山东省丙型肝炎病毒分离株NS5区核苷酸序列分析

目的：了解山东省丙型肝炎病毒（HCV）分离株的基因型及其基因的变异情况，方法：应用德国UBI HCV EIA 4．0诊断试剂盒筛选山东省部分地区64例临床检验为抗－HCV阳性的血清标本，有54例阳性，随意抽取其中28例，应用逆转录套式－聚合酶链反应（RT－nested-PCR)扩增319bp的HCV NS5区基因片段，结果12例出现特异性条带，随后将这12个NS5区片段直接进行T载体克隆，并用Sanger法对克隆成功的10个NS5区基因片段进行序列测定，将所得到的10个序列与GenBank中所有的HCV分离株进行同源性比较。结果：10例山东省部分地区HCV分离株的基因型均属于HCV－1b型，对获得的10个NS5区片段进行是性分析发现，所有的核苷酸变化都是由于替代作用引起的，没有碱基的插入和缺失；大部分的突变属于同义突变，占突变总数的74％。RNA－依赖性RNA聚合酶的G－D－D基序和所有的半胱氨酸都完全保守，结论：本研究证明了HCV－1b型是山东省部分地区主要的基因型。     

Hepatitis C virus (HCV) NS5A protein: role in HCV replication and resistance to interferon-alpha

The hepatitis C virus (HCV) non-structural (NS) 5A protein appears to play an important regulatory role on viral replication and could also be involved in viral pathogenesis. HCV resistance to interferon is a complex mechanism involving multiple causes, among which certain NS5A functions could play a role.     

The HCV NS5B nucleoside and non-nucleoside inhibitors

This article introduces one of the most diverse classes of direct-acting antivirals for hepatitis C, the nucleoside and non-nucleoside NS5B polymerase inhibitors. Through a systematic review of the published literature, we describe their structure, mechanism of action, issues with resistance, and clinical effectiveness shown in the latest clinical trials. Direct-acting antiviral combination trials that have already shown some early promising results even in the setting of interferon-sparing antiviral regimens are discussed.     

Selection of HCV NS5A quasispecies during IFN therapy in patients with chronic HCV

Interferon treatment of chronic hepatitis C virus infection is successful in a minority of patients. The sequence of the interferon sensitivity determining region (ISDR) of the NS5A protein may determine the outcome of therapy in patients infected with HCV genotype 1. To determine whether IFN treatment caused selection of ISDR quasispecies and whether sequences bearing the putative IFN-resistance motif (HCV-J sequence) were selected, we examined amino acid changes in the ISDR in patients with HCV of different genotypes with and without therapy. We found that the ISDR sequence was highly variable and variability was greatest in patients with HCV of genotype 1. IFN treatment was found to exert a selection pressure on ISDR quasispecies, but the putative interferon-resistant variant was not enriched in patients of any genotype. Hence factors other than the sequence of the ISDR region played a role in the IFN resistance of these patients.     

HCV NS5B在人肝癌细胞系Huh7细胞内的表达及活性分析

目的为靶向抗HCV药物筛选奠定基础。方法使用脂质体将包含HCV非结构蛋白5B（NS5B）基因的重组载体pcDNA-5B转染至人肝癌细胞系Huh7细胞,分别采用RT-PCR和Western Blot鉴定NS5B mRNA和蛋白表达,采用荧光素酶试验检测NS5B细胞内RNA依赖的RNA聚合酶（RdRp）活性。结果转染的Huh7细胞出现1．8kb的特异性NSSB mRNA目的带及一条相对分子质量约66kD的蛋白目的带,且后者具有细胞内RdRp活性。结论人肝癌细胞系Huh7细胞可成功表达具有细胞内RdRp活性的NS5B,以其为靶标的抗HCV药物可为临床治疗提供新思路。     

Hepatitis C virus (HCV) NS5A protein: role in HCV replication and resistance to interferon-α

The hepatitis C virus (HCV) non-structural (NS) 5A protein appears to play an important regulatory role on viral replication and could also be involved in viral pathogenesis. HCV resistance to interferon is a complex mechanism involving multiple causes, among which certain NS5A functions could play a role.     

