Influenza A Virus NS1 Protein Structural Flexibility Analysis According to Its Structural Polymorphism Using Computational Approaches

Influenza A viruses are highly contagious RNA viruses that cause respiratory tract infections in humans and animals. Their non-structural protein NS1, a homodimer of two 230-residue chains, is the main viral factor in counteracting the antiviral defenses of the host cell. Its RNA-binding domain is an obligate dimer that is connected to each of the two effector domains by a highly flexible unstructured linker region of ten amino acids. The flexibility of NS1 is a key property that allows its effector domains and its RNA binding domain to interact with several protein partners or RNAs. The three-dimensional structures of full-length NS1 dimers revealed that the effector domains could adopt three distinct conformations as regards their mutual interactions and their orientation relative to the RNA binding domain (closed, semi-open and open). The origin of this structural polymorphism is currently being investigated and several hypotheses are proposed, among which one posits that it is a strain-specific property. In the present study, we explored through computational molecular modeling the dynamic and flexibility properties of NS1 from three important influenza virus A strains belonging to three distinct subtypes (H1N1, H6N6, H5N1), for which at least one conformation is available in the Protein Data Bank. In order to verify whether NS1 is stable in three forms for the three strains, we constructed homology models if the corresponding forms were not available in the Protein Data Bank. Molecular dynamics simulations were performed in order to predict the stability over time of the three distinct sequence variants of NS1, in each of their three distinct conformations. Our results favor the co-existence of three stable structural forms, regardless of the strain, but also suggest that the length of the linker, along with the presence of specific amino acids, modulate the dynamic properties and the flexibility of NS1.     

禽流感病毒NS1A蛋白诱导人肺癌细胞SPC-A1的凋亡

目的研究禽流感病毒NS1A蛋白对人肺癌细胞SPC-A1增殖的影响。方法将含有NS1A基因的重组质粒pVAX1-NS1A经脂质体介导转染人肺癌细胞SPC-A1,用MTT检测其对细胞增殖的抑制作用;Annexin VFITC-PI结合流式细胞仪检测细胞凋亡率;PI结合流式细胞仪检测细胞周期的变化;对转染细胞的表达产物进行Western blot分析。结果NS1A蛋白能抑制人肺癌细胞SPC-A1增殖且呈时间依赖性。随着作用时间延长,重组质粒转染组细胞凋亡率可达38.17%,细胞周期中G0/G1比例升高,G1期阻滞。Western blot分析显示,重组质粒转染组细胞在相对分子质量约30000处可见特异性反应条带,与NS1A蛋白产物大小相符。结论禽流感病毒NS1A蛋白能够抑制SPC-A1细胞增殖,并诱导SPC-A1细胞凋亡。     

人甲型流感病毒H1N1亚型NS1蛋白的原核表达及纯化

目的表达并纯化人甲型流感病毒H1N1亚型NS1蛋白。方法用RT-PCR法从人甲型流感病毒株A/PR/8/34(H1N1)中扩增NS1基因,克隆入原核表达载体pTXB1,构建重组原核表达质粒pTXB1/NS1,经酶切鉴定后,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析表达形式和表达量。经几丁质柱亲和层析纯化表达蛋白,串联飞行时间质谱仪检测其相对分子质量。结果所构建的重组表达质粒pTXB1/NS1序列完整,插入的基因片段全长690bp。以1.0mmol/LIPTG37℃诱导4h,重组蛋白表达量最高,占菌体总蛋白的50%以上。破菌上清及沉淀中均有目的蛋白表达。纯化的NS1蛋白纯度达95%以上,相对分子质量约为26000。结论已成功表达并纯化了人甲型流感病毒H1N1亚型NS1蛋白,为其进一步的研究奠定了基础。     

Glycyrrhizic Acid Derivatives Bearing Amino Acid Residues in the Carbohydrate Part as Dengue Virus E Protein Inhibitors: Synthesis and Antiviral Activity

