The dengue virus NS2B–NS3 protease retains the closed conformation in the complex with BPTI

The C-terminal β-hairpin of NS2B (NS2Bc) in the dengue virus NS2B–NS3 protease is required for full enzymatic activity. In crystal structures without inhibitor and in the complex with bovine pancreatic trypsin inhibitor (BPTI), NS2Bc is displaced from the active site. In contrast, nuclear magnetic resonance (NMR) studies in solution only ever showed NS2Bc in the enzymatically active closed conformation. Here we demonstrate by pseudocontact shifts from a lanthanide tag that NS2Bc remains in the closed conformation also in the complex with BPTI. Therefore, the closed conformation is the best template for drug discovery.     

Backbone 1H, 13C and 15N resonance assignments of dengue virus NS2B–NS3p in complex with aprotinin

Dengue virus, belongs to Flaviviridae, is an arthropod transmitted virus that threatens millions of people’s lives. As with other flaviviruses, a positive single-stranded 11-kilobases RNA in the dengue virus genome encodes three structural proteins (capsid protein C, membrane protein M, and envelope protein E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The two component protease NS2B–NS3p is essential for viral replication and is believed to be a potential antiviral drug target. Aprotinin, a native inhibitor, is proved to retard the activity of NS2B–NS3p. The backbone assignments of NS2B–NS3p will be essential for determining the high resolution solution structure of NS2B–NS3p and screening new antiviral drugs. Herein, we report the backbone ¹H, ¹⁵N, ¹³C resonance assignments of the N terminal fragment of NS2B (4.8 kDa) and NS3p (18.5 kDa) in complex with aprotinin (6.5 kDa) by high resolution NMR.     

3D-QSAR pharmacophore-based virtual screening,molecular docking and molecular dynamics simulation toward identifying lead compounds for NS2B–NS3 protease inhibitors

NS2B–NS3 protease has been identified to serve as lead drug design target due to its significant role in West Nile viral (WNV) and dengue virus (DENV) reproduction and replication. There are currently no approved chemotherapeutic drugs and effective vaccines to inhibit DENV and WNV infections. In this work, 3D-QSAR pharmacophore model has been developed to discover potential inhibitory candidates. Validation through Fischer’s model and decoy test indicate that the developed 3D pharmacophore model is highly predictive for DENV inhibitors, which was then employed to screen ZINC chemical library to obtain reasonable hits. Following ADMET filtering, 15 hits were subjected to further filter through molecular docking and CoMFA modeling. Finally, top three hits were identified as lead compounds or potential inhibitory candidates with IC₅₀ values of ～0.4637?µM and fitness of ～57.73. It is implied from CoMFA modeling that substituents at the side site of benzotriazole such as a p-nitro group (e.g. biphenyl head) and a carbonyl (e.g. carboxylate function) at the side site of furan or amino group may improve bioactivity of ZINC85645245, respectively. Molecular dynamics simulations (MDS) were performed to discover new interactions and reinforce the binding modes from docking for the hits also. The QSAR and MDS results obtained from this work should be useful in determining structural requirements for inhibitor development as well as in designing more potential inhibitors for NS2B–NS3 protease.     

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 2: Synthesis and structure–activity relationships of benzothiazine-substituted quinolinediones

A new series of benzothiazine-substituted quinolinediones were evaluated as inhibitors of HCV polymerase NS5B. SAR studies on this series revealed a methyl sulfonamide group as a high affinity feature. Analogues with this group showed submicromolar potencies in the HCV cell based replicon assay. Pharmacokinetic and toxicology studies were also performed on a selected compound (34) to evaluate in vivo properties of this new class of inhibitors of HCV NS5B polymerase.     