A functional role for NS5ATP9 in the induction of HCV NS5A‐mediated autophagy

Autophagy has been shown to facilitate replication of hepatitis C virus (HCV); however, the mechanism by which HCV induces autophagy has not been fully established. NS5A, a nonstructural protein expressed by HCV, regulates numerous cellular pathways, including autophagy, by up‐regulating Beclin 1; however, the underlying mechanism remains unclear. To obtain new insights into HCV‐regulated autophagy, NS5ATP9 was overexpressed in HepG2 and L02 cells, resulting in up‐regulation of endogenous Beclin 1 mRNA and protein levels, respectively. The luciferase‐reporter assay results showed that both NS5A and NS5ATP9 could transactivate Beclin 1 promoter activity, but that NS5A could not transactivate the Beclin 1 promoter in NS5ATP9‐silenced HepG2 and L02 cells. Up‐regulation of Beclin 1 mRNA and protein expression by NS5A could also be attenuated by NS5ATP9 knock‐down. Furthermore, the HepG2 and L02 cells that transiently overexpressed NS5ATP9 had enhanced accumulation of vacuoles carrying the autophagy marker LC3, consistent with the conversion of endogenous LC3‐I to LC3‐II. In contrast, the conversion of endogenous LC3‐I to LC3‐II could not be enhanced by NS5A in NS5ATP9‐silenced HepG2 cells. These results highlight an important potential role for NS5ATP9 in HCV NS5A‐induced hepatocyte autophagy.     

HCV NS5A结合蛋白顺乌头酸酶1的验证研究

目的筛选人肝脏cDNA文库中与HCV NS5A的结合蛋白基因,验证其中顺乌头酸酶1与HCV NS5A的相互作用。方法应用酵母双杂交系统3筛选人肝脏cDNA文库中的HCV NS5A结合蛋白基因,应用哺乳动物双杂交及免疫共沉淀技术验证其中顺乌头酸酶1蛋白与HCV NS5A之间的相互作用。结果成功筛选出人肝脏cD-NA文库中与HCV NS5A存在相互作用的蛋白基因,哺乳动物双杂交及免疫共沉淀实验结果证实HCV NS5A与顺乌头酸酶1蛋白在HepG2细胞内存在相互作用。结论本实验成功筛选人肝脏cDNA文库中的HCV NS5A结合蛋白基因,并且在体外水平即细胞内证实HCV NS5A与其中的顺乌头酸酶1蛋白之间的相互作用,为进一步细胞内及体内的糖、脂类代谢等功能研究奠定基础。     

丙型肝炎病毒非结构基因5A反式激活基因4A结合蛋白基因的筛选

目的 筛选并克隆HCV非结构基因NS5A反式激活基因4剪切体NS5ATP4A的结合蛋白基因,为研究NS5ATP4A的生物学功能提供线索. 方法 构建HCV NS5ATP4A蛋白诱饵酵母质粒,转化酵母AH109后与含文库质粒的酵母Y187进行配合,在营养缺陷培养基上进行双杂交筛选.选择既能在4重营养缺陷培养基(SD/-Trp/-Leu/-Ade/-His)上生长,又能在涂有X-α-半乳糖的四缺培养平皿上生长的蓝色菌落,提取此酵母克隆的质粒,转化大肠杆菌后进行测序,并进行生物信息学分析.结果 筛选出7个基因,其中已知功能基因6个,未知功能基因1个,这些基因与RNA合成、蛋白质翻译、细胞周期及肿瘤免疫有关. 结论 HCV NS5ATP4A结合蛋白基因的成功筛选,提示了HCV NS5ATP4A新的信号转导途径,为HCV致病机制的进一步研究提供了依据.     

丙型肝炎病毒NS5B的体外表达、纯化和RNA多聚酶活性鉴定

目的 研究丙型肝炎病毒NS5B蛋白的表达及其生物学功能。方法 构建长度为1676bp(7261-9036bp,编码558aa)的HCV NS5B编码区cDNA,定向克隆到表达质粒pET16b中,并在大肠杆菌BL21中表达。应用合成的多聚A或寡聚U为模板进行~3H-UTP掺入实验分析纯化蛋白的活性。结果 表达的NS5B蛋白分子量为65KD,以包涵体形式存在细胞内,改变部分表达条件未能减少包涵体的形成及可溶性蛋白的增加,该包涵体蛋白在6M尿素浓度、pH:10及500mM NaCl的溶液中完全溶解。~3H-UTP掺入实验表明纯化的蛋白具有RNA多聚酶活性并依赖多聚A的存在。结论 对NS5B蛋白生物学活性的研究,有助于了解HCV的复制及抗病毒药物的开发。     

The conserved lysine 151 of HCV NS5B modulates viral genome replication and infectious virus production

Summary. Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA‐dependent RNA polymerase (RdRp) that is involved in genome replication and virus assembly. NS5B contains a distinct loop (loop Λ2) at the beginning of the nucleoside triphosphate tunnel with a highly conserved lysine (K151). In this study, reverse genetic analysis revealed that substitution of Jc1 NS5B K151 for alanine (K151A) and aspartic acid (K151D) affected genome replication and infectious virus production. However, genome replication and virus production by Jc1 containing NS5B K151R remained unaltered. A major deletion in loop Λ2 abolished RNA replication, suggesting a role for this structural domain in NS5B polymerase activity. In conclusion, this study demonstrated that the conserved K151 modulates infectious virus production; and loop Λ2 is essential for the polymerase activity of NS5B.     

