呼吸道合胞病毒非结构蛋白NS1、NS2的研究进展

呼吸道合胞病毒(reespiratory syncytial virus,RSV)是引起婴幼儿和老年人下呼吸道感染的重要病原体之一.由于该病毒的致病机理还不太清楚导致目前尚无有效治疗RSV的方法.研究表明,呼吸道合胞病毒的非结构蛋白NS1、NS2具有抗细胞凋亡的作用,同时可以逃避宿主免疫系统(IFN)对病毒的干扰,有利于病毒复制.敲除这两种基因的减毒活疫苗和袁达沉默NS1的小干扰RNA(siRNA)的质粒研究已经取得了一定的进展.对非结构蛋白功能的深入研究有助于了解RSV的致病机理,同时为预防和治疗RSV感染奠定理论基础.     

呼吸道合胞病毒F蛋白研究进展

人类呼吸道合胞病毒(RSV)融合蛋白(F蛋白)为病毒表面结构蛋白,可以刺激机体产生保护性抗体。因此对F蛋白的深入研究将有助于了解病毒感染的机理,从而为诊断试剂和亚单位疫苗的研制提供帮助。本文就F蛋白结构及结构与功能关系的研究进展进行简要综述。     

呼吸道合胞病毒疫苗研究进展

呼吸道合胞病毒(RSV)是引起严重下呼吸道感染的重要病原体,尽管经历了半个多世纪的努力,至今仍未有安全有效的RSV疫苗上市。近年来在RSV F蛋白结构生物学方面的研究进展为新一代RSV疫苗的开发提供了新方向,同时更多的采用不同技术、或针对不同人群的RSV侯选疫苗也在迅速发展,尤其是针对婴幼儿及老年人的RSV侯选苗已有60多种在研究中,大部分已处于临床前研究阶段,18种侯选苗已进入临床试验。我们简要介绍RSV疫苗的最新研究进展。     

人呼吸道合胞病毒M蛋白非复制型重组腺病毒的构建和鉴定

人呼吸道合胞病毒（human respiratory syncytial virus, RSV)基质蛋白（matrix protein,M）在RSV形态发生上具有重要作用,因含有CTL抗原表位,在疫苗研究上具有一定意义。为此,应用RT-PCR 方法从感染RSV的HEp-2 细胞中扩增获得M蛋白基因,构建了含M基因的非复制型重组腺病毒并进行表达和鉴定。基因序列分析显示RSV M基因仅有一处碱基发生错义突变。非复制型重组腺病毒DNA分子FGAd/RSVM转染293细胞,观察到细胞出现CPE,RT-PCR发现M基因有转录,Western blotting及间接免疫荧光分析检测到M蛋白。成功克隆A亚型RSV Long株M基因,并获得一株可表达A亚型RSV M蛋白的非复制型重组腺病毒FGAd/RSVM,可用于体内研究观察其免疫效果及免疫保护作用。     

呼吸道合胞病毒G蛋白的相关研究

呼吸道合胞病毒G蛋白介导病毒附着在宿主细胞表面,且具有亚型间抗原结构变异大的特点,其抗原变异推测是呼吸道合胞病毒逃避已存在免疫监视,引起反复感染的原因,了解G蛋白与免疫反应的关系对研制呼吸道合胞病毒亚单位疫苗,有效地防治该病毒感染具有重要意义。     

呼吸道合胞病毒保护性抗原的研究进展

对呼吸道合胞病毒（ＲＳＶ）保护性免疫机理更为详尽的了解将有助于研制安全有效的疫苗。本文介绍ＲＳＶ的蛋白组成以及单一蛋白诱生保护性免疫的能力,研究结果显示,Ｆ和Ｇ蛋白为体液免疫保护性抗原,Ｍ２、Ｆ和Ｎ为细胞免疫保护性抗原。综合诱生保护性体液和细胞免疫的能力,ＲＳＶ蛋白在保护性免疫中的重要性为：Ｆ〉Ｇ〉Ｍ２〉Ｎ。     

呼吸道合胞病毒在北京地区分离株G蛋白的基因分析

从经单克隆抗体证实为Ａ亚型的北京地区呼吸道合胞病毒（ＲＳＶ）分离株Ｂ７９中,用ＲＴ－ＰＣＲＴ扩增出编码Ｇ蛋白的基因片段,克隆至载体ｐＴＺ１８Ｒ中,经核苷酸序列测定证明,我国北京地区分离的Ａ亚型株Ｂ７９与ＲＳＶＡ亚型原型株（Ａ２株）Ｇ蛋白基因的核苷酸同源性为９３．８％,核苷酸的有义突变率为６５％,由核苷酸推导出氨基酸序列的同源性为８９．６％,氨基酸的变异主要集中在胞外区一个高度保守区的两端,而胞内区     