HCV NS5B蛋白对HCV RNA的模板特异性研究

在大肠杆菌菌株BL21(DE3)中表达并纯化HCV的依赖于RNA的RNA多聚酶(RNAdependentRNApolymerase,RdRp,NS5B蛋白)。以HCV正、负链RNA3′末端的序列为模板,体外研究NS5B蛋白催化的RNA合成。结果显示,正链RNA在体外不能指导RNA合成,而负链RNA模板可以产生一条全长的正链RNA产物,表明NS5B对负链RNA具有模板特异性。NS5B对负链RNA的特异性在模板竞争性实验中得到进一步证实,正链RNA的存在和竞争对以负链为模板的RNA合成没有影响。这样,就合理解释了在HCVRNA复制时正链RNA的数量远比负链RNA多这一问题。同时,本实验的结果也为进一步研究病毒或其它细胞因子参与以正链RNA为模板进行的RNA合成,以及有关负链RNA模板特性的研究奠定了基础。     

丙型肝炎病毒NS3-5B转基因小鼠模型的构建

目的:构建表达丙型肝炎病毒(HCV)NS3-5B蛋白的转基因小鼠模型。方法:显微注射线性化p IRES2-EGFP-NS3-5B载体到小鼠受精卵,制备并传代筛选HCV NS3-5B转基因小鼠;通过血清谷丙转氨酶(ALT)、谷草转氨酶(AST)检测和肝脏HE染色,对6周龄转基因小鼠进行肝功能评价。结果:PCR、RT-PCR和Western印迹结果表明HCV NS3-5B转基因小鼠构建成功;6周龄部分转基因小鼠血清ALT和AST值升高,但差别无统计学意义,肝组织形态无改变。结论:构建了HCV NS3-5B转基因小鼠模型,为进一步在体研究HCV复制复合体建立了平台。     

HCV NS5B蛋白对HCV RNA的模板特异性研究

在大肠杆菌菌株BL21(DE3)中表达并纯化HCV的依赖于RNA的RNA多聚酶(RNA-dependent RNA polymerase,RdRp,NS5B蛋白).以HCV正、负链RNA 3′末端的序列为模板,体外研究NS5B蛋白催化的RNA合成.结果显示,正链RNA在体外不能指导RNA合成,而负链RNA模板可以产生一条全长的正链RNA产物,表明NS5B对负链RNA具有模板特异性.NS5B对负链RNA的特异性在模板竞争性实验中得到进一步证实,正链RNA的存在和竞争对以负链为模板的RNA合成没有影响.这样,就合理解释了在HCV RNA复制时正链RNA的数量远比负链RNA多这一问题.同时,本实验的结果也为进一步研究病毒或其它细胞因子参与以正链RNA为模板进行的RNA合成,以及有关负链RNA模板特性的研究奠定了基础.     

Characterization of interactions between hepatitis C virus NS5B polymerase,annexin A2 and RNA – effects on NS5B catalysis and allosteric inhibition

Background

Direct acting antivirals (DAAs) provide efficient hepatitis C virus (HCV) therapy and clearance for a majority of patients, but are not available or effective for all patients. They risk developing HCV-induced hepatocellular carcinoma (HCC), for which the mechanism remains obscure and therapy is missing. Annexin A2 (AnxA2) has been reported to co-precipitate with the non-structural (NS) HCV proteins NS5B and NS3/NS4A, indicating a role in HCC tumorigenesis and effect on DAA therapy.

Methods

Surface plasmon resonance biosensor technology was used to characterize direct interactions between AnxA2 and HCV NS5B, NS3/NS4 and RNA, and the subsequent effects on catalysis and inhibition.

Results

No direct interaction between AnxA2 and NS3/NS4A was detected, while AnxA2 formed a slowly dissociating, high affinity (K _D?=?30 nM), complex with NS5B, decreasing its catalytic activity and affinity for the allosteric inhibitor filibuvir. The RNA binding of the two proteins was independent and AnxA2 and NS5B interacted with different RNAs in ternary complexes of AnxA2:NS5B:RNA, indicating specific preferences.

Conclusions

The complex interplay revealed between NS5B, AnxA2, RNA and filibuvir, suggests that AnxA2 may have an important role for the progression and treatment of HCV infections and the development of HCC, which should be considered also when designing new allosteric inhibitors.

