HCV 1b亚型NS5A氨基酸变异对慢性丙型肝炎抗病毒治疗的影响
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摘要
研究背景和目的
丙型肝炎病毒(Hepatitis C virus, HCV)感染可导致慢性肝病、肝硬化、肝细胞癌等,是肝脏疾病的主要原因之一。WHO评估目前全球约有1.8亿人口感染HCV(感染率：3%)。我国人群抗-HCV阳性率约为3.2%,全国约有4000万人感染HCV。聚乙二醇化干扰素(Pegylated interferon, PEG-IFN)联合利巴韦林(Ribavirin, RBV)治疗是目前慢性丙型肝炎(Chronic hepatitis C,以下简称为CHC或慢性丙肝)患者的标准方案。但此方案对HCV1b亚型的疗效欠佳,持续病毒学应答率(SVR)仅约为50%。
在对HCV1b型基因序列的研究中,许多报道发现HCV NS5A蛋白多个区域的氨基酸(amino acid, aa)序列突变与IFN、IFN/RBV的疗效有关,这些区域包括ISDR (aa2209-2248)、PKRBD (aa2209-2274)、V3(aa2356-2379)、IRRDR (aa2334-2379)。
ISDR:1995年日本学者研究发现HCV的非结构蛋白5A区(NS5A) C端2209-2248位氨基酸突变数目可作为HCV1b型干扰素疗效的独立预测因素,干扰素应答组患者的ISDR变异数目高于无应答组病人。遂把HCV NS5A2209-2248氨基酸序列命名为干扰素敏感性决定区(IFN sensitivity-determining region, ISDR)。其重要的研究结论为：HCV1b型患者ISDR区氨基酸序列与干扰素疗效密切相关,与HCV-J相比,突变型(氨基酸变异≥4个)对干扰素敏感,中间型(氨基酸变异1-3个)和野生型(未变异)不敏感。此结论被日本的诸多后续研究证实,但是来自欧洲和美国的多项研究结果与这一结论存在矛盾之处。
PKRBD:1997、1998年Gale等通过体外实验证实,HCV抵抗IFN a主要是通过NS5A和PKR蛋白的相互作用,PKR蛋白是主要的宿主抗病毒蛋白之一。这种相互作用需要ISDR以及其后的26个氨基酸残基,该区域位于aa2209-2274,被称为PKR-binding domain (PKRBD). HCV1b型PKRBD变异与IFN a或IFNa/PEG-IFN a联合RBV的持续病毒学应答明显相关已有报道。
V3和IRRDR:近些时候,Veillon和El-Shamy等研究发现HCV NS5A的V3区(aa2356-2379),以及V3加上其N端侧翼区,被称为Interferon/ribavirin resistance determining region (IRRDR; aa2334-2379)存在高度变异现象,V3和IRRDR的氨基酸变异与IFNa和RBV治疗的应答有关。更有2009年Bouzgarrou等报道NS5A从ISDR至V3(aa2209-2379)的氨基酸变异能够影响抗病毒治疗的应答。
IFN a的经典信号通路是通过JAK/STAT途径。Ⅰ型IFN与相应的受体结合,激活信号级联反应,从而调节一系列干扰素刺激基因(ISGs)的转录翻译,而翻译的一些蛋白因子具有抗病毒活性,如PKR、MxA、ISG15等。蛋白激酶PKR在RNA病毒基因复制时可与dsRNA结合而激活,激活后能够磷酸化翻译启始因子eIF-2a亚单位的51位丝氨酸残基,使磷酸化的eIF-2a无法参与蛋白多肽链的翻译过程,从而抑制病毒或细胞蛋白的合成,达到抗病毒的效果。而Gale等通过体外实验证明NS5A蛋白通过PKRBD,能够结合IFN a诱导产生的PKR。HCV NS5A蛋白与宿主细胞内蛋白激酶PKR的相互作用对细胞内干扰素刺激基因(ISGs)的翻译起着重要抑制作用。综上所述,HCV1b型NS5A关键区域发生氨基酸突变的数目不同,则干扰NS5A-PKR蛋白相互作用的程度不同,进而不同程度地影响干扰素发挥其疗效。
除过日本,来自亚洲其他国家和地区的相关研究报道极少,尤其是中国大陆的研究报道。所以,本研究的目的是首先确定是否治疗前HCV1b亚型感染患者的NS5A的氨基酸变异与PEG-IFN联合RBV治疗疗效有关。分析聚焦于NS5A区的ISDR、PKRBD、IRRDR和V3四个功能区域。同时筛选NS5A关键区域变异数目不同的合适的临床毒株构建HCV重组replicon,建立HCV复制子细胞培养系统,对HCV NS5A不同氨基酸变异数目与以IFN为基础抗病毒治疗应答的机理进行初步探讨。
第一部分HCV1b型NS5A全长基因的扩增和克隆构建
研究方法
采用RT-PCR技术从53例临床丙型肝炎患者血标本中扩增HCV1b型NS5A全长基因,测序分析,MEGA4软件比对其与HCV J株(GenBank注册号：D90208)病毒NS5A蛋白PKRBD、ISDR、V3和IRRDR区的氨基酸突变数目和位点,了解广东HCV1b亚型NS5A氨基酸突变情况。同时根据NS5A测序结果,挑选合适的NS5A基因片段与pMD18-T载体连接,构建pMD18-NS5A克隆。
统计分析软件应用SPSS17.0,统计描述采用频数表示,计量资料采用均数±标准差(x±s)表示,组间比较采用两独立样本t检验,进行Levene方差齐性分析,方差齐同条件下采用t检验,方差不齐条件下采用Satterthwaite近似t检验。P<0.05有统计学意义。
结果
1. HCV1b亚型NS5A基因成功扩增,全长1477bp, pMD18-NS5A克隆成功构建。
2.广东地区HCV1b亚型NS5A的ISDR区氨基酸突变率为73.6%(39/53),ISDR突变数目超过4个以上的仅占1.9%(1/53),其余均为为野生型和中间型。PKRBD氨基酸残基突变数目5.7±1.4,V3氨基酸残基突变数目4.9±1.1,IRRDR氨基酸残基突变数目5.7±1.5。
3. HCV lb型NS5A氨基酸突变数目与性别、年龄和传播途径的统计结果显示：年龄分组(≥50岁/<50岁)与V3区氨基酸变异数目有显著统计学差异(t=0.555,P=0.033),年龄分组(≥50岁/<50岁)与IRRDR区氨基酸变异数目有显著统计学差异(t=0.288,P=0.041),说明感染HCV年纪较大的患者IRRDR和V3区氨基酸变异较多,而ISDR和PKRBD区无显著差异(P=0.222和P=0.081)。NS5A氨基酸突变数目与性别和传播途径无关(P>0.05)。
第二部分HCV NS5A变异对Peg-IFN/RBV治疗1b亚型CHC疗效的影响
研究方法
对广东地区53例HCV1b亚型感染的慢性丙型肝炎患者,经PEG-IFN/RBV标准方案(40例经180μg/次或135μg/次pegylated IFN-a-2a联合RBV800-1200mg/日方案,13例经80μg/次pegylated IFN-a-2b联合RBV800-1200mg/日方案)治疗48周随访24周,进行回顾性研究。分析HCV NS5A aa变异及其影响抗病毒治疗应答的特点。分析主要聚焦于NS5A区的ISDR、PKRBD、IRRDR和V3功能区域。
统计分析软件应用SPSS17.0,统计描述采用频数表示,计量资料采用均数±标准差(x±s)表示,组间比较采用两独立样本t检验；分类变量比较采用X2检验；二分类Logistic回归用于分析与SVR有关的各种因素。P<0.05有统计学意义。
结果
1.53例慢性丙型肝炎患者采用PEG-IFN联合RBV治疗48周并随访24周,EVR、ETVR和SVR分别是69.8%(37/53)、73.6%(39/53)和50.9%(27/53)。治疗前基线特征中年龄与SVR有显著统计学差异(t=3.499,P=0.001),PLT与SVR有显著统计学差异(t=-2.611,P=0.0120),EVR与SVR有显著统计学差异(χ2=6.173,P=0.013), ETVR与SVR有显著统计学差异(X2=19.757,P=0.000)。EVR和ETVR作为IFN的预测因素已被广泛接受。本研究显示PLT与SVR相关(P=0.012),分析原因是由于患者基线特征的差异所致,因为获得SVR的27例患者中有6例为肝硬化(22.2%),而26例未获得SVR的患者中有10例为肝硬化(38.5%)。进一步年龄分组比对,≥50岁组的CHC患者SVR率比<50岁组的SVR率低(P=0.008),但是两组都可以获得较高的EVR和ETVR。提示对于获得了ETVR的≥50岁的CHC患者,延长疗程至72周或更长,可能提高SVR。
2. ISDR aa突变数目与SVR有显著统计学差异(1.3±1.1对0.6±0.5；t=-3.095,P=0.003)。进一步分析发现ISDR aa突变数目≥2组与<2组之间SVR率存在显著统计学差异(X2=7.767,P=0.005)。研究结果显示采用PEG-IFN/RBV抗病毒治疗,ISDR氨基酸突变数目不仅能够预测SVR,更为重要的是,ISDR aa变异数目由既往Enomoto报道的4个降为2个即可对SVR进行有效预测。经与Meta分析结果对比发现,采用PEG-IFN/RBV联合方案,主要提高了既往HCV1b亚型感染患者ISDR aa突变中间型的SVR。
