登革病毒衣壳蛋白靶向核酸酶表达系统的建立及应用

根据登革 2型病毒衣壳蛋白C基因和葡萄球菌核酸酶SN基因序列设计引物 ,从构建的原核表达载体pLEX D2C SN中扩增获得编码登革病毒衣壳蛋白和葡萄球菌核酸酶的融合基因D2C SN ,将其插入到真核表达载体pcDNA6 V5 His中 ,筛选获得重组质粒pcDNA D2C SN .电穿孔转染BHK细胞后 ,5mg Lblasticidin压力筛选 ,通过RT PCR、间接免疫荧光和免疫印迹鉴定表达的蛋白 ,体外DNA消化试验检测核酸酶活性 .结果表明 ,融合蛋白D2C SN在BHK细胞中获得了稳定表达 ,表达的融合蛋白能够被抗登革病毒衣壳蛋白的单克隆抗体特异识别 ,并具有良好的核酸酶活性 ,能够对DNA进行切割 .同时 ,BHK细胞中稳定表达的融合蛋白D2C SN能够有效抑制登革病毒的增殖 ,使其感染性降低 10 3 ～ 10 4倍 .这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于人类抗登革病毒感染奠定了基础     

衣壳蛋白靶向灭活 一种新型抗病毒策略

衣壳蛋白靶向灭活（CTVI）是一种新型抗病毒策略,它是通过将病毒衣壳蛋白与核酸酶（金黄色葡萄球菌和酸梅、核糖核酸酶Barnase、大肠杆菌RNase HI等）的融合蛋白装配到病毒粒子中,使核酸酶接触并降解病毒核酸,从而达到抑制病毒复制的目的。该策略已经在人免疫缺陷病毒、鼠白血病病毒、乙肝病毒、登革病毒等病毒的抗病毒研究中取得良好效果,展示了广阔的应用前景。     

PTEN 通过下调自噬活性抑制登革病毒2型感染

为探讨PTEN、细胞自噬与登革病毒2型（DENV-2）感染之间的关系及可能机制,本研究将DENV-2以不同感染复数（MOI）和不同持续时间感染人脐静脉血管内皮细胞（HUVEC）,蛋白免疫印迹法检测PTEN表达及指示细胞自噬的LC3型别转换情况;进一步构建pLenti-PTEN和pLenti-shPTEN表达质粒,包装成慢病毒,感染 HUVEC以建立稳定表达细胞系,并检测 PTEN过表达及敲低对自噬标记尤其是LC3Ⅱ／LC3Ⅰ比值、DENV-2 capsid蛋白和mRNA水平及子代病毒颗粒感染性的影响。结果显示,PTEN下调可抑制细胞自噬水平,继而降低DENV-2 capsid蛋白表达和子代病毒颗粒释放,提示 DENV-2感染 HUVEC发生的PTEN蛋白表达下调很可能是宿主细胞本身的一种针对DENV-2感染的保护性反应。     

草鱼呼肠孤病毒在CIK细胞中复制及形态发生的研究

以草鱼呼肠孤病毒(GCRV)感染的草鱼肾细胞系(CIK)为模型,进行了草鱼呼肠孤病毒在细胞内的形态发生的研究.当病毒以感染复数为5～10PFU/CELL感染CIK细胞时,在病毒感染细胞4h以内的切片中,可观察到脱去部分外层衣壳的不完整病毒颗粒.感染细胞8h,可观察到浆胞内病毒发生基质,其内含有大量的直径约50nm的亚病毒颗粒,无外层蛋白结构.感染12～16h后,这些亚病毒颗粒装配上外层蛋白结构,形成直径为72nm左右的成熟的病毒粒子.病毒感染细胞8h后,开始出现典型的病毒包含体,16～20h小时病毒包含体裂解,继而释放出有感染性的子代病毒颗粒.该结果有助于对GCRV致病机理的了解.     

猪瘟病毒衣壳蛋白靶向核酸酶表达系统的建立及鉴定

根据猪瘟病毒衣壳蛋白(C)基因序列设计一对引物,RT-PCR扩增获得编码猪瘟病毒衣壳蛋白的C基因,将其插入到含有葡萄球菌核酸酶(SN)基因的真核表达载体pcDNA-SN中,筛选获得重组质粒pcDNA-C-SN。脂质体转染猪肾细胞(PK-15),并经G418稳定筛选,通过RT-PCR、免疫印迹和间接免疫荧光鉴定表达的融合蛋白,体外DNA消化试验检测核酸酶活性。结果表明融合蛋白C-SN在PK-15细胞中获得了稳定表达,能够被兔抗猪瘟病毒衣壳蛋白多抗所识别,并具有良好的核酸酶活性,能够对DNA进行切割。同时,稳定表达融合蛋白C-SN的PK-15细胞系中能够有效抑制猪瘟野毒的增殖,使其感染性降低102～103倍。这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于抵抗猪瘟病毒感染奠定了基础。     

