasmids containing the prM/M and E gene regions for DENV-1, -3, and -4 that express and secrete VLPs when electroporated into Chinese hamster ovary cells. Constructs containing the full-length DENV-1 E protein gene did not secrete VLPs into tissue culture fluid effectively. However, a 16-fold increase in ELISA titers of DENV-1 VLPs was achieved after replacing the carboxy-terminal 20% region of DENV-1 E protein gene with the corresponding sequence of Japanese encephalitis virus (JEV). DENV-3 plasmids containing either the full-length DENV-3 E protein gene or the 20% JEV sequence replacement secreted VLPs to similarly high levels. Whereas DENV-4 VLPs were secreted to high levels by plasmids containing the full-length DENV-4 E protein gene but not by the chimeric plasmid containing 20% JEV E replacement. Domain substitutions by replacing prM/M protein stem-anchor region with the corresponding prM/M stem-anchor region of JEV or DENV-2 in the chimeric DENV-4 construct failed to promote the secretion of DENV-4 VLPs. Using the DENV-2 chimeric plasmid with carboxy-terminal 10% of JEV E gene, the sequence responsible for intracellular localization of E protein was mapped onto the E-H1 alpha-helix domain of DENV-2 E protein. Substitution of three amino acids from the DENV-2 sequence to the corresponding amino acids in the JEV sequence (I398L, M401A, and M412L) in the E-H1 was sufficient to promote extracellular secretion and resulted in detectable titers of DENV-2 VLP secretion.     

Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal

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.     

A strong endoplasmic reticulum retention signal in the stem-anchor region of envelope glycoprotein of dengue virus type 2 affects the production of virus-like particles

Recombinant virus-like particles (VLPs) of flaviviruses have been shown to be produced efficiently by co-expressing the precursor membrane (PrM) and envelope (E) proteins with few exceptions, such as dengue virus type 2 (DENV2). It was reported previously that chimeric DENV2 PrM/E construct containing the stem-anchor region of E protein of Japanese encephalitis virus (JEV) produced VLPs efficiently (Chang, G. J., Hunt, A. R., Holmes, D. A., Springfield, T., Chiueh, T. S., Roehrig, J. T., and Gubler, D. J. 2003. Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus. Virology 306, 170-180.). We investigated the mechanisms involved and reported that compared with authentic DENV2 PrM/E-expressing cells, E protein in chimeric DENV2 PrM/E-expressing cells was also present in an endoglycosidase H (endo H)-resistant compartment and has shifted more to the pellets of the soluble fraction. Replacement of the transmembrane and cytoplasmic domains of CD4 with the stem-anchor of DENV2 (CD4D2) or JEV (CD4JEV) rendered the chimeric CD4 retained predominantly in the endoplasmic reticulum (ER). Flow cytometry revealed higher proportion of CD4JEV than CD4D2 expressed on the cell surface. Together, these findings suggested that the stem-anchor of DENV2 contained an ER retention signal stronger than that of JEV, which might contribute to the inefficient production of DENV2 VLPs. Moreover, co-expression of C protein can enhance the production of DENV2 VLPs, suggesting a mechanism of facilitating viral particle formation during DENV2 replication.     

JC病毒在肾移植受者中的感染状况

目的 探究多瘤JC病毒(JC virus,JCV)在肾移植受者中的感染情况及其感染对移植肾功能的影响,并初步探索JCV感染的影响因素.方法 采用巢式PCR方法检测49例肾移植病人术后尿液标本中的JCV DNA,并以24例健康体检者为对照;定性PCR方法检测病人尿液标本中的巨细胞病毒(cytomegalovirus,CMV)DNA;Binary Logistic回归方法分析JCV感染的可能影响因素:病人的年龄、性别、免疫抑制方案、CMV感染;以肾小球滤过率(glomerular filtration rate,GFR)作为肾功能的指标,t检验方法比较JCV感染和非感染者GFR有无差别.结果 JCV在肾移植病人中的感染率为42.9%,健康体检者为4.2%.移植后CMV感染和强的松+MMF+环孢素三联免疫抑制方案是JCV感染的危险因素(OR=10.101,P=0.049;OR=6.286,P=0.008).JCV感染者和非感染者的GFR分别为86.470±29.990和84.060±33.729,两者间的差异无统计学意义(t=0.259,P=0.797).结论 JCV在肾移植病人中有很高的感染率,CMV感染和使用强的松+MMF+环孢素三联免疫抑制方案可明显增加JCV感染的危险性;JCV感染对移植肾功能无影响.