四川省丙型肝炎患者HCV基因分型及基线HCV NS5A耐药突变位点分析

目的检测四川省丙型肝炎患者HCV基因分型及HCV NS5A耐药突变位点的分布特征。方法收集2017年-2018年在四川省人民医院确诊的来自四川省的丙型肝炎患者160例,采集外周静脉血标本,使用Sanger测序法鉴定每一样本的HCV基因型及其亚型和HCV NS5A的基因序列。结果 160例丙型肝炎患者共测出1、2、3、6四种基因型和1b、2a、3a、3b、6a、6n六种基因亚型,其中以1b亚型检出率最高,占86.25%(138/160),其次为3b、6a亚型,均各占3.75%(6/160),余下6n型占2.50%(4/160),2a、3a型均各占1.875%(3/160)。在HCV 1b亚型丙型肝炎患者在中,HCV NS5A基因耐药突变检出率为18.84%(26/138),其中以Y93H耐药突变率较高,占17.39%(24/138),其次L31M耐药突变率为1.45%(2/138)。结论分析四川省丙型肝炎患者HCV基因型及HCV NS5A耐药突变位点的分布特点,可为该地区丙型肝炎患者基因型分布及耐药性研究和个体化抗病毒治疗提供指导依据。     

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。     

NS5A-TP5蛋白结合蛋白1的基因克隆化研究

目的 :应用酵母双杂交技术及生物信息学 (bioinformatics)技术筛选并克隆NS5A TP5蛋白结合蛋白基因1(NBP1)。方法 :应用酵母双杂交系统 3 ,构建NS5A TP5诱饵质粒 ,转化酵母AH10 9,与含人肝细胞cDNA文库质粒的酵母Y 187进行配合 ,于涂有x α gal营养缺陷型培养基 (SD/Trp Leu His Ade)上筛选生长。 结果 :挑选蓝色克隆 ,提取此酵母克隆的质粒转化大肠杆菌提取质粒DNA后进行测序 ,然后进行生物信息学分析 ,新基因的开放读码框架长度为 2 40个核苷酸 (nt) ,编码产物由 79个氨基酸残基 (aa)组成 ,命名为NBP1,GenBank注册号为AY 45 92 91。结论 :NBP1的筛选与克隆 ,可为进一步研究HCVNS5A TP5蛋白相互作用的分子生物学机制和探索新型治疗技术奠定基础     

酵母双杂交技术筛选与克隆白细胞中与NS5ATP7蛋白结合的蛋白编码基因

目的 应用酵母双杂交体系寻找与丙型肝炎病毒(HCV)非结构蛋白5A反式激活蛋白7(human gene 5 transactivated by nonstructural protein 5A of hepatitis C virus,NS5ATP7)相互作用的白细胞蛋白,以探讨SN5ATP7的生物功能。方法 应用酵母双杂交系统3,构建NS5ATP7诱饵质粒,转化酵母AH109,与含人白细胞cDNA文库质粒的酵母Y187进行配合,于涂有X-α-gal营养缺陷型培养基(SD／-Trp-Leu-His-Ade)上筛选生长。挑选蓝色克隆,提取此酵母克隆的质粒转化大肠杆菌提取质粒。DNA后进行测序,然后进行生物信息学分析。结果 筛选出15个与NS5ATP7特异性相互作用的克隆,其中包括人类RhoGDF分裂抑制剂α、人类细胞色素b558a亚单位、人类MLL(MLL)基因外显子、人类S100钙结合蛋白A9(钙粒蛋白B)、人类移动抑制因子相关蛋白14变体E(S100A9)、人类金属硫蛋白2A、人类染色体序列及人类cDNA序列等,1个是未知功能基因。结论 初步克隆了NSSATP7与白细胞结合蛋白基因,根据所克隆到的基因,对以后研究NS5ATP7的功能有一定的提示作用;为以后研究这些能与NS5ATP7相互作用的基因在白细胞中的生理功能奠定了基础。     