丙型肝炎病毒5＇端非编码区和NS3蛋白酶共调控外分泌性碱性磷酸酶表达细胞模型的建立及意义

目的 构建丙型肝炎病毒5'端非编码区(HCV 5'NCR)和NS3丝氨酸蛋白酶共调控外分泌性碱性磷酸酶(SEAP)表达细胞模型,并分析其用于抗HCV药物筛选和评价的可行性。 方法 用聚合酶链反应技术扩增HCV 5'NCR和NS3/4A片段,定向克隆至表达质粒pSEAP2-Control的SEAP基因上游,构建含HCV 5'NCR-NS3/4A-SEAP嵌合基因的重组表达质粒pNCR-NS3/4A-SEAP。将重组质粒转染至肝细胞株QSG7701,用化学发光法检测SEAP的表达,并观察HCV 5'NCR区对应的反义寡聚核苷酸(ASODN)和丝氨酸蛋白酶抑制剂TPCK对SEAP表达的影响。 结果 重组质粒pNCR-NS3/4A-SEAP有高强度SEAP表达,5μmol/L、10μmol/L ASODN和100μmol/L TPCK对SEAP表达有显著抑制作用(t值分别为4.315、6.985、6.949,P值均<0.01)。 结论 重组质粒pNCR-NS3/4A-SEAP的SEAP表达受HCV 5'NCR和NS 3蛋白酶共调控,建立的细胞模型可用于以HCV 5'NCR和NS3蛋白酶为靶位点的药物筛选和评价。     

丙型肝炎NS5ATP6基因原核表达载体的构建和表达纯化及多克隆抗体的制备

目的构建丙型肝炎病毒NS5A蛋白反式激活蛋白6基因的原核表达载体并进行表达、鉴定。方法从已构建的pGBKT7-NS5ATP6质粒上切取NS5ATP6基因,再克隆入pET32a( )质粒,构建pET32a( )-NS5ATP6表达重组体。结果以pET32a( )-NS5ATP6重组体分别转化DH5α和Rosseta大肠埃希菌后,经IPTG诱导,pET32a( )-NS5ATP6表达出分子量为41KD左右的重组蛋白。免疫动物并经Westernblot检测证实其具有良好的抗原性。结论成功地构建了原核表达载体pET32a( )-NS5ATP6,诱导表达和纯化了NS5ATP6融合蛋白,并制备了该蛋白的多克隆抗体,为下一步该基因功能研究奠定了基础。     

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蛋白NS5A对PI3K/Akt通路的分子调节作用

目的研究丙肝病毒（HCV）蛋白NS5A对PI3K/Akt信号的调节机制及其意义。方法 HepG2细胞分别转染NS5A质粒和对照载体。提取总蛋白,用Western blotting法分析PI3K信号Akt磷酸化水平,并用免疫沉淀法检测p85酪氨酸磷酸化水平及p85与p110蛋白间的相互作用。结果 NS5A转染细胞p-Akt蛋白水平上调,同时p85酪氨酸磷酸化水平显著提高,但催化亚基p110与调节亚基p85的结合作用没有明显变化。结论丙肝病毒（HCV）蛋白NS5A可以和PI3Kp85亚基结合而调节PI3K/Akt信号通路,但其机制可能有p85/p110以外的机制。这可能为临床丙肝IFN敏感性的诊断与治疗提供依据。     

Relation of pretreatment sequence diversity in NS5A region of HCV genotype 1 with immune response between pegylated‐INF/ribavirin therapy outcomes

Summary. Hepatitis C virus (HCV) infection frequently persists despite substantial virus‐specific immune responses and the combination of pegylated interferon (INF)‐α and ribavirin therapy. Major histocompatibility complex class I restricted CD8⁺ T cells are responsible for the control of viraemia in HCV infection, and several studies suggest protection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated α‐INF with ribavirin therapy and test evidence of natural selection on the virus in those who failed treatment, using five maximum likelihood evolutionary models from PAML package. The group of sustained virological responders showed three epitopes with frequencies higher than Non‐responders group, all had statistical support, and we observed evidence of selection pressure in the last group. No escape mutation was observed. Interestingly, the epitope VLSDFKTWL was 100% conserved in SVR group. These results suggest that the response to treatment can be explained by the increase in immune pressure, induced by interferon therapy, and the presence of those epitopes may represent an important factor in determining the outcome of therapy.     