     

寨卡病毒NS2B蛋白的表达纯化及单克隆抗体制备

目的:应用大肠杆菌重组表达并纯化寨卡病毒NS2B蛋白,制备抗NS2B蛋白的单克隆抗体。方法:构建携带NS2B基因的重组原核表达质粒,采用大肠杆菌ER2566作为表达菌株,以IPTG诱导表达,通过亲和层析纯化获得NS2B目的蛋白。用纯化后的NS2B蛋白免疫BALB/c小鼠,筛选制备抗NS2B蛋白的单克隆抗体,并初步分析抗体的反应活性和特异性。结果:表达并纯化获得寨卡病毒NS2B蛋白,筛选获得可与NS2B蛋白结合并具有较好反应活性的单克隆抗体2H11,该单抗可识别寨卡病毒感染细胞后表达的NS2B蛋白。结论:筛选获得靶向寨卡病毒NS2B蛋白的单克隆抗体,可为后期深入开展寨卡病毒NS2B蛋白的功能和相关抗病毒药物研究提供支持。     

The discovery and structure–activity relationships of pyrano[3,4-b]indole based inhibitors of hepatitis C virus NS5B polymerase

We describe the structure–activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.     

丙型肝炎病毒非结构蛋白NS4B抑制IFN介导的抗病毒反应

丙型肝炎病毒(Hepatitis C Virus,HCV)非结构蛋白NS4B的功能仍不很清楚。本实验旨在研究NS4B对干扰素介导的抗病毒反应的影响。建立稳定表达NS4B的细胞系后,用空斑实验研究NS4B在不同浓度的IFN_α下对水泡口炎病毒(VSV)的影响,利用代表2308个信号传导相关的基因的微点阵研究其对细胞基因表达的影响,和流式细胞仪分析IFNGR1的荧光强度。结果显示HCV_NS4B能微弱地抑制IFN_α介导的抗病毒反应,可能原因是HCV_NS4B抑制一些与免疫反应相关的基因的表达,特别是与IFN_γ信号传导有关的基因。因此,NS4B对HCV耐受干扰素治疗起一定的作用。     

丙型肝炎病毒非结构蛋白NS4B诱导细胞非折叠蛋白反应

用RT-PCR和免疫印迹的方法检测稳定表达NS4B的HeLa细胞中的XBP1;通过RT-PCR的方法在表达NS4B的HeLa和Huh-7细胞中检测ATF6,Grp78和caspase-12的转录,并且通过报告基因的方法分析XBP1和Grp78启动子活性.实验结果表明在表达NS4B的HeLa细胞中检测到XBP1的两种形式(剪接和未剪接),此外,在细胞中ATF6、Grp78的转录水平和XBP1、Grp78启动子的荧光素酶活性较没有表达NS4B的HeLa和Huh-7细胞中的量有所增加;通过染色质免疫沉淀实验(ChIP)分析,这些增加可能是由于XBP1结合到了这些基因的启动子上引起的.总之,实验结果可提示HCV NS4B通过ATF6或XBP1途径引起内质网压力,导致UPR反应.NS4B可能在HCV的致病性中起着重要的作用,特别是在慢性肝炎,甚至肝细胞癌中.     

丙型肝炎病毒非结构蛋白NS4B诱导细胞非折叠蛋白反应

用RT-PCR和免疫印迹的方法检测稳定表达NS4B的HeLa细胞中的XBP1;通过RT-PCR的方法在表达NS4B的HeLa和Huh-7细胞中检测ATF6,Grp78和caspase-12的转录,并且通过报告基因的方法分析XBP1和Grp78启动子活性。实验结果表明:在表达NS4B的HeLa细胞中检测到XBP1的两种形式(剪接和未剪接),此外,在细胞中ATF6、Grp78的转录水平和XBP1、Grp78启动子的荧光素酶活性较没有表达NS4B的HeLa和Huh-7细胞中的量有所增加;通过染色质免疫沉淀实验(ChIP)分析,这些增加可能是由于XBP1结合到了这些基因的启动子上引起的。总之,实验结果可提示HCVNS4B通过ATF6或XBP1途径引起内质网压力,导致UPR反应。NS4B可能在HCV的致病性中起着重要的作用,特别是在慢性肝炎,甚至肝细胞癌中。     

丙型肝炎病毒NS2蛋白在Huh-7细胞中抑制NF-κB活性

将丙型肝炎病毒(HCV)非结构蛋白NS2基因的全长序列插入到真核表达载体pCDNA3.1(-)CMV启动子下游,构建成重组质粒pCNS2.用脂质体LipoVecTM转染Huh-7细胞,转染细胞内可检出NS2的mRNA和蛋白质,表明构建的pCNS2可在Huh-7细胞内成功表达;把不同剂量的pCNS2质粒DNA与报告质粒pNF-κ B-Luc共转染Huh-7细胞,48h后检测荧光素酶活性,结果显示与pCNS2共转染的细胞中pNF-κ B-Luc表达出的荧光素酶的活性比对照细胞降低了约2～4倍,并呈明显的剂量相关性.表明HCV NS2对NF-κ B激活转录活性有明显的抑制作用.这可能与HCV慢性持续性感染的致病性有一定的相关性.     