丙型肝炎病毒NS5A蛋白功能的研究进展

丙型肝炎病毒（HCV）可引起肝炎、肝硬化和肝癌等一系列肝脏疾病,且慢性化几率较高。目前全世界HCV感染人数仍呈增加趋势,因此科学家对HCV的研究高度重视。HCV有一个大的开放阅读框编码约三千多个氨基酸的多聚蛋白,经蛋白酶裂解为十个多肽片段;氨基末端为结构蛋白包括有核心蛋白、包膜蛋白E1和E2、P7蛋白;羧基末端为非结构蛋白包括有NS2、NS3、NS4A、NS4B、NS5A和NS5B蛋白。     

丙型肝炎病毒5''-非编码区与NS 5b区酶切分型对比研究

探讨北京与河南地区丙型肝炎病毒(HCV)基因型分布情况以及HCV5’—NCR和HCV NS5b两区域分型结果之间的差异。对56例慢性丙型肝炎和80例有偿供血员的HCV RNA阳性血清，同时进行HCV5’—NCR和HCV NS5b酶切分型研究。HCV5’—NCR和HCV NS5b两区域的Ⅱ／1b型感染率北京地区分别占85．7％和87．5％，河南地区分别占68．8％和70．0％。HCV Ⅱ／1b型为北京和河南地区HCV感染的优势株，同时HCV5’—NCR和HCV NS5b区酶切分型结果问具有良好的相互对应关系。     

丙型肝炎病毒非结构蛋白NS5A反式激活基因NS5ATP9的克隆化研究

目的 筛选并克隆丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)反式激活新型靶基因，研究HCV NS5A反式激活作用的分子生物学机制。方法 应用抑制性消减杂交技术(SSH)及生物信息学技术，以HCV NS5A表达质粒pcDNA3．1(-)-NS5A转染HepG2细胞，以空载体pcDNA3．1(-)为平行对照，提取mRNA并进行抑制性消减杂交分析。对于所获基因片段序列分析表明，其中之一为新型基因片段，与GenBank中注册的已知功能基因序列没有同源性，利用表达序列标签(EST)序列数据库的搜索和比对，进行电子拼接，根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷酸信号序列，确定新型基因序列。从HepG2细胞提取总RNA，以逆转录多聚酶链反应(RT-PCR)技术扩增获得该新基因的全长序列，并测序证实，命名为NS5ATP9，在GenBank中注册，注册号为AF529370。结果 Ns5ATP9基因的编码序列全长为336个核苷酸(nt)，编码产物由111个氨基酸残基(aa)组成，并成功的克隆化。结论 HCVNS5A反式激活新型靶基因NS5ATP9的筛选与克隆，为进一步研究HcvNs5A反式激活作用的分子生物学机制开辟了新的研究方向。     

丙型肝炎病毒非结构蛋白NS4A肝细胞结合蛋白基因的筛选与克隆

目的筛选并克隆人肝细胞cDNA文库中与丙型肝炎病毒（HCV）非结构蛋白4A（NS4A）相互作用蛋白的基因，明确其具体作用机制。方法应用酵母双杂交系统3，将聚合酶链反应法扩增的HCV NS4A基因连接入酵母表达载体PGBKT7中构建诱饵质粒，转化酵母细胞AH109并在其内表达，然后与转化了人肝cDNA文库质粒PACT2的酵母细胞Y187进行配合，在营养缺陷型培养基和X—α-半乳糖上进行双重筛选阳性菌落，提取阳性酵母菌落的质粒转化大肠杆菌，接种在氨苄青霉素-LB平板上，选择生长菌落，提取质粒酶切鉴定，测序并在GenBank中进行生物信息学分析。结果成功克隆出HCV NS4A基因，构建表达载体并在酵母细胞中表达，与肝文库配合后选出既能在四缺培养基又能在铺有X—α-半乳糖的四缺培养基上生长，并变成蓝色的真阳性菌落22个，序列分析显示，筛选到的肝细胞蛋白编码基因参与细胞能量代谢、蛋白翻译合成等多种生物学过程。结论成功克隆出HCV NS4A蛋白在肝细胞内的结合蛋白，为进一步研究NS4A蛋白的功能、阐明HCV致病的分子生物学机制提供了新线索。     

HCV NS3/4A丝氨酸蛋白酶和NS5B RNA聚合酶抑制剂的临床研究进展

丙型肝炎病毒(hepatitis C virus.HCV)NS3/4A丝氨酸蛋白酶和NSSB RNA依赖的RNA聚合酶是病毒蛋白前体加工成熟和复制过程中十分重要的两个酶,是抗HCV治疗的理想靶点.近年来,关于HCV NS3/4A蛋白酶和NSSBRNA聚合酶抑制剂的研究是抗HCV研究最为活跃的方向,本文综述了他们在临床试验中的最新研究进展.     