Genetic Determinants in a Critical Domain of NS5A Correlate with Hepatocellular Carcinoma in Cirrhotic Patients Infected with HCV Genotype 1b

HCV is an important cause of hepatocellular carcinoma (HCC). HCV NS5A domain-1 interacts with cellular proteins inducing pro-oncogenic pathways. Thus, we explore genetic variations in NS5A domain-1 and their association with HCC, by analyzing 188 NS5A sequences from HCV genotype-1b infected DAA-naïve cirrhotic patients: 34 with HCC and 154 without HCC. Specific NS5A mutations significantly correlate with HCC: S3T (8.8% vs. 1.3%, p = 0.01), T122M (8.8% vs. 0.0%, p < 0.001), M133I (20.6% vs. 3.9%, p < 0.001), and Q181E (11.8% vs. 0.6%, p < 0.001). By multivariable analysis, the presence of ≥1 of them independently correlates with HCC (OR (95%CI): 21.8 (5.7–82.3); p < 0.001). Focusing on HCC-group, the presence of these mutations correlates with higher viremia (median (IQR): 5.7 (5.4–6.2) log IU/mL vs. 5.3 (4.4–5.6) log IU/mL, p = 0.02) and lower ALT (35 (30–71) vs. 83 (48–108) U/L, p = 0.004), suggesting a role in enhancing viral fitness without affecting necroinflammation. Notably, these mutations reside in NS5A regions known to interact with cellular proteins crucial for cell-cycle regulation (p53, p85-PIK3, and β-catenin), and introduce additional phosphorylation sites, a phenomenon known to ameliorate NS5A interaction with cellular proteins. Overall, these results provide a focus for further investigations on molecular bases of HCV-mediated oncogenesis. The role of these NS5A domain-1 mutations in triggering pro-oncogenic stimuli that can persist also despite achievement of sustained virological response deserves further investigation.     

流行性乙型脑炎病毒NS5蛋白的表达

目的 在大肠杆菌中表达并纯化流行性乙型脑炎病毒NS5蛋白,为进一步研究乙脑病毒NS5蛋白的结构与功能奠定基础。方法 根据乙脑病毒NS5蛋白的基础序列,设计两对PCR引物,采用RT-PCR法分别扩增乙脑病毒JaOH0566株部分及全长NS5基因。将扩增产物分别克隆到表达载体pQE30。筛选重组质粒,转化大肠杆菌表达重组蛋白。重组蛋白经金属柱亲和层析纯化。结果与结论 获得了含部分及全长乙脑病毒NS5基因的重组质粒,重组质粒经限制性酶双酶切、DNA序列分析及PCR扩增筛选证实。在大肠杆菌中表现出了带6个组氨酸头的部分及全长的重组乙脑病毒NS5蛋白。经SDS-PAGE及Western-blot分析证实表达蛋白确定是重组乙脑病毒NS5蛋白。     

丙型肝炎病毒5''端非编码区的5''端序列对其翻译启动活性的影响

目的分析丙型肝炎病毒(HCV)5’端非编码区(5’NCR)的5’端序列对其翻译启动活性的影响。方法用PCR技术扩增获得全长和5’端17个碱基缺失的HCV5’NCR片段,定向克隆至表达质粒pSEAP2Control的SEAP基因上游,分别构建成全长和5’端截短的HCV5’NCR调控SEAP表达的重组质粒pNCRSEAP和pdNCRSEAP。用脂质体方法将重组质粒转染至肝细胞株QSG7701,并用化学发光法检测SEAP的表达。结果酶切和测序结果表明,重组质粒pNCRSEAP和pdNCRSEAP构建成功。表达的发光强度分别为390482±42856和635265±52285RLU,二者有差异显著性(P<0.01)。结论HCV5’NCR的5’端碱基缺失提高了它对SEAP基因的翻译启动活性,为进一步分析HCVIRES的结构提供了实验基础。     

Resistance Patterns Associated with HCV NS5A Inhibitors Provide Limited Insight into Drug Binding

Direct-acting antivirals (DAAs) have significantly improved the treatment of infection with the hepatitis C virus. A promising class of novel antiviral agents targets the HCV NS5A protein. The high potency and broad genotypic coverage are favorable properties. NS5A inhibitors are currently assessed in advanced clinical trials in combination with viral polymerase inhibitors and/or viral protease inhibitors. However, the clinical use of NS5A inhibitors is also associated with new challenges. HCV variants with decreased susceptibility to these drugs can emerge and compromise therapy. In this review, we discuss resistance patterns in NS5A with focus prevalence and implications for inhibitor binding.