Comparative Virus-Host Protein Interactions of the Bluetongue Virus NS4 Virulence Factor

Bluetongue virus (BTV) is the etiologic agent of a non-contagious arthropod-borne disease transmitted to wild and domestic ruminants. BTV induces a large panel of clinical manifestations ranging from asymptomatic infection to lethal hemorrhagic fever. Despite the fact that BTV has been studied extensively, we still have little understanding of the molecular determinants of BTV virulence. In our report, we have performed a comparative yeast two-hybrid (Y2H) screening approach to search direct cellular targets of the NS4 virulence factor encoded by two different serotypes of BTV: BTV8 and BTV27. This led to identifying Wilms’ tumor 1-associated protein (WTAP) as a new interactor of the BTV-NS4. In contrast to BTV8, 1, 4 and 25, NS4 proteins from BTV27 and BTV30 are unable to interact with WTAP. This interaction with WTAP is carried by a peptide of 34 amino acids (NS4²²⁻⁵⁵) within its putative coil-coiled structure. Most importantly, we showed that binding to WTAP is restored with a chimeric protein where BTV27-NS4 is substituted by BTV8-NS4 in the region encompassing residue 22 to 55. We also demonstrated that WTAP silencing reduces viral titers and the expression of viral proteins, suggesting that BTV-NS4 targets a cellular function of WTAP to increase its viral replication.     

HCV耐药突变在三个Ⅰb期临床试验中对DAA单药给药后短期疗效影响的分析

耐药相关突变(Resistance-associated substitutions,RASs)是丙型肝炎病毒(Hepatitis C virus,HCV)治疗的关键因素,并与一些直接抗病毒药物(Direct acting antivirals,DAAs)为基础的治疗方案的治疗结果相关.在本研究中,我们主要分析了三个Ⅰ...     

乙型脑炎病毒NS5蛋白原核表达与亚细胞定位研究

目的对乙型脑炎病毒NS5蛋白进行原核表达和亚细胞定位研究。方法通过PCR扩增乙型脑炎病毒NS5基因,并将之克隆至原核表达载体pET-28a（＋）中,构建的重组表达载体转化大肠杆菌感受态细胞,经IPTG诱导表达目的蛋白。将表达产物纯化后,免疫BALB/c小鼠,制备NS5蛋白特异性的多克隆抗体。同时,构建NS5基因与绿色荧光蛋白（EG-FP）融合表达的真核表达质粒pcDNA-NS5-EGFP,将之转染BHK-21细胞,利用激光共聚焦荧光显微镜观察NS5蛋白在BHK-21细胞中的亚细胞定位。结果 NS5蛋白在大肠杆菌中获得了高效表达,表达蛋白分子量大小约为103kD,该蛋白在真核细胞中的表达呈颗粒状不均匀分布于整个细胞,且颗粒状主要分布在细胞质中。结论成功表达乙型脑炎病毒NS5蛋白,并对NS5蛋白的亚细胞定位进行了分析,为进一步研究NS5蛋白提供依据。     

HCV NS5A基因在hepG2细胞中的表达以及对PI3K的影响

目的：探讨HCV—NS5A对PI3K表达的影响。方法：应用PCR技术从含有HCV全长开放阅读框的质粒中获得NS5A全长基因片段，利用基因重组技术将其克隆至真核表达载体pcDNA3．0（-）中。通过酶切、PCR及测序鉴定，NS5A基因已正确插入到pcDNA3．0（-）中，再利用脂质体转染HepG2细胞。结果：经RT—PCR及Western blot检测，HCV的NS5A基因在HepG2细胞中获得表达，而且在表达重组NS5A的转染HepG2细胞中，检测到PI3K蛋白的表达。结论：NS5A可在体外激活PI3K及其信号通路。     

丙型肝炎病毒非结构蛋白NS5A反式激活基因的克隆化研究

应用抑制性消减杂交技术构建丙型肝炎病毒非结构蛋白5A（HCV NS5A）反式激活基因差异表达的cDNA消减文库，克隆HCV NS5A蛋白反式激活相关基因。以HCV NS5A表达质粒pcDNA3.1(－）-NS5A转染HepG2细胞，以空载体pcDNA3.1(－）为对照，制备转染后的细胞裂解液，提取mRNA并逆转录为cDNA，经RsaI酶切后，将实验组cDNA分成两组，分别与两种不同的接头衔接，再与对照组cDNA进行消减杂交及两次抑制性PCR，将产物与T／A载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆PCR扩增后进行测序及同源性分析。文库扩增后得到121个阳性克隆，经菌落PCR分析，得到115个200－1000bp的插入片段，对其中的90个片段测序，并进行同源性分析，显示31种在基因编码蛋白和15种未知功能基因序列，包括一些与细胞周期、细胞凋亡、信号传导及肿瘤发生等细胞生长调节密切相关的蛋白编码基因，可能是NS5A反式激活靶基因。结果提示，成功构建了HCV NS5A反式激活基因差异表达的cDNA消减文库，该文库的建立为进一步阐明HCV NS5A反式调节的靶基因及致肝细胞癌发生的分子生物学机制提供了理论依据。     