Dengue virus (DENV) is one of the most geographically distributed mosquito-borne flaviviruses, like Japanese encephalitis virus (JEV), and Zika virus (ZIKV). In this study, a library of the known and novel Glycyrrhizic acid (GL) derivatives bearing amino acid residues or their methyl/ethyl esters in the carbohydrate part were synthesized and studied as DENV inhibitors in vitro using the cytopathic effect (CPE), viral infectivity and virus yield assays with DENV1 and DENV-2 in Vero E6 and A549 cells. Among the GL conjugates tested, compound hits GL-D-ValOMe 3, GL-TyrOMe 6, GL-PheOEt 11, and GL-LysOMe 21 were discovered to have better antiviral activity than GL, with IC50 values ranging from <0.1 to 5.98 μM on the in vitro infectivity of DENV1 and DENV2 in Vero E6 and A549 cells. Compound hits 3, 6, 11, and 21 had a concentration-dependent inhibition on the virus yield in Vero E6, in which GL-D-ValOMe 3 and GL-PheOEt 11 were the most active inhibitors of DENV2 yield. Meanwhile, the time-of-addition assay indicated that conjugates GL-D-ValOMe 3 and GL-PheOEt 11 exhibited a substantial decrease in the DENV2 attachment stage. Subsequently, chimeric single-round infectious particles (SRIPs) of DENV2 C-prM-E protein/JEV replicon and DENV2 prM-E/ZIKV replicon were utilized for the DENV envelope I protein-mediated attachment assay. GL conjugates 3 and 11 significantly reduced the attachment of chimeric DENV2 C-prM-E/JEV and DENV2 prM-E/ZIKV SRIPs onto Vero E6 cells in a concentration-dependent manner but did not impede the attachment of wild-type JEV CprME/JEV and ZIKV prM-E/ZIKV SRIPs, indicating the inhibition of Compounds 3 and 11 on DENV2 E-mediated attachment. Molecular docking data revealed that Compounds 3 and 11 have hydrophobic interactions within a hydrophobic pocket among the interfaces of Domains I, II, and the stem region of the DENV2 envelope (E) protein. These results displayed that Compounds 3 and 11 were the lead compounds targeting the DENV E protein. Altogether, our findings provide new insights into the structure–activity relationship of GL derivatives conjugated with amino acid residues and can be the new fundamental basis for the search and development of novel flavivirus inhibitors based on natural compounds.     

38例甲型H1N1流感患者病毒核酸阴转时限的观察

目的观察甲型H1N1流感患者病毒核酸阴转时限。方法选择2009年9月16～27日我院收治的甲型H1N1流感确诊病例38例,经同一名医生进行咽拭子采集,采用RT-PCR方法检测甲型通用、甲1通用、季节性流感、甲型H1N1亚型4个病毒亚型,以甲型通用、甲1通用、甲型H1N1亚型均阴转作为判定病毒核酸阴转的标准。结果 38例甲型H1N1流感患者病毒核酸阴转的时限最短1 d,最长14 d,平均4.5 d;病毒核酸阴转时间主要集中在第3、4、5天,第5天与第4天相比,甲型通用、甲1通用和甲型H1N1的阴转比例均明显增加(P<0.05);发病36 h内接受抗病毒治疗者,病毒核酸阴转平均时间为4.1 d;37～72 h接受抗病毒治疗者,病毒核酸阴转平均时间为5.2 d;有3例甲型通用、甲1通用、甲型H1N1亚型病毒核酸未同时阴转。结论甲型H1N1流感抗病毒治疗1周,大部分患者病毒核酸阴转,不具有传染性;尽早(36 h内)接受抗病毒治疗,可以缩短病毒核酸阴转时间;甲型通用、甲1通用、甲型H1N1亚型具有较好的一致性,对三者未同时阴转的情况,应注意是否同时合并其他甲型流感病毒亚型感染。