3. PKRBD aa突变数目与SVR有显著统计学差异(6.1±1.7对5.3±0.7；t=-2.315,P=0.027)。进一步分析发现PKRBD aa突变≥6组与<6组之间的SVR率存在显著统计学差异(X2=4.259,P=0.039)。研究结果说明PKRBD aa序列的变异数与PEG-IFN/RBV疗效明显正相关,变异数≥6的病例所获得的SVR显著高于其他病例,从临床的角度支持Gale等的研究结果。
4.对于V3、IRRDR以及整个NS5A (ISDR-V3)区,统计分析未发现其氨基酸突变与病毒学应答有显著统计学差异(P=0.819、P=0.949和P=0.244)。
5.广东HCV1b亚型NS5A区单个氨基酸突变的常见位点为2218、2257、2259、2260、2262、2265、2268、2270、2271、2336、2351、2356、2360、2367、2368、2372、2373、2375和2378。统计分析未发现上述单个氨基酸位点的突变与PEG-IFN/RBV治疗疗效有统计学差异(P>0.05),有必要增加病例数进行深入研究。
6. Logistic回归分析结果显示：影响SVR的最主要因素是ISDR aa突变数目(OR=9.2,P=0.006),其次为年龄(OR=0.9,P=0.006)。
第三部分HCV1b亚型NS5A插入式复制子重组质粒的构建
研究方法
以能够高效复制的HCV1b replicon质粒为骨架,构成带有MIu Ⅰ和Bel Ⅰ双酶切位点的拉链质粒,前期采用逆转录-聚合酶链反应(RT-PCR)从不同CHC患者血清中扩增获得HCV1b亚型NS5A全长片段,克隆到pMD-18载体中,测序分析其中的PKRBD、ISDR、V3和IRRDR区域的变异情况,挑选重要区域aa突变数目不同的NS5A片段,插入到此HCV replicon拉链质粒中。扩增产物中无MIu Ⅰ和Bel Ⅰ酶切序列的,在引物中引入相关酶切序列后再扩增,双酶切后将NS5A区段置换入HCV1b replicon骨架中,构建包含中国NS5A区段的插入式复制子重组质粒。
结果
分析Dr. Z. Huang惠赠的HCV1b replicon质粒,发现其NS5A区具有MIuⅠ和Bcl Ⅰ的单一酶切位点,双酶切移去质粒中原有的NS5A后(此时暂称为HCV replicon拉链质粒),可以方便地插入从具有不同临床抗病毒治疗结局的CHC患者血清标本中克隆出的HCV NS5A,进而在细胞培养中深入分析不同来源的NS5A的生物学特点,阐明其与抗病毒药物疗效之间的关系。本研究从克隆的NS5A全长片段中挑选重要区域aa突变数目不同的3个片段,插入到HCVreplicon拉链质粒中,成功构建了包含中国NS5A区段的插入式复制子重组质粒,为研究中国HCV1b亚型NS5A蛋白的生物学功能、难治性CHC干扰素抵抗的机制以及个体化抗病毒治疗相关因素的分析奠定了的基础。
第四部分HCV NS5A变异与以IFN为基础抗病毒治疗应答机理的初步探讨
研究方法
通过将各个HCV1b亚型NS5A插入式复制子重组质粒进行Sca Ⅰ单酶切线性化,体外转录成RNA,转染Huh7.5.1细胞,利用荧光素酶检测鉴定不同时间点HCV RNA的瞬时表达,经G418筛选得到含有HCV RNA稳定复制的Huh7.5.1细胞,利用RT-PCR法和免疫荧光检测鉴定细胞中HCV NS5A的基因和蛋白表达,验证各重组replicon质粒是否能够正常工作。
用干扰素α-2b0IU/ml、25IU/ml、50IU/ml、100IU/ml、200IU/ml和400IU/ml; RBV0μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml和80μg/ml; IFN/RBV不同方案：IFN25IU/ml+RBV10μg/ml、IFN50IU/ml+RBV10μg/ml和IFN100IU/ml+RBV10μg/ml,分别干预负载有重组replicon的Huh7.5.1细胞48h后,收集细胞裂解液,测定荧光素酶活性,从而通过体外实验验证临床研究的结果,对其机理做初步探讨。
统计分析软件用SPSS17.0,实验数据均采用均数±标准差(x±s)表示,不同浓度和方案的药物干预Huh7.5.1,三组间采用析因设计资料的方差分析进行显著性检验统计分析,同时对每个浓度药物、每个方案干预Huh7.5.1的结果采用one-way ANOVA方法进行显著性检验统计分析,方差齐同条件下,采用LSD法对各组数据进行多重比较,方差不齐条件下,采用Dunnett'T3方法对各组数据进行多重比较,P<0.05为差异有统计学意义。
结果
1.经检测不同时间点各重组replicon转染Huh7.5.1的瞬时表达效率,通过G418筛选得到含有HCV RNA稳定复制的Huh7.5.1细胞,利用RT-PCR法和免疫荧光检测鉴定细胞中HCV NS5A的基因和蛋白表达,证明重组的HCV replicon能够正常工作,可用于进一步的实验研究。
2.用不同浓度的IFN和RBV分别干预负载有重组replicon的Huh7.5.1细胞,发现三组的荧光素酶活性均呈现剂量依赖性的下降,说明IFN和RBV对HCV RNA复制的抑制作用呈剂量依赖性增加。以不同浓度的IFN处理HCV复制子细胞后,析因设计资料的方差分析结果显示：不同组间有显著差异(F=5.432,P=0.009)。不同药物浓度间有显著差异(F=583.930,P=0.000)。对每个浓度干预结果统计分析时,三组的one-way ANOVA分析显示：在100IU/ml浓度时,三组有显著性差异(F=6.588,P=0.031),在200IU/ml浓度时,三组有显著性差异(F=34.220,P=0.010)。多重比较发现经过这2个药物浓度干预后,NS5A-3组的荧光素酶活性都是最低水平。说明HCV NS5A关键区域PKRBD/ISDR区发生氨基酸突变较多毒株,对IFN治疗的反应敏感。
3.用不同方案的IFN联合RBV进行了干预实验,采用不同方案干预Huh7.5.1,析因设计资料的方差分析结果显示：不同组间有显著差异(F=10.579,P=0.001)。对每个浓度干预时,三组的one-way ANOVA分析显示：IFN50IU/ml+RBV10μg/ml方案下,三组比较有显著性差异(F=8.565,P=0.017),多重比较发现NS5A-1组病毒量显著高于NS5A-2组和NS5A-3组,说明HCV NS5A关键区域PKRBD/ISDR区发生氨基酸突变的replicon,对联合用药反应敏感。而而IFN100IU/ml+RBV10μg/ml方案下,三组比较有显著性差异(F=14.317,P=0.005),多重比较显示组间两两比较有统计学差异,而NS5A-3组的病毒量最低,NS5A-2组居中。说明联合用药方案情况下,HCV对以IFN为基础的联合抗病毒治疗的反应敏感,并且HCV NS5A关键区域PKRBD/ISDR区变异数目越多,对药物的反应越好,这些发现与我们前期的研究结果相符合。
结论
1.本研究表明：HCV lb亚型NS5A氨基酸变异与以干扰素为基础的抗病毒治疗应答密切相关,关键区域集中在NS5A的ISDR和PKRBD区。并且采用PEG-IFN联合RBV标准方案治疗,ISDR氨基酸突变对治疗应答的预测可由4个降为2个。而实验部分也证明：对于HCV1b型NS5A区PKRBD/ISDR变异数目较多的病毒株,采用IFN联合RBV用药干预较单用IFN干预,治疗反应会更加敏感,并且HCV NS5A关键区域PKRBD/ISDR区变异数目越多,对联合治疗的反应越好。
2.本研究提示：对于HCV1b型NS5A蛋白与抗病毒治疗的研究应给予足够重视,在基础实验研究中,利用本实验构建的重组replicon细胞培养系统,针对NS5A区不同区域氨基酸突变如何影响IFN信号转导通路的实验研究值得深入探索。
Background and Objection:
Hepatitis C virus (HCV) is one of the major causes of chronic liver disease and the patients frequently progress to cirrhosis and hepatocellular carcinoma (HCC). WHO estimates that approximately180million people, representing3%of the world's population, are infected with HCV. HCV infection is also a serious public health problem in China, the infection rate was estimated to be3.2%. The total number of Chinese people who had detectable HCV antibodies might exceed40million. The combination therapy of pegylated interferon (PEG-IFN) and ribavirin (RBV) is the current standard of care for patients with chronic hepatitis C. Unfortunately, approximately50%of patients infected with hepatitis C virus genotype1b (HCV-lb) are resistant to this treatment regimen.