NS5B与HCV负链RNA 3＇末端特异性结合的分析

NS5B是RNA依赖性RNA聚合酶,在病毒RNA合成过程中起到中心催化酶的作用.在大肠杆菌中表达和提纯了GST-NS5B融合蛋白,应用紫外交联试验(UVcross-linking)检测NS5B与丙型肝炎病毒(HCV)负链RNA3＇末端的结合,确定NS5B是否参与HCV负链RNA3＇末端复制体的形成.NS5B可与HCV负链RNA3＇末端发生结合,这种结合存在量效关系,比与正链RNA3＇UTRX区的结合强约10倍,超大量的非同源性RNA和蛋白质不能竞争抑制NS5B与负链RNA3＇末端的结合,证明这种结合存在特异性.结果提示NS5B是HCV负链RNA3＇末端复制体的成分之一.     

B型流感病毒NS1蛋白羧基端缺失对病毒致病性的影响分析

本研究利用基因工程技术探索B型流感病毒NS1蛋白羧基端缺失对病毒致病性的影响,并利用反向遗传学技术从体外拯救NS1蛋白羧基端缺失的B型流感病毒,评估去除NS1蛋白羧基端对B型流感病毒致病性的影响,探究B型流感病毒致病性变异的分子基础。通过全基因合成和反向遗传学技术从体外拯救NS1蛋白羧基端发生171个氨基酸缺失的B型流感病毒B/Yamagata/16/88株,命名为B-L5。将实验室之前拯救的母本株B/Yamagata/16/88(以下简称B-S9)和B-L5以3×105 EID5 0的攻毒剂量分别人工感染BALB/c小鼠,通过体重变化、生存率、肺脏病毒滴度等方面进行致病性分析。成功构建了B型流感病毒B/Yamagata/16/88NS1蛋白羧基端缺失株反向遗传平台。母本株B-S9和NS1蛋白羧基端缺失株B-L5均能够人工感染BALB/c小鼠,但不致死,对BALB/c小鼠均呈现低致病性;B-S9感染小鼠后,从攻毒后第3d至第7d,体重持续下降,相反,B-L5感染小鼠后,只在攻毒后第2d出现体重下降,攻毒后第3d体重开始回升;B-S9和B-L5感染小鼠后均能在小鼠肺部进行复制,但攻毒后第3d,B-L5感染小鼠肺脏滴度要比B-S9低7 900倍,攻毒后第6d,B-L5感染小鼠肺脏已检测不到病毒复制。实验结果表明,NS1蛋白羧基端缺失可以明显降低B型流感病毒对小鼠的致病性。B型流感病毒致病性变异的分子基础还有待继续研究,该反向遗传操作系统的建立为B型流感病毒分子机制的研究奠定了基础,同时也为B型流感减毒活疫苗的研究开辟了新途径。     

NS5B与HCV负链RNA 3′末端特异性结合的分析

NS5B是RNA依赖性RNA聚合酶，在病毒RNA合成过程中起到中心催化酶的作用，在肠杆菌中表达和提纯了GST－NS5B融合蛋白，应用紫外交联试验（UV cross-linking）检测NS5B与丙型肝炎病毒（HCV）负链RNA3′末端的结合，确定NS5B是否参与HCV负链与HCN负链RNA3′末端复制体的形成。NS5B可与HCV负链RNA3′末端发生结合，这种结合存在量效关系， 比与正链RNA3′UTR X区的结合强约10倍，超大量的非同源性RNA和蛋白质不能竞争抑制S5B与负链RNA3′末端的结合，证明这种结合存在特异性。结果提示NS5B是HCV负链RNA3′末端复制体的成分之一。     

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.