呼吸道合胞病毒诱导细胞凋亡与自噬

呼吸道合胞病毒感染与细胞凋亡、自噬的关系错综复杂。研究发现呼吸道合胞病毒感染细胞后,既能产生促细胞凋亡作用,也能产生抗细胞凋亡作用,还能诱导细胞发生自噬。研究这些过程机理,能帮助我们更好地认识呼吸道合胞病毒感染发病机制,为预防和治疗呼吸道合胞病毒感染提供一些新的方向。     

呼吸道合胞病毒F蛋白抗体的研究进展

呼吸道合胞病毒(respiratory syncytial virus,RSV)感染,是造成婴幼儿、学龄前儿童、免疫缺陷患者、老年人等高危群体住院治疗及死亡的重要病因。目前,多个预防RSV感染的候选疫苗正处于研发中,尚无安全、有效的疫苗面世。对RSV感染的处理仍以治疗为主,使用帕利珠单抗(Palivizumab)是当前仅有的预防药物。在过去数年间出现的新型抗体药物,如多克隆抗体、单克隆抗体、纳米抗体等有些已进入了临床前或I、II、III期临床试验阶段。融合蛋白(fusion protein,F蛋白)在RSV感染过程中是不可或缺的,它介导病毒包膜与宿主细胞膜的融合。在感染过程中,F蛋白从亚稳态的融合前构象状态(prefusion fusion protein,pre F)转变为热力学稳定的融合后状态(postfusion fusionprotein,post F)。近年来,研究人员通过不断筛选,获得了多株针对pre F的抗体。与结合post F的抗体相比,这些抗体具有更强的RSV中和活性。一些更新的抗体药物候选品,在实验中显示出了效力强、药代动力学特征明显、半衰期长等特点,并能以其他途径给药,而且能降低其制备成本。现就抗RSV pre F的抗体研究进展作一概述。     

呼吸道合胞病毒细胞免疫的研究进展

本文通过介绍单核吞噬细胞、T辅助细胞、细胞毒性T细胞、NK细胞和K细胞在呼吸道合胞病毒感染免疫中的作用,对呼吸道合胞病毒的细胞免疫机制作一概述。     

可调控表达人博卡病毒1型非结构蛋白NS1稳定细胞系的建立及其反式转录激活作用初探

人博卡病毒1型(Human bocavirus 1,HBoV1)非结构蛋白NS1是多功能蛋白,对病毒复制有重要作用,同时可诱导宿主细胞凋亡。在研究NS1蛋白功能时,降低NS1蛋白对宿主细胞的毒性作用是急需解决的问题。基于此,文中建立了可调控表达HBoV1非结构蛋白NS1的稳定细胞系。构建NS1重组慢病毒质粒(含可调控启动子),应用转染试剂将NS1重组慢病毒质粒转染至HEK293T细胞。通过嘌呤霉素筛选抗性细胞、多西环素诱导NS1表达,建立可稳定表达NS1-100、NS1-70蛋白的HEK 293T细胞系,利用荧光标记蛋白和Western blotting检测,确定NS1蛋白的表达。并在稳定表达NS1细胞系中转染HBoV1启动子-荧光素酶基因的质粒,分析NS1的反式转录激活活性。结果表明NS1蛋白可在建立的细胞系中稳定表达,且稳定表达NS1蛋白对HBoV1启动子有较强的激活活性,为进一步研究非结构蛋白NS1的功能及人博卡病毒致病机理奠定了良好的基础。     

Purification and characterization of recombinant human respiratory syncytial virus nonstructural protein NS1

We report here the first biochemical and structural characterization of the respiratory syncytial virus (RSV) NS1 protein. We have used a pET-ubiquitin expression system to produce respiratory syncytial virus (RSV) NS1 protein in E. coli that contains a hexahistidine-tag on either the amino- or carboxyl-terminus (His(6)-NS1 and NS1-His(6), respectively). We have been able to isolate milligram quantities of highly purified His(6)-NS1 and NS1-His(6) by nickel affinity chromatography. Generation of recombinant RSV indicated that addition of the hexahistidine tag to the C-terminus of NS1 slightly decreased viral replication competence whereas addition of the tag to the N-terminus had no observable effect. Therefore, we performed a comprehensive biochemical and biophysical characterization on His(6)-NS1. His(6)-NS1 is monodisperse in solution as determined by dynamic light scattering analysis. Both gel filtration and analytical ultracentrifugation showed that His(6)-NS1 is predominantly a monomer. In agreement with theoretical predictions, circular dichroism spectroscopy showed that His(6)-NS1 contains 21% alpha-helices, 34% beta-sheets, and 45% undefined structure. Immunization with purified His(6)-NS1 generated an antiserum that specifically recognizes NS1 by immunoprecipitation from HEp-2 cells infected by RSV, indicating that His(6)-NS1 resembles native NS1. The availability of purified RSV NS1 will permit biochemical and structural investigations providing insight into the function of NS1 in viral replication and interferon antagonism.     

Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1

The non-structural (NS) proteins of parvoviruses are involved in essential steps of the viral life cycle. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to the protein NS1. Compared with the non-structural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been well characterized. Here, we describe the biochemical properties of NS1 of Periplaneta fuliginosa densovirus (PfDNV). We have expressed and purified NS1 using a baculovirus system and analyzed its enzymatic activity. The purified recombinant NS1 protein possesses ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg(2+) or Mn(2+) as a cofactor. The ATPase activity of NS1 can be efficiently stimulated by single-stranded DNA. The ATPase coupled helicase activity was detected on blunt-ended double-stranded oligonucleotide substrate. Using South-Western and Dot-spot assays, we identified a DNA fragment that is recognized specifically by the recombinant NS1 protein. The fragment consists of (CAC)(4) and is located on the hairpin region of the terminal palindrome. The domain for DNA binding was defined to the amino-terminal region (amino acids 1-250). In addition, we found that NS1 can form oligomeric complexes in vivo and in vitro. Mutagenesis analysis showed that ATP binding is necessary for oligomerization. Based on these results, it seems that PfDNV NS1, a multifunctional protein, plays an important role in viral DNA replication comparable to those of vertebrate parvovirus initiator proteins.     

Expression and characterization of a multivalent human respiratory syncytial virus protein

Respiratory syncytial virus (RSV) has been recognized as one of the most common causes of severe respiratory tract infection in infants worldwide. As yet, a safe and effective vaccine has not been developed to protect humans from RSV. The F and G surface proteins have been widely investigated due to their potential to induce protective immunity. In addition, the M2 protein has been shown to be important in inducing a T-cell response. Our project involved the cl oning of the immunodominant regions of the RSV F, M2 and G proteins into a bacterial vector, pET-32a(+). The recombinant RFM2G protein was expressed in Escherichia coli and purified using His Bind columns. The purified rRFM2G protein was analyzed by poly-acrylamide gel electrophoresis and Western blotting. The predicted structure of the recombinant protein built by the Swiss PDB Viewer program suggested a rod shape with a distinct swollen head and neck which was confirmed by transmission electron microscopy and atomic force microscopy. BALB/c female mice were immunized with either RSV, rRFM2G alone, or rRFM2G in combination with flagellin as a mucosal adjuvant. Serum was collected on days 0, 14, 28 and 49 to assess the immune response by Enzyme-linked immunosorbent assay. Intranasal immunization of mice with the rRFM2G protein yielded significantly high serum IgG titers. Co-administration of the rRFM2G protein with flagellin did not augment the serum antibody response.     

可调控表达甲型流感病毒M2蛋白的哺乳动物细胞系的建立与鉴定

甲型流感病毒M2蛋白是一种具有离子通道功能的跨膜蛋白,其氨基酸序列非常保守,可用于流感通用疫苗的研究。为了构建可调控的稳定表达甲型流感病毒M2蛋白的哺乳动物细胞系,首先应用PCR方法从含有流感病毒PR8株第七节段全长基因的质粒中扩增得到M2基因。将该片段亚克隆到真核表达载体pcDNA5/FRT/TO上,用BamHⅠ和NotⅠ双酶切鉴定正确后将重组质粒与表达Flp重组酶的pOG44质粒共转染Flp-In T-REx-293细胞,使目的基因整合到宿主细胞染色体。筛选具有Hygromycin B抗性的细胞株。在该细胞的培养基中加入四环素以诱导目的基因表达,48 h后通过间接免疫荧光方法检测到M2蛋白的表达。共得到16株高表达M2蛋白的重组细胞株,这些细胞株在传10代后仍能稳定表达目的蛋白。未加四环素诱导的细胞没有检测到M2蛋白,说明四环素调控系统严格控制着目的基因的表达。今后,该细胞系可用于流感病毒M2蛋白的功能研究、流感候选疫苗的免疫学评价以及流感病毒减毒活疫苗的研制。     

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.     

四环素诱导的丙型肝炎病毒非结构蛋白5B稳定细胞株的构建和检测

丙型肝炎病毒(HCV)感染个体后在宿主细胞内长时间保持低水平复制,与慢性肝炎、肝硬化及肝细胞肝癌的发生密切相关.目前,HCV感染后肝细胞发生转化的具体机制还不清楚.非结构蛋白5B(NS5B)是HCV编码的非结构蛋白之一,具有RNA依赖的RNA聚合酶活性(RdRp),是病毒复制所需的关键酶.除参与病毒复制外,NS5B通过...     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.