Studies have shown that mutations in several subgenomic regions of non-structural protein5A (NS5A) of hepatitis C virus (HCV) genotype1b, which include the interferon sensitivity-determining region (ISDR, aa2209-2248), the PKR-binding domain (PKRBD, aa2209-2274), the variable region3(V3, aa2356-2379), and the interferon/ribavirin resistance-determining region (IRRDR, aa2334-2379), are associated with response to interferon (IFN) or IFN/RBV therapy.
ISDR:In1995, Enomoto et al. first reported a strong correlation between the number of mutations in the so-called IFN sensitivity-determining region (ISDR; amino acids2209-2248) and the efficacy of IFN monotherapy in Japanese patients with HCV genotype lb infection. Their important conclusion is that patients infected with genotype-lb HCV harbouring more than three mutated sites in this region are more likely to become sustained responders than those whose HCV quasispecies harbour few ISDR mutations. There were three groups according to their ISDR pattern into:no mutation (wild-type; WT); one to three mutations (intermediate-type; IT); more than three mutations (mutant-type; MT). This finding was later confirmed by several other Japanese studies. However, the results of studies from Europe and the United States concerning the correlation between ISDR mutations and treatment response are conflicting.
PKRBD:In1997and1998, Gale et al. indicated that not only the sequence of ISDR, but also an additional26amino acids after the carboxy terminal of ISDR, is the key for the interaction between NS5A and the RNA-dependent protein kinase (PKR), which is one of the most important host antiviral proteins. This region was termed the PKR-binding domain (PKRBD; amino acids2209-2274). Subsequent studies established a significant association between mutations within the PKRBD of HCV genotype1and a long-term sustained response to IFN and IFN/PEG-IFN plus ribavirin.
V3and IRRDR:Recently, Veillo et al, and El-Shamy et al, reported that a number of residue substitutions within the variable region3(V3; amino acids2356-2379), or within the V3region plus its N-terminally flanking region, called interferon/ribavirin resistance determining region (IRRDR; amino acids2334-2379), have also been found to be associated with treatment outcome. In addition, in2009, the mutation number of entire region from ISDR to V3of NS5A (amino acids2209-2379) was believed to influence the treatment response was reported by Bouzgarrou.
The classic IFNa signaling pathway is JAK/STAT pathway. By binding to their specific receptors on the surface of target cells, Type I IFNs stimulates a cascade of intracellular signaling pathways that result in the expression of a large number of interferon-stimulated genes (ISGs), Among the products of numerous ISGs, are the IFN-induced, doublestranded (ds) RNA-dependent protein kinase (PKR), the MxA protein, and ISG15et al, of which the antiviral activities have been well characterized. PKR is a double-stranded RNA-dependent serine/threonine protein kinase. Viral dsRNA binds and activates the enzyme through dimerization and phosphorylation. The activated PKR phosphorylates eIF-2a to limit mRNA translation. As a result, viral replication is blocked at the level of protein synthesis. Gale et al. found that the interaction of NS5A and PKR is a potential mechanism of IFN treatment resistance. This interaction requires I SDR plus26additional amino acids at its C-terminal extremity called PKRBD. It results in a disruption of PKR dimerization and, consequently, an inhibition of PKR-mediated eIF-2alpha phosphorylation. So HCV phosphoprotein NS5A binds PKR and confers resistance to the virus against IFNa.
Nevertheless, except Japan, the effect of mutations in NS5A on IFN efficacy has not yet been studied well in other nations or regions in Asia, especially in Mainland China. In this study, we examined the effect of amino acid substitutions in NS5A on the outcome of combined interferon/ribavirin therapy in Chinese patients with chronic HCV-lb infection. The analyses focused on ISDR, PKRBD, IRRDR and V3regions of NS5A protein. Meanwile, we chose suitable HCV NS5A fragments to construct the recombinant HCV-lb replicon, and to set up cell culture system with those recombinant HCV-lb replicons. Finlly, we explored the mechanism that the effect of different numbers amino acid substitutions in NS5A on the outcome of IFN-a based antivirus therapy.
Part I. Amplification of the full length NS5A fragment of HCV-1b and construction of pMD18-NS5A
METHODS:
A RT-PCR was used to amplify the full-length NS5A genes from clinic blood samples using LA Taq DNA Polymerase. The amplified PCR products were sequenced directly, and the amino acid sequences of PKRBD, ISDR, V3and IRRDR were analysed using MEGA4software with HCV-J as a reference sequence (Access. No. D90208). Meanwile, we chose suitable HCV NS5A fragments to construct the pMD18-NS5A plasmids.
The SPSS software (version17.0, SPSS Inc, USA) was used to analyse the data. The continuous variables were summarized as mean±standard deviation (SD), and comparisons were performed with the two-sample t-test. A P-value<0.05was considered statistically significant. All P values described were two-sided.
RESULTS:
1. The full length fragments of NS5A of HCV-1b were obtained, cloned and sequenced succefully from CHC patients.
2. The mutations rate of ISDR within NS5A of HCV-lb is73.6%in Guangdong area. According to the classification by Enomoto et al, only one patient was infected with the mutant-type strain (≥4mutations)(1.9%),14of53patients (26.4%) were infected with the wild-type ISDR strains (0mutations),38patients (71.7%) were infected with the intermediate-type ISDR strains (1-3mutations). The mutation numbers of PKRBD is5.7±1.4. The mutation numbers of V3is4.9±1.1. The mutation numbers of IRRDR is5.7±1.5.
3. The analyses of the mutation of NS5A of HCV-1b and age group, gender and route of transmission showed that:the number of amino acid substitutions in V3and IRRDR were significantly higher in patients≥50years than in those<50years (P=0.041/P=0.033), to reflect that the olders with HCV-lb infection may have more mutation numbers in V3and IRRDR. However, the variability of amino acid sequence in ISDR and PKRBD regions was similar between patients≥50years and those<50years (P=0.222/P=0.081). There were no significant differences between the the number of amino acid substitutions in NS5A and gender and route of transmission (P>0.05).
Part II. Impact of mutations in NS5A of HCV genotype lb on the efficacy of the combined PEG-IFN/RBV therapy in Chinese patients with chronic hepatitis C
METHODS:
A total of53patients with chronic HCV lb infection who received the combination therapy were analyzed retrospectively. All of the patients completed a48-week course of a combination therapy of the pegylated IFN with ribavirin and a24-week follow up. Forty of the53patients were treated with180μg or135μg of pegylated IFN-a-2a subcutaneously once weekly and800-1000mg of ribavirin orally daily. The remaining13patients were treated with80μg of pegylated IFN-a-2b subcutaneously once weekly and800-1000mg of ribavirin orally daily. To analyse the characteristic of impact of mutations in NS5A of HCV genotype lb on the response of the combined therapy, and focus on the analyses of ISDR, PKRBD, IRRDR and V3regions in NS5A protein.