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.     

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.     

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.     

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.     

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.     

Characterization of demographics and NS5A genetic diversity for hepatitis C virus genotype 4‐infected patients with or without cirrhosis treated with ombitasvir/paritaprevir/ritonavir

Hepatitis C virus (HCV) genotype 4 (GT4) is genetically diverse with 17 confirmed and 4 provisional subtypes. In this report, HCV GT4‐infected patient samples from Phase 2/3 clinical studies were analysed to characterize global demographics and genetic diversity of GT4 infection among patients treated with ombitasvir (OBV, NS5A inhibitor) plus paritaprevir/r (NS3/4A inhibitor codosed with ritonavir). Among 17 subtypes isolated from GT4‐infected patients in the PEARL‐I and AGATE‐I studies, subtype prevalence by country of enrolment and country of origin suggested that subtypes 4a and 4d were likely circulating in Europe, while heterogeneous GT4 subtypes and a portion of GT4a detected in European and North American countries were likely due to immigration of HCV‐infected patients from Africa. The distributions of birth cohort and race were also significantly different across GT4 subtypes 4a, 4d, and non‐4a/4d. In addition, phylogenetic analyses of NS5A sequences revealed clustering within subtype 4a which segregated by the patient‐reported country of origin and the presence of the L30R/S polymorphism. HCV NS5A sequences derived from GT4a‐infected patients who originated from Europe and the United States clustered separately from sequences derived from patients who originated from Egypt, suggesting that genetically distinct strains of subtype 4a may be circulating globally. Finally, NS5A baseline polymorphisms were frequently detected at amino acid positions of interest for the inhibitor‐class and OBV retained activity against 37 of 39 NS5A GT4 clinical isolates, with no impact on treatment outcome in the PEARL‐I and AGATE‐I studies.     

A phase 1, randomized,dose‐ranging study of GS‐5816, a once‐daily NS5A inhibitor,in patients with genotype 1–4 hepatitis C virus

GS‐5816 is an inhibitor of the hepatitis C virus (HCV) NS5A protein that has demonstrated pan‐genotypic activity and a high barrier to resistance in HCV replicon assays. The aim of this study was to evaluate the safety, antiviral activity and pharmacokinetics of once‐daily doses of GS‐5816 in patients with genotype 1–4 HCV infection. Patients with genotype 1–4 HCV infection were randomized to 3 days of GS‐5816 at doses ranging from 5 to 150 mg or placebo. Adverse events were recorded, and plasma samples obtained for analysis of pharmacokinetics, HCV RNA and NS5A sequencing studies. GS‐5816 5–150 mg for 3 days was well tolerated and resulted in rapid declines in HCV RNA that were sustained over the dosing period. In patients treated with the 150 mg dose of GS‐5816, the mean maximal HCV RNA declines were 4.0, 4.0, 4.4, 3.3 and 3.5 log₁₀ IU/mL in patients with genotype 1a, 1b, 2, 3 and 4 HCV infection, respectively. Pretreatment NS5A resistance‐associated polymorphisms were detected in 31% (22/70) of patients. Genotype 1 and 3 HCV‐infected patients without pretreatment NS5A resistance‐associated polymorphisms had greater declines in HCV RNA than patients with resistance‐associated polymorphisms. Plasma pharmacokinetics were supportive of once‐daily dosing. GS‐5816 demonstrated pangenotypic antiviral activity in patients with genotype 1‐4 HCV infection. It will be further evaluated in combination with other pangenotypic direct‐acting antivirals to achieve the goal of developing a well‐tolerated, highly effective treatment for all HCV genotypes.