风疹病毒衣壳蛋白与宿主细胞相互作用蛋白的初步筛选及分析

风疹病毒(Rubella virus,RV)的衣壳蛋白(Capsid protein,CP)不仅是病毒颗粒的重要组成部分,而且还可以通过与宿主蛋白之间的相互作用来调控病毒的转录和复制.为了系统研究衣壳蛋白与宿主蛋白之间的相互作用关系,我们从RV基因组中克隆获得衣壳蛋白基因序列,将该序列导入含有eXact-6His串联亲和纯化标签的慢病毒表达载体中,并构建了稳定表达eXact-6His-Capsid融合蛋白的293T细胞系.通过eXact和6His标签的两次亲和纯化获得衣壳蛋白相互作用蛋白复合物,质谱检测并筛选后发现22个可能与衣壳蛋白相互作用的宿主蛋白.随后构建衣壳蛋白的相互作用网络并进行功能学分析,发现其相互作用蛋白主要参与病毒感染、RNA剪切、细胞凋亡及酶相关通路等过程.     

SINDBIS病毒对宿主细胞基因表达的影响

Sindbis病毒(SBV)的感染能迅速地抑制宿主细胞的基因表达(mRNA合成与蛋白质合成),但细胞rRNA的合成水平与正常细胞接近.同时SBV还诱导产生一种细胞特异的核基质结合蛋白P105.用放线菌素D处理细胞,导致感染细胞中病毒结构蛋白的合成量及有感染力的子代病毒产量明显下降.实验结果不仅显示了SBV对宿主细胞基因表达的复杂调控关系,而且还表明SBV的非结构蛋白nsP2和衣壳蛋白C可能直接参与这一过程.     

登革2型病毒衣壳蛋白在哺乳动物细胞中的表达与鉴定

从构建的重组质粒pLEX—C中高保真PCR获得编码登革2型病毒43株C基／E／（D2C）DNA片段,通过基因重组的方法将其克隆入真核表达载体pcDNA6／V5-His获得了重组真核表达载体pc／D2C。经电穿孔的方法转染BHK21细胞后,分别通过RT—PCR、免疫荧光和western印迹鉴定表达的蛋白。结果重组蛋白在BHK21细胞中获得表达,表达的蛋自主要存在于胞浆中,并具有较好的抗原性,能够被抗登革病毒衣壳蛋白单克隆抗体特异识别。此研究为深入了解登革病毒衣壳蛋白在病毒复制及组装过程中的生物学功能奠定了基础。     

细胞松弛素D对棉铃虫核型多角体病毒复制和装配的影响

杆状病毒感染引起宿主细胞肌动蛋白骨架的构象变化 ,使之形成缆绳结构 .棉铃虫核型多角体病毒 (HaNPV)的衣壳蛋白也能使宿主昆虫的肌动蛋白发生凝聚 ,用细胞松弛素D抑制宿主肌动蛋白形成纤丝结构 ,病毒感染Hz AM1,空斑计数表明 ,0 1μg/ml细胞松弛素D可使棉铃虫核型多角体病毒的增殖下降 10 4倍 ,细胞松弛素D浓度增高到 0 5 μg/ml则测不到子代病毒粒子 .Western印迹分析表明 ,细胞松弛素D并不影响受染细胞中肌动蛋白的含量 .斑点印迹 (dotblot)也表明 ,病毒DNA的合成也没有受到影响 ,推测宿主细胞的肌动蛋白纤丝结构与病毒的复制有关 .在电子显微镜下观察超薄切片发现 ,在 0 5 μg/ml细胞松弛素D处理细胞中形成的病毒粒子形态与正常形态明显不同 ,提示细胞松弛素D抑制HaNPV的增殖是由于抑制病毒组装成完整有感染性的病毒粒子 .从而可以认为宿主昆虫细胞的丝状肌动蛋白对子代病毒的复制和组装是必需的 .     

登革病毒衣壳蛋白在大肠杆菌中的表达及其自组装活性的研究

采用高保真RT-PCR自登革2型病毒43株基因组RNA中扩增全长C基因及缺失羧基端Cv片段,分别构建可表达C及Cv的重组质粒pLEX—C和pLEX—Cv,转化E．coliGI724后用色氨酸诱导表达。经SDS—PAGE分析,表达的C及Cv蛋白相对分子质量分别约为12000和10000,分别约占菌体蛋白总量的19％和13％。Western印迹检测表明重组表达的C蛋白均可被特异识别登革病毒衣壳蛋白的单克隆抗体特异识别。表达的蛋白经过硫酸铵沉淀和蔗糖密度梯度离心后,通过琼脂糖凝胶电泳和负染电镜均未能检测到衣壳样颗粒的存在,说明登革病毒衣壳蛋白可能不具体外自组装活性。     

Therapeutic effect of Gag-nuclease fusion protein on retrovirus-infected cell cultures.

Capsid-targeted viral inactivation is a novel protein-based strategy for the treatment of viral infections. Virus particles are inactivated by targeting toxic fusion proteins to virions, where they destroy viral components from within. We have fused Staphylococcus nuclease (SN) to the C-terminal end of Moloney murine leukemia virus Gag and demonstrated that expression of this fusion protein in chronically infected chicken embryo fibroblasts resulted in its incorporation into virions and subsequent inactivation of the virus particles by degradation of viral RNA. Release of particles incorporating Gag-SN fusion proteins into the extracellular milieu activates the nuclease and results in destruction of the virion from within. By comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of SN, on the infectivity of virus particles, we have clearly demonstrated that nucleolytic activity is the antiviral mechanism. Expression of Gag-SN fusion proteins as a therapeutic agent causes a stable reduction of infectious titers by 20- to 60-fold. The antiviral effect of capsid-targeted viral inactivation in our model system, using both prophylactic and therapeutic approaches, suggests that a similar anti-human immunodeficiency virus strategy might be successful.     

Development of cell lines stably expressing staphylococcal nuclease fused to dengue 2 virus capsid protein for CTVI

Dengue virus (DV) is one of the most significanthuman viral pathogens transmitted by arthropod vectorsand now present in over 100 countries. Half of the world’spopulation live in areas at risk of dengue virus infection[1]. The infection of DV causes a spectrum of diseasesranging from a debilitating, self-limited illness (denguefever), and life-threaten syndromes (dengue haemorrhagicfever/dengue shock syndrome). Annually, the fourserotypes of DV collectively cause 50–100 million casesof inf…     

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.     

Expression of a murine leukemia virus Gag-Escherichia coli RNase HI fusion polyprotein significantly inhibits virus spread.

The antiviral strategy of capsid-targeted viral inactivation (CTVI) was designed to disable newly produced virions by fusing a Gag or Gag-Pol polyprotein to a degradative enzyme (e.g., a nuclease or protease) that would cause the degradative enzyme to be inserted into virions during assembly. Several new experimental approaches have been developed that increase the antiviral effect of the CTVI strategy on retroviral replication in vitro. A Moloney murine leukemia virus (Mo-MLV) Gag-Escherichia coli RNase HI fusion has a strong antiviral effect when used prophylactically, inhibiting the spread of Mo-MLV and reducing virus titers 1,500- to 2,500-fold. A significant (approximately 100-fold) overall improvement of the CTVI prophylactic antiviral effect was produced by a modification in the culture conditions which presumably increases the efficiency of delivery and expression of the Mo-MLV Gag fusion polyproteins. The therapeutic effect of Mo-MLV Gag-RNase HI polyproteins is to reduce the production of infectious Mo-MLV up to 18-fold. An Mo-MLV Gag-degradative enzyme fusion junction was designed that can be cleaved by the Mo-MLV protease to release the degradative enzyme.     

Therapeutic Effect of a Gag-Nuclease Fusion Protein against Retroviral Infection In Vivo

Recently, remarkable progress has been made in developing effective combination drug therapies that can control but not cure retroviral replication. Even when effective, these drug regimens are toxic, they require demanding administration schedules, and resistant viruses can emerge. Thus the need for new gene-based therapies continues. In one such approach, capsid-targeted viral inactivation (CTVI), nucleases fused to viral coat proteins are expressed in infected cells and become incorporated during virion assembly. CTVI can eliminate infectious murine retrovirus titer in tissue culture. Here we describe transgenic mice expressing fusions of the Moloney murine leukemia virus (Mo-MuLV) Gag protein to staphylococcal nuclease. This work tests the protective effect and demonstrates in vivo proof-of-principle of CTVI in transgenic mice expressing endogenous proviral copies of Mo-MuLV. The antiviral protein-expressing mice are phenotypically normal, attesting to the lack of toxicity of the fusion protein. The Mo-MuLV infection was much less virulent in transgenic littermates than in nontransgenic littermates. Gag-nuclease expression reduced infectious titers in blood up to 10-fold, decreased splenomegaly and leukemic infiltration, and increased life spans up to 2.5-fold in transgenic relative to nontransgenic infected animals. These results suggest that gene therapies based on similar fusion proteins, designed to attack human immunodeficiency virus or other retroviruses, could provide substantial therapeutic benefits.