The correlations between the mutations of amino acid sequences in NS5A genes of HCV and the clinical outcomes of patients treated with PEG-IFN/RBV were analysised using the SPSS software (version17.0, SPSS Inc, USA). The continuous variables were summarized as mean±standard deviation (SD), and comparisons were performed with the two-sample t-test. The Chi square test was used in comparisons of categorical variables. Binary logistic analyses were performed to identify independent factors associated with a SVR. A P-value<0.05was considered statistically significant. All P values described were two-sided.
RESULTS:
1. Among53patients enrolled in this study,37(69.8%),39(73.6%) and27(50.9%) patients achieved EVR, ETVR and SVR, respectively. We identified that age, PLT, EVR and ETVR were related to SVR (P=0.001,P=0.012, P=0.013and P=0.000). The EVR and ETVR as predictors of the SVR to IFN therapy have been generally acknowledged. PLT as a predictor for SVR could be explained by the difference at the baseline characteristics of patients. Of27patients with SVR,6had cirrhosis (22.2%). In contrast10of the26non-SVR patients had cirrhosis (38.5%). This study suggests that the older patients (>50years old) achieved SVR with lower frequency. But for EVR and ETVR, no significant association between younger patients (<50years old) and those older patients could be observed (P>0.05). This result demonstrated that the younger patients and the older patients could achieve EVR and ETVR with the same frequency. However, during follow-up, the relapse rate of older patients is higher, resulting in a lower SVR at last. Therefore, the older patients with ETVR should be treated with72weeks or more extended therapy to improve SVR.
2. The number of amino acid substitutions in ISDR was significantly higher in patients with SVR than in those with non-SVR (1.3±1.1vs.0.6±0.5; P=0.003). patients with≥2mutations in ISDR was more likely to achieve SVR (P=0.005). we demonstrated that the number of mutations in ISDR is closely associated with response to a more effective PEG-IFN/RBV combination therapy in Chinese patients infected with chronic HCV-lb. Interestingly, the mutation number of ISDR as a predictor of SVR was found to be2in our study as compared with4by previous studies. Comparing with the two Meta-analysises reports, it is very encouraging that we found PEG-IFN/RBV therapy showed substantial improvement on the SVR of CHC patients with intermediate type of HCV ISDR.
3. The number of amino acid substitutions in PKRBD was also significantly higher in patients with SVR than in those who did not respond to antiviral therapy (6.1±1.7vs.5.3±0.7; P=0.027). patients with≥6mutations in PKRBD was more likely to achieve SVR (P=0.039). The mutations within the PKRBD of HCV-1are associated with a sustained virological response to PEG-IFN/RBV therapy. The presence of more than six mutations within PKRBD in the pretreatment sera was a useful factor to predict a sustained virological response to combination therapy.
4. Despite the high variability in V3, IRRDR and NS5A (ISDR-V3), there was no significant correlation was observed between the number of mutations within these regions and the treatment response from the present study (P=0.819, P=0.949and P=0.244).
5. In our study, none of amino acid mutations of NS5A protein was correlated with a sustained virological response (P>0.05). However, frequent amino acid substitutions at positions2218,2257,2259,2260,2262,2265,2268,2270,2271, 2336,2351,2356,2360,2367,2368,2372,2373,2375and2378were observed in Chinese patients.
6. Binary logistic analyses were performed to identify predictors of SVR among patients who received PEG-IFN/RBV. It was found that the number of amino acid mutations within ISDR (odds ratio,9.2; P=0.006), and age (odds ratio,0.9; P=0.006) were two independent predictive factors of SVR.
Part Ⅲ. Construction of recombinant HCV-1b replicon by replacing NS5A region from patients'serum samples
METHODS:
The on-off plasmid containing sequences of restriction endonucleases of MIu I and Bcl I was designed based on the backbone of robust HCV1b replicon. The full length fragments of HCV NS5A were amplified from different CHC patients, by RT-PCR, cloned into pMD-18vector, and sequenced for analysis of amino acid (aa) mutation of ISDR, PKRBD, V3and IRRDR within NS5A region. If the amplicon from patient without cutting sequences of both MIu I and Bcl I, the NS5A fragment would be re-amplified using primers containing them for double restriction enzyme cutting and inserted into the replicon for replacement. To construct the recombinant HCV-lb replicon by replacing NS5A region from chronic hepatitis C (CHC) patients' serum samples in Southern China.
RESULTS:
The on-off plasmid was obtained by cutting with restriction endonucleases of MIu I and Bcl I on the backbone of robust HCV1b replicon. The NS5A fragment would be cutting with double restriction enzyme and inserted into the replicon for replacement. The core region of ISDR-V3of NS5A was replaced into the HCV replicon plasmid and showed right sequencing result. The plug-in typy recombinant HCV replicon for replacement of NS5A region from CHC patients has been successfully constructed, which provides a basis for further research on the biological characteristics of NS5A protein, mechanism of interferon-resistance and antiviral therapy of difficult-to-treat CHC.
Part IV. Mechanism of the effect of amino acid substitutions in NS5A on the outcome of IFN-a based antiviral therapy
METHODS:
HCV lb recombinant replicon plasmids containing different NS5A fragments were cutted with restriction endonuclease Scal. HCV subgenomic replicon RNA was obtained by transcription and then transfect into Huh7.5.1cell. To detect the transient replication efficiency of recombinant replicons were transfected into Huh7.5.1cells by luciferase assay. After screening the cells in the medium containing G418, we detected the HCV NS5A genes in the obtained cell colonies by RT-PCR and the HCV NS5A protein by immunofluorescence.
The cell colonies which contain HCV RNA were treated with various concentrations of IFNa-2b (0IU/ml,25IU/ml,50IU/ml,100IU/ml,200IU/ml and400IU/ml), RBV (0μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml and80μg/ml), and treated with different schemes of IFN/RBV (IFN25IU/ml+RBV10μg/ml, IFN50IU/ml+RBV10μg/ml and IFN100IU/ml+RBV10μg/ml) for48hours. Then, we detected the luciferase activity of cell colonies treated with IFNa-2b, RBV and IFN/RBV. At last, to validate our hypothesis and to explore the possible mechanism.
Datas that the cell colonies were treated with various concentrations of IFNa-2b, RBV and IFN/RBV were analysised using the SPSS software (version17.0, SPSS Inc, USA). The variables were summarized as mean±standard deviation (SD). The comparisons of three groups were performed with analysis of variance of factorial design and one-way AN OVA test. Multiple comparisons were performed to identify between-groups differences by LSD or Dunnett'T3methods. A P-value<0.05was considered statistically significant. All P values described were two-sided.
RESULTS:
1. We detected the transient replication efficiency of recombinant replicons after transfection at4,24,48and72hour sites. After screening the cells in the medium containing G418, we detected the HCV NS5A genes in the obtained cell colonies by RT-PCR and the HCV NS5A protein by immunofluorescence. These datas certified recombinant replicons could express normally.
2. The cell colonies which contain HCV replicon RNA were treated with various concentrations of IFNa-2b and RBV. After that, we found luciferase activity of three groups showed a dose dependent decreasion, accounting that IFN and RBV all could inhibit HCV RNA in replicon cells with a dose dependent enhancement. Interestingly, when cells treated with100IU/ml and200IU/ml of IFNa-2b, luciferase activity of three groups were significant difference (P=0.031and P=0.010). Meanwile, multiple comparisons were performed and showed luciferase activity of NS5A-3group was lowest than those in other groups. This experiment part demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb was sensitive response to IFN therapy.
3. The cell colonies which contain HCV replicon RNA were treated with three schemes of IFN/RBV. After that, we detected the luciferase activity of three groups, when cells treated with IFN50IU/ml plus RBV10μg/ml, luciferase activity of three groups were significant difference (P=0.017). Meanwile, multiple comparisons were performed and showed the luciferase activity of NS5A-1group was highest than those in other groups. It demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb was sensitive response to IFN/RBV therapy. when cells treated with IFN100IU/ml plus RBV10μg/ml, luciferase activity of three groups were significant difference (P=0.005). Meanwile, multiple comparisons were performed and showed significant differences between every two groups. The luciferase activity of NS5A-3group was lowest than those in other groups, and the luciferase activity of NS5A-2group was higher than that in NS5A-1group. This experiment part demonstrated that HCV was more sensitivity in combined IFN/RBV therapy. And the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb, the better response to IFN/RBV therapy.