以AdMax载体系统构建携带HCV NS5B基因的重组腺病毒表达载体

目的构建表达丙型肝炎病毒(HCV)非结构基因NS5B的重组腺病毒表达载体,并对其相关指标进行鉴定,为进一步研究防止丙型肝炎感染的基因免疫和基因治疗奠定实验基础。方法应用基因工程技术将HCVNS5B基因定向克隆至穿梭质粒pDC316上,利用脂质体介导的方法将AdMax腺病毒包装系统的骨架质粒pBHGloxΔE1、3Cre和穿梭质粒pDC316-NS5B共转染293细胞,进行同源重组,产生重组腺病毒pAd-NS5B并进行鉴定、反复感染293细胞进行扩增,扩增后测定重组病毒滴度。结果重组病毒颗粒pAd-NS5B经双引物PCR、凝胶电泳证明插入片段与HCV非结构基因NS5B片段大小相符;经测序证明其插入序列与设计HCV NS5B基因序列完全一致,扩增后重组病毒滴度达到2.3×1013IU/L。结论成功构建了表达HCV NS5B基因的重组腺病毒载体。     

HCV NS5A反式激活基因 NS5ATP13在毕氏酵母中的表达研究

目的 探讨NS5AATP13能否在毕氏酵母中成功表达.方法 用Bst X I将pPICZαANS5ATP13线性化后,电转化毕氏酵母菌株KM71H,用Zeocin筛选高拷贝数的酵母菌落,在BMGY和BMMY培养基中进行诱导表达,以甲醇诱导,30℃培养4 d,表达产物进行SDS-PAGE和Western blot 分析.结果 预期分子量大小的NS5ATP13蛋白分泌至培养液.结论 NS5ATP13在毕氏酵母中成功表达.     

丙肝病毒基因在肝门部胆管癌组织中的表达及其意义

目的 探讨丙肝病毒核糖核酸（ＨＣＶ ＲＮＡ）及蛋白在肝门部胆管癌组织中的表达及其生物学意义。方法 应用套式逆转录、多聚酶链反应（ｎｅｓｔｅｄ ＲＴ－ＰＣＲ）和免疫组织化学技术检测３６例肝门部胆管癌患者癌组织和癌旁组织中ＨＣＶ ＲＮＡ、非结构蛋白（ＮＳＳ）蛋白水平的表达。结果 ３６例肝门部这癌患者癌组织和癌旁组织中ＨＣＶ ＲＮＡ和ＮＳ５蛋白阳性表达率为８３．３％（３０／３６）４７．２％（１７／３６）     

Inflammatory markers neopterin and alanine aminotransferase in HCV patients treated with HCV NS3•4A protease inhibitor telaprevir (VX-950) and/or peginterferon alfa-2a

Objective. Neopterin is a marker of monocyte/macrophage activity. Alanine aminotransferase (ALAT) is a marker of hepatocyte injury. The aim of this study was to determine changes in neopterin and ALAT levels, as markers of inflammation, in two ancillary studies during two-phase 1b trials of hepatitis C virus (HCV) NS3?4A protease inhibitor telaprevir (VX-950), with or without peginterferon alfa-2a (Peg-IFN). Material and methods. Fifty-four chronic hepatitis C patients (genotype 1) received placebo or telaprevir, with or without Peg-IFN, for 14 days in two multiple-dose studies. Results. During administration of telaprevir, every patient demonstrated a >2-log decrease in HCV RNA. Mean neopterin and ALAT levels decreased in all four groups receiving telaprevir alone. In contrast, mean neopterin levels increased and ALAT levels decreased in the Peg-IFN plus telaprevir and Peg-IFN plus placebo groups. Conclusions. These data suggest that treatment of chronic hepatitis C patients with an HCV NS3?4A protease inhibitor ameliorates inflammation. The increase in neopterin levels and the decrease in ALAT levels during administration of Peg-IFN with or without telaprevir are in accordance with earlier observations showing that IFN reduces hepatocyte injury but increases monocyte/macrophage activity. The IFN-mediated immunomodulatory effects appear to remain intact when IFN is combined with telaprevir.