CONCLUSION:
1. Our results indicated the mutations in NS5A of HCV genotype1b could influence the efficacy of the IFN-a based antivirus therapy in Chinese patients, and the critical regions are ISDR and PKRBD within NS5A protein. We demonstrated that the number of mutations in ISDR was closely associated with response to a more effective PEG-IFN/RBV combination therapy. The mutation number of ISDR as a predictor of SVR was found to be2in our study as compared with4by previous studies. Our experiment part also demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb, the better response to IFN/RBV therapy than IFN therapy.
2. Our results suggested a special attention should be paid to the potential accuracy mechanism of the effect of different numbers amino acid substitutions in NS5A on the outcome of IFN-a based antivirus therapy.
丙型肝炎病毒(Hepatitis C virus, HCV)感染可导致慢性肝病、肝硬化、肝细胞癌等,是肝脏疾病的主要原因之一。WHO评估目前全球约有1.8亿人口感染HCV(感染率：3%)。我国人群抗-HCV阳性率约为3.2%,全国约有4000万人感染HCV。聚乙二醇化干扰素(Pegylated interferon, PEG-IFN)联合利巴韦林(Ribavirin, RBV)治疗是目前慢性丙型肝炎(Chronic hepatitis C,以下简称为CHC或慢性丙肝)患者的标准方案。但此方案对HCV1b亚型的疗效欠佳,持续病毒学应答率(SVR)仅约为50%。
在对HCV1b型基因序列的研究中,许多报道发现HCV NS5A蛋白多个区域的氨基酸(amino acid, aa)序列突变与IFN、IFN/RBV的疗效有关,这些区域包括ISDR (aa2209-2248)、PKRBD (aa2209-2274)、V3(aa2356-2379)、IRRDR (aa2334-2379)。
ISDR:1995年日本学者研究发现HCV的非结构蛋白5A区(NS5A) C端2209-2248位氨基酸突变数目可作为HCV1b型干扰素疗效的独立预测因素,干扰素应答组患者的ISDR变异数目高于无应答组病人。遂把HCV NS5A2209-2248氨基酸序列命名为干扰素敏感性决定区(IFN sensitivity-determining region, ISDR)。其重要的研究结论为：HCV1b型患者ISDR区氨基酸序列与干扰素疗效密切相关,与HCV-J相比,突变型(氨基酸变异≥4个)对干扰素敏感,中间型(氨基酸变异1-3个)和野生型(未变异)不敏感。此结论被日本的诸多后续研究证实,但是来自欧洲和美国的多项研究结果与这一结论存在矛盾之处。
PKRBD:1997、1998年Gale等通过体外实验证实,HCV抵抗IFN a主要是通过NS5A和PKR蛋白的相互作用,PKR蛋白是主要的宿主抗病毒蛋白之一。这种相互作用需要ISDR以及其后的26个氨基酸残基,该区域位于aa2209-2274,被称为PKR-binding domain (PKRBD). HCV1b型PKRBD变异与IFN a或IFNa/PEG-IFN a联合RBV的持续病毒学应答明显相关已有报道。
V3和IRRDR:近些时候,Veillon和El-Shamy等研究发现HCV NS5A的V3区(aa2356-2379),以及V3加上其N端侧翼区,被称为Interferon/ribavirin resistance determining region (IRRDR; aa2334-2379)存在高度变异现象,V3和IRRDR的氨基酸变异与IFNa和RBV治疗的应答有关。更有2009年Bouzgarrou等报道NS5A从ISDR至V3(aa2209-2379)的氨基酸变异能够影响抗病毒治疗的应答。
IFN a的经典信号通路是通过JAK/STAT途径。Ⅰ型IFN与相应的受体结合,激活信号级联反应,从而调节一系列干扰素刺激基因(ISGs)的转录翻译,而翻译的一些蛋白因子具有抗病毒活性,如PKR、MxA、ISG15等。蛋白激酶PKR在RNA病毒基因复制时可与dsRNA结合而激活,激活后能够磷酸化翻译启始因子eIF-2a亚单位的51位丝氨酸残基,使磷酸化的eIF-2a无法参与蛋白多肽链的翻译过程,从而抑制病毒或细胞蛋白的合成,达到抗病毒的效果。而Gale等通过体外实验证明NS5A蛋白通过PKRBD,能够结合IFN a诱导产生的PKR。HCV NS5A蛋白与宿主细胞内蛋白激酶PKR的相互作用对细胞内干扰素刺激基因(ISGs)的翻译起着重要抑制作用。综上所述,HCV1b型NS5A关键区域发生氨基酸突变的数目不同,则干扰NS5A-PKR蛋白相互作用的程度不同,进而不同程度地影响干扰素发挥其疗效。
除过日本,来自亚洲其他国家和地区的相关研究报道极少,尤其是中国大陆的研究报道。所以,本研究的目的是首先确定是否治疗前HCV1b亚型感染患者的NS5A的氨基酸变异与PEG-IFN联合RBV治疗疗效有关。分析聚焦于NS5A区的ISDR、PKRBD、IRRDR和V3四个功能区域。同时筛选NS5A关键区域变异数目不同的合适的临床毒株构建HCV重组replicon,建立HCV复制子细胞培养系统,对HCV NS5A不同氨基酸变异数目与以IFN为基础抗病毒治疗应答的机理进行初步探讨。
第一部分HCV1b型NS5A全长基因的扩增和克隆构建
研究方法
采用RT-PCR技术从53例临床丙型肝炎患者血标本中扩增HCV1b型NS5A全长基因,测序分析,MEGA4软件比对其与HCV J株(GenBank注册号：D90208)病毒NS5A蛋白PKRBD、ISDR、V3和IRRDR区的氨基酸突变数目和位点,了解广东HCV1b亚型NS5A氨基酸突变情况。同时根据NS5A测序结果,挑选合适的NS5A基因片段与pMD18-T载体连接,构建pMD18-NS5A克隆。
统计分析软件应用SPSS17.0,统计描述采用频数表示,计量资料采用均数±标准差(x±s)表示,组间比较采用两独立样本t检验,进行Levene方差齐性分析,方差齐同条件下采用t检验,方差不齐条件下采用Satterthwaite近似t检验。P<0.05有统计学意义。
结果
1. HCV1b亚型NS5A基因成功扩增,全长1477bp, pMD18-NS5A克隆成功构建。
2.广东地区HCV1b亚型NS5A的ISDR区氨基酸突变率为73.6%(39/53),ISDR突变数目超过4个以上的仅占1.9%(1/53),其余均为为野生型和中间型。PKRBD氨基酸残基突变数目5.7±1.4,V3氨基酸残基突变数目4.9±1.1,IRRDR氨基酸残基突变数目5.7±1.5。
3. HCV lb型NS5A氨基酸突变数目与性别、年龄和传播途径的统计结果显示：年龄分组(≥50岁/<50岁)与V3区氨基酸变异数目有显著统计学差异(t=0.555,P=0.033),年龄分组(≥50岁/<50岁)与IRRDR区氨基酸变异数目有显著统计学差异(t=0.288,P=0.041),说明感染HCV年纪较大的患者IRRDR和V3区氨基酸变异较多,而ISDR和PKRBD区无显著差异(P=0.222和P=0.081)。NS5A氨基酸突变数目与性别和传播途径无关(P>0.05)。
第二部分HCV NS5A变异对Peg-IFN/RBV治疗1b亚型CHC疗效的影响
研究方法
对广东地区53例HCV1b亚型感染的慢性丙型肝炎患者,经PEG-IFN/RBV标准方案(40例经180μg/次或135μg/次pegylated IFN-a-2a联合RBV800-1200mg/日方案,13例经80μg/次pegylated IFN-a-2b联合RBV800-1200mg/日方案)治疗48周随访24周,进行回顾性研究。分析HCV NS5A aa变异及其影响抗病毒治疗应答的特点。分析主要聚焦于NS5A区的ISDR、PKRBD、IRRDR和V3功能区域。
统计分析软件应用SPSS17.0,统计描述采用频数表示,计量资料采用均数±标准差(x±s)表示,组间比较采用两独立样本t检验；分类变量比较采用X2检验；二分类Logistic回归用于分析与SVR有关的各种因素。P<0.05有统计学意义。
结果
1.53例慢性丙型肝炎患者采用PEG-IFN联合RBV治疗48周并随访24周,EVR、ETVR和SVR分别是69.8%(37/53)、73.6%(39/53)和50.9%(27/53)。治疗前基线特征中年龄与SVR有显著统计学差异(t=3.499,P=0.001),PLT与SVR有显著统计学差异(t=-2.611,P=0.0120),EVR与SVR有显著统计学差异(χ2=6.173,P=0.013), ETVR与SVR有显著统计学差异(X2=19.757,P=0.000)。EVR和ETVR作为IFN的预测因素已被广泛接受。本研究显示PLT与SVR相关(P=0.012),分析原因是由于患者基线特征的差异所致,因为获得SVR的27例患者中有6例为肝硬化(22.2%),而26例未获得SVR的患者中有10例为肝硬化(38.5%)。进一步年龄分组比对,≥50岁组的CHC患者SVR率比<50岁组的SVR率低(P=0.008),但是两组都可以获得较高的EVR和ETVR。提示对于获得了ETVR的≥50岁的CHC患者,延长疗程至72周或更长,可能提高SVR。
2. ISDR aa突变数目与SVR有显著统计学差异(1.3±1.1对0.6±0.5；t=-3.095,P=0.003)。进一步分析发现ISDR aa突变数目≥2组与<2组之间SVR率存在显著统计学差异(X2=7.767,P=0.005)。研究结果显示采用PEG-IFN/RBV抗病毒治疗,ISDR氨基酸突变数目不仅能够预测SVR,更为重要的是,ISDR aa变异数目由既往Enomoto报道的4个降为2个即可对SVR进行有效预测。经与Meta分析结果对比发现,采用PEG-IFN/RBV联合方案,主要提高了既往HCV1b亚型感染患者ISDR aa突变中间型的SVR。
3. PKRBD aa突变数目与SVR有显著统计学差异(6.1±1.7对5.3±0.7；t=-2.315,P=0.027)。进一步分析发现PKRBD aa突变≥6组与<6组之间的SVR率存在显著统计学差异(X2=4.259,P=0.039)。研究结果说明PKRBD aa序列的变异数与PEG-IFN/RBV疗效明显正相关,变异数≥6的病例所获得的SVR显著高于其他病例,从临床的角度支持Gale等的研究结果。
4.对于V3、IRRDR以及整个NS5A (ISDR-V3)区,统计分析未发现其氨基酸突变与病毒学应答有显著统计学差异(P=0.819、P=0.949和P=0.244)。
5.广东HCV1b亚型NS5A区单个氨基酸突变的常见位点为2218、2257、2259、2260、2262、2265、2268、2270、2271、2336、2351、2356、2360、2367、2368、2372、2373、2375和2378。统计分析未发现上述单个氨基酸位点的突变与PEG-IFN/RBV治疗疗效有统计学差异(P>0.05),有必要增加病例数进行深入研究。
6. Logistic回归分析结果显示：影响SVR的最主要因素是ISDR aa突变数目(OR=9.2,P=0.006),其次为年龄(OR=0.9,P=0.006)。
第三部分HCV1b亚型NS5A插入式复制子重组质粒的构建
研究方法
以能够高效复制的HCV1b replicon质粒为骨架,构成带有MIu Ⅰ和Bel Ⅰ双酶切位点的拉链质粒,前期采用逆转录-聚合酶链反应(RT-PCR)从不同CHC患者血清中扩增获得HCV1b亚型NS5A全长片段,克隆到pMD-18载体中,测序分析其中的PKRBD、ISDR、V3和IRRDR区域的变异情况,挑选重要区域aa突变数目不同的NS5A片段,插入到此HCV replicon拉链质粒中。扩增产物中无MIu Ⅰ和Bel Ⅰ酶切序列的,在引物中引入相关酶切序列后再扩增,双酶切后将NS5A区段置换入HCV1b replicon骨架中,构建包含中国NS5A区段的插入式复制子重组质粒。
结果
分析Dr. Z. Huang惠赠的HCV1b replicon质粒,发现其NS5A区具有MIuⅠ和Bcl Ⅰ的单一酶切位点,双酶切移去质粒中原有的NS5A后(此时暂称为HCV replicon拉链质粒),可以方便地插入从具有不同临床抗病毒治疗结局的CHC患者血清标本中克隆出的HCV NS5A,进而在细胞培养中深入分析不同来源的NS5A的生物学特点,阐明其与抗病毒药物疗效之间的关系。本研究从克隆的NS5A全长片段中挑选重要区域aa突变数目不同的3个片段,插入到HCVreplicon拉链质粒中,成功构建了包含中国NS5A区段的插入式复制子重组质粒,为研究中国HCV1b亚型NS5A蛋白的生物学功能、难治性CHC干扰素抵抗的机制以及个体化抗病毒治疗相关因素的分析奠定了的基础。
第四部分HCV NS5A变异与以IFN为基础抗病毒治疗应答机理的初步探讨
研究方法
通过将各个HCV1b亚型NS5A插入式复制子重组质粒进行Sca Ⅰ单酶切线性化,体外转录成RNA,转染Huh7.5.1细胞,利用荧光素酶检测鉴定不同时间点HCV RNA的瞬时表达,经G418筛选得到含有HCV RNA稳定复制的Huh7.5.1细胞,利用RT-PCR法和免疫荧光检测鉴定细胞中HCV NS5A的基因和蛋白表达,验证各重组replicon质粒是否能够正常工作。
用干扰素α-2b0IU/ml、25IU/ml、50IU/ml、100IU/ml、200IU/ml和400IU/ml; RBV0μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml和80μg/ml; IFN/RBV不同方案：IFN25IU/ml+RBV10μg/ml、IFN50IU/ml+RBV10μg/ml和IFN100IU/ml+RBV10μg/ml,分别干预负载有重组replicon的Huh7.5.1细胞48h后,收集细胞裂解液,测定荧光素酶活性,从而通过体外实验验证临床研究的结果,对其机理做初步探讨。
统计分析软件用SPSS17.0,实验数据均采用均数±标准差(x±s)表示,不同浓度和方案的药物干预Huh7.5.1,三组间采用析因设计资料的方差分析进行显著性检验统计分析,同时对每个浓度药物、每个方案干预Huh7.5.1的结果采用one-way ANOVA方法进行显著性检验统计分析,方差齐同条件下,采用LSD法对各组数据进行多重比较,方差不齐条件下,采用Dunnett'T3方法对各组数据进行多重比较,P<0.05为差异有统计学意义。
结果
1.经检测不同时间点各重组replicon转染Huh7.5.1的瞬时表达效率,通过G418筛选得到含有HCV RNA稳定复制的Huh7.5.1细胞,利用RT-PCR法和免疫荧光检测鉴定细胞中HCV NS5A的基因和蛋白表达,证明重组的HCV replicon能够正常工作,可用于进一步的实验研究。
2.用不同浓度的IFN和RBV分别干预负载有重组replicon的Huh7.5.1细胞,发现三组的荧光素酶活性均呈现剂量依赖性的下降,说明IFN和RBV对HCV RNA复制的抑制作用呈剂量依赖性增加。以不同浓度的IFN处理HCV复制子细胞后,析因设计资料的方差分析结果显示：不同组间有显著差异(F=5.432,P=0.009)。不同药物浓度间有显著差异(F=583.930,P=0.000)。对每个浓度干预结果统计分析时,三组的one-way ANOVA分析显示：在100IU/ml浓度时,三组有显著性差异(F=6.588,P=0.031),在200IU/ml浓度时,三组有显著性差异(F=34.220,P=0.010)。多重比较发现经过这2个药物浓度干预后,NS5A-3组的荧光素酶活性都是最低水平。说明HCV NS5A关键区域PKRBD/ISDR区发生氨基酸突变较多毒株,对IFN治疗的反应敏感。
3.用不同方案的IFN联合RBV进行了干预实验,采用不同方案干预Huh7.5.1,析因设计资料的方差分析结果显示：不同组间有显著差异(F=10.579,P=0.001)。对每个浓度干预时,三组的one-way ANOVA分析显示：IFN50IU/ml+RBV10μg/ml方案下,三组比较有显著性差异(F=8.565,P=0.017),多重比较发现NS5A-1组病毒量显著高于NS5A-2组和NS5A-3组,说明HCV NS5A关键区域PKRBD/ISDR区发生氨基酸突变的replicon,对联合用药反应敏感。而而IFN100IU/ml+RBV10μg/ml方案下,三组比较有显著性差异(F=14.317,P=0.005),多重比较显示组间两两比较有统计学差异,而NS5A-3组的病毒量最低,NS5A-2组居中。说明联合用药方案情况下,HCV对以IFN为基础的联合抗病毒治疗的反应敏感,并且HCV NS5A关键区域PKRBD/ISDR区变异数目越多,对药物的反应越好,这些发现与我们前期的研究结果相符合。
结论
1.本研究表明：HCV lb亚型NS5A氨基酸变异与以干扰素为基础的抗病毒治疗应答密切相关,关键区域集中在NS5A的ISDR和PKRBD区。并且采用PEG-IFN联合RBV标准方案治疗,ISDR氨基酸突变对治疗应答的预测可由4个降为2个。而实验部分也证明：对于HCV1b型NS5A区PKRBD/ISDR变异数目较多的病毒株,采用IFN联合RBV用药干预较单用IFN干预,治疗反应会更加敏感,并且HCV NS5A关键区域PKRBD/ISDR区变异数目越多,对联合治疗的反应越好。
2.本研究提示：对于HCV1b型NS5A蛋白与抗病毒治疗的研究应给予足够重视,在基础实验研究中,利用本实验构建的重组replicon细胞培养系统,针对NS5A区不同区域氨基酸突变如何影响IFN信号转导通路的实验研究值得深入探索。
Background and Objection:
Hepatitis C virus (HCV) is one of the major causes of chronic liver disease and the patients frequently progress to cirrhosis and hepatocellular carcinoma (HCC). WHO estimates that approximately180million people, representing3%of the world's population, are infected with HCV. HCV infection is also a serious public health problem in China, the infection rate was estimated to be3.2%. The total number of Chinese people who had detectable HCV antibodies might exceed40million. The combination therapy of pegylated interferon (PEG-IFN) and ribavirin (RBV) is the current standard of care for patients with chronic hepatitis C. Unfortunately, approximately50%of patients infected with hepatitis C virus genotype1b (HCV-lb) are resistant to this treatment regimen.
Studies have shown that mutations in several subgenomic regions of non-structural protein5A (NS5A) of hepatitis C virus (HCV) genotype1b, which include the interferon sensitivity-determining region (ISDR, aa2209-2248), the PKR-binding domain (PKRBD, aa2209-2274), the variable region3(V3, aa2356-2379), and the interferon/ribavirin resistance-determining region (IRRDR, aa2334-2379), are associated with response to interferon (IFN) or IFN/RBV therapy.
ISDR:In1995, Enomoto et al. first reported a strong correlation between the number of mutations in the so-called IFN sensitivity-determining region (ISDR; amino acids2209-2248) and the efficacy of IFN monotherapy in Japanese patients with HCV genotype lb infection. Their important conclusion is that patients infected with genotype-lb HCV harbouring more than three mutated sites in this region are more likely to become sustained responders than those whose HCV quasispecies harbour few ISDR mutations. There were three groups according to their ISDR pattern into:no mutation (wild-type; WT); one to three mutations (intermediate-type; IT); more than three mutations (mutant-type; MT). This finding was later confirmed by several other Japanese studies. However, the results of studies from Europe and the United States concerning the correlation between ISDR mutations and treatment response are conflicting.
PKRBD:In1997and1998, Gale et al. indicated that not only the sequence of ISDR, but also an additional26amino acids after the carboxy terminal of ISDR, is the key for the interaction between NS5A and the RNA-dependent protein kinase (PKR), which is one of the most important host antiviral proteins. This region was termed the PKR-binding domain (PKRBD; amino acids2209-2274). Subsequent studies established a significant association between mutations within the PKRBD of HCV genotype1and a long-term sustained response to IFN and IFN/PEG-IFN plus ribavirin.
V3and IRRDR:Recently, Veillo et al, and El-Shamy et al, reported that a number of residue substitutions within the variable region3(V3; amino acids2356-2379), or within the V3region plus its N-terminally flanking region, called interferon/ribavirin resistance determining region (IRRDR; amino acids2334-2379), have also been found to be associated with treatment outcome. In addition, in2009, the mutation number of entire region from ISDR to V3of NS5A (amino acids2209-2379) was believed to influence the treatment response was reported by Bouzgarrou.
The classic IFNa signaling pathway is JAK/STAT pathway. By binding to their specific receptors on the surface of target cells, Type I IFNs stimulates a cascade of intracellular signaling pathways that result in the expression of a large number of interferon-stimulated genes (ISGs), Among the products of numerous ISGs, are the IFN-induced, doublestranded (ds) RNA-dependent protein kinase (PKR), the MxA protein, and ISG15et al, of which the antiviral activities have been well characterized. PKR is a double-stranded RNA-dependent serine/threonine protein kinase. Viral dsRNA binds and activates the enzyme through dimerization and phosphorylation. The activated PKR phosphorylates eIF-2a to limit mRNA translation. As a result, viral replication is blocked at the level of protein synthesis. Gale et al. found that the interaction of NS5A and PKR is a potential mechanism of IFN treatment resistance. This interaction requires I SDR plus26additional amino acids at its C-terminal extremity called PKRBD. It results in a disruption of PKR dimerization and, consequently, an inhibition of PKR-mediated eIF-2alpha phosphorylation. So HCV phosphoprotein NS5A binds PKR and confers resistance to the virus against IFNa.
Nevertheless, except Japan, the effect of mutations in NS5A on IFN efficacy has not yet been studied well in other nations or regions in Asia, especially in Mainland China. In this study, we examined the effect of amino acid substitutions in NS5A on the outcome of combined interferon/ribavirin therapy in Chinese patients with chronic HCV-lb infection. The analyses focused on ISDR, PKRBD, IRRDR and V3regions of NS5A protein. Meanwile, we chose suitable HCV NS5A fragments to construct the recombinant HCV-lb replicon, and to set up cell culture system with those recombinant HCV-lb replicons. Finlly, we explored the mechanism that the effect of different numbers amino acid substitutions in NS5A on the outcome of IFN-a based antivirus therapy.
Part I. Amplification of the full length NS5A fragment of HCV-1b and construction of pMD18-NS5A
METHODS:
A RT-PCR was used to amplify the full-length NS5A genes from clinic blood samples using LA Taq DNA Polymerase. The amplified PCR products were sequenced directly, and the amino acid sequences of PKRBD, ISDR, V3and IRRDR were analysed using MEGA4software with HCV-J as a reference sequence (Access. No. D90208). Meanwile, we chose suitable HCV NS5A fragments to construct the pMD18-NS5A plasmids.
The SPSS software (version17.0, SPSS Inc, USA) was used to analyse the data. The continuous variables were summarized as mean±standard deviation (SD), and comparisons were performed with the two-sample t-test. A P-value<0.05was considered statistically significant. All P values described were two-sided.
RESULTS:
1. The full length fragments of NS5A of HCV-1b were obtained, cloned and sequenced succefully from CHC patients.
2. The mutations rate of ISDR within NS5A of HCV-lb is73.6%in Guangdong area. According to the classification by Enomoto et al, only one patient was infected with the mutant-type strain (≥4mutations)(1.9%),14of53patients (26.4%) were infected with the wild-type ISDR strains (0mutations),38patients (71.7%) were infected with the intermediate-type ISDR strains (1-3mutations). The mutation numbers of PKRBD is5.7±1.4. The mutation numbers of V3is4.9±1.1. The mutation numbers of IRRDR is5.7±1.5.
3. The analyses of the mutation of NS5A of HCV-1b and age group, gender and route of transmission showed that:the number of amino acid substitutions in V3and IRRDR were significantly higher in patients≥50years than in those<50years (P=0.041/P=0.033), to reflect that the olders with HCV-lb infection may have more mutation numbers in V3and IRRDR. However, the variability of amino acid sequence in ISDR and PKRBD regions was similar between patients≥50years and those<50years (P=0.222/P=0.081). There were no significant differences between the the number of amino acid substitutions in NS5A and gender and route of transmission (P>0.05).
Part II. Impact of mutations in NS5A of HCV genotype lb on the efficacy of the combined PEG-IFN/RBV therapy in Chinese patients with chronic hepatitis C
METHODS:
A total of53patients with chronic HCV lb infection who received the combination therapy were analyzed retrospectively. All of the patients completed a48-week course of a combination therapy of the pegylated IFN with ribavirin and a24-week follow up. Forty of the53patients were treated with180μg or135μg of pegylated IFN-a-2a subcutaneously once weekly and800-1000mg of ribavirin orally daily. The remaining13patients were treated with80μg of pegylated IFN-a-2b subcutaneously once weekly and800-1000mg of ribavirin orally daily. To analyse the characteristic of impact of mutations in NS5A of HCV genotype lb on the response of the combined therapy, and focus on the analyses of ISDR, PKRBD, IRRDR and V3regions in NS5A protein.
The correlations between the mutations of amino acid sequences in NS5A genes of HCV and the clinical outcomes of patients treated with PEG-IFN/RBV were analysised using the SPSS software (version17.0, SPSS Inc, USA). The continuous variables were summarized as mean±standard deviation (SD), and comparisons were performed with the two-sample t-test. The Chi square test was used in comparisons of categorical variables. Binary logistic analyses were performed to identify independent factors associated with a SVR. A P-value<0.05was considered statistically significant. All P values described were two-sided.
RESULTS:
1. Among53patients enrolled in this study,37(69.8%),39(73.6%) and27(50.9%) patients achieved EVR, ETVR and SVR, respectively. We identified that age, PLT, EVR and ETVR were related to SVR (P=0.001,P=0.012, P=0.013and P=0.000). The EVR and ETVR as predictors of the SVR to IFN therapy have been generally acknowledged. PLT as a predictor for SVR could be explained by the difference at the baseline characteristics of patients. Of27patients with SVR,6had cirrhosis (22.2%). In contrast10of the26non-SVR patients had cirrhosis (38.5%). This study suggests that the older patients (>50years old) achieved SVR with lower frequency. But for EVR and ETVR, no significant association between younger patients (<50years old) and those older patients could be observed (P>0.05). This result demonstrated that the younger patients and the older patients could achieve EVR and ETVR with the same frequency. However, during follow-up, the relapse rate of older patients is higher, resulting in a lower SVR at last. Therefore, the older patients with ETVR should be treated with72weeks or more extended therapy to improve SVR.
2. The number of amino acid substitutions in ISDR was significantly higher in patients with SVR than in those with non-SVR (1.3±1.1vs.0.6±0.5; P=0.003). patients with≥2mutations in ISDR was more likely to achieve SVR (P=0.005). we demonstrated that the number of mutations in ISDR is closely associated with response to a more effective PEG-IFN/RBV combination therapy in Chinese patients infected with chronic HCV-lb. Interestingly, the mutation number of ISDR as a predictor of SVR was found to be2in our study as compared with4by previous studies. Comparing with the two Meta-analysises reports, it is very encouraging that we found PEG-IFN/RBV therapy showed substantial improvement on the SVR of CHC patients with intermediate type of HCV ISDR.
3. The number of amino acid substitutions in PKRBD was also significantly higher in patients with SVR than in those who did not respond to antiviral therapy (6.1±1.7vs.5.3±0.7; P=0.027). patients with≥6mutations in PKRBD was more likely to achieve SVR (P=0.039). The mutations within the PKRBD of HCV-1are associated with a sustained virological response to PEG-IFN/RBV therapy. The presence of more than six mutations within PKRBD in the pretreatment sera was a useful factor to predict a sustained virological response to combination therapy.
4. Despite the high variability in V3, IRRDR and NS5A (ISDR-V3), there was no significant correlation was observed between the number of mutations within these regions and the treatment response from the present study (P=0.819, P=0.949and P=0.244).
5. In our study, none of amino acid mutations of NS5A protein was correlated with a sustained virological response (P>0.05). However, frequent amino acid substitutions at positions2218,2257,2259,2260,2262,2265,2268,2270,2271, 2336,2351,2356,2360,2367,2368,2372,2373,2375and2378were observed in Chinese patients.
6. Binary logistic analyses were performed to identify predictors of SVR among patients who received PEG-IFN/RBV. It was found that the number of amino acid mutations within ISDR (odds ratio,9.2; P=0.006), and age (odds ratio,0.9; P=0.006) were two independent predictive factors of SVR.
Part Ⅲ. Construction of recombinant HCV-1b replicon by replacing NS5A region from patients'serum samples
METHODS:
The on-off plasmid containing sequences of restriction endonucleases of MIu I and Bcl I was designed based on the backbone of robust HCV1b replicon. The full length fragments of HCV NS5A were amplified from different CHC patients, by RT-PCR, cloned into pMD-18vector, and sequenced for analysis of amino acid (aa) mutation of ISDR, PKRBD, V3and IRRDR within NS5A region. If the amplicon from patient without cutting sequences of both MIu I and Bcl I, the NS5A fragment would be re-amplified using primers containing them for double restriction enzyme cutting and inserted into the replicon for replacement. To construct the recombinant HCV-lb replicon by replacing NS5A region from chronic hepatitis C (CHC) patients' serum samples in Southern China.
RESULTS:
The on-off plasmid was obtained by cutting with restriction endonucleases of MIu I and Bcl I on the backbone of robust HCV1b replicon. The NS5A fragment would be cutting with double restriction enzyme and inserted into the replicon for replacement. The core region of ISDR-V3of NS5A was replaced into the HCV replicon plasmid and showed right sequencing result. The plug-in typy recombinant HCV replicon for replacement of NS5A region from CHC patients has been successfully constructed, which provides a basis for further research on the biological characteristics of NS5A protein, mechanism of interferon-resistance and antiviral therapy of difficult-to-treat CHC.
Part IV. Mechanism of the effect of amino acid substitutions in NS5A on the outcome of IFN-a based antiviral therapy
METHODS:
HCV lb recombinant replicon plasmids containing different NS5A fragments were cutted with restriction endonuclease Scal. HCV subgenomic replicon RNA was obtained by transcription and then transfect into Huh7.5.1cell. To detect the transient replication efficiency of recombinant replicons were transfected into Huh7.5.1cells by luciferase assay. After screening the cells in the medium containing G418, we detected the HCV NS5A genes in the obtained cell colonies by RT-PCR and the HCV NS5A protein by immunofluorescence.
The cell colonies which contain HCV RNA were treated with various concentrations of IFNa-2b (0IU/ml,25IU/ml,50IU/ml,100IU/ml,200IU/ml and400IU/ml), RBV (0μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml and80μg/ml), and treated with different schemes of IFN/RBV (IFN25IU/ml+RBV10μg/ml, IFN50IU/ml+RBV10μg/ml and IFN100IU/ml+RBV10μg/ml) for48hours. Then, we detected the luciferase activity of cell colonies treated with IFNa-2b, RBV and IFN/RBV. At last, to validate our hypothesis and to explore the possible mechanism.
Datas that the cell colonies were treated with various concentrations of IFNa-2b, RBV and IFN/RBV were analysised using the SPSS software (version17.0, SPSS Inc, USA). The variables were summarized as mean±standard deviation (SD). The comparisons of three groups were performed with analysis of variance of factorial design and one-way AN OVA test. Multiple comparisons were performed to identify between-groups differences by LSD or Dunnett'T3methods. A P-value<0.05was considered statistically significant. All P values described were two-sided.
RESULTS:
1. We detected the transient replication efficiency of recombinant replicons after transfection at4,24,48and72hour sites. After screening the cells in the medium containing G418, we detected the HCV NS5A genes in the obtained cell colonies by RT-PCR and the HCV NS5A protein by immunofluorescence. These datas certified recombinant replicons could express normally.
2. The cell colonies which contain HCV replicon RNA were treated with various concentrations of IFNa-2b and RBV. After that, we found luciferase activity of three groups showed a dose dependent decreasion, accounting that IFN and RBV all could inhibit HCV RNA in replicon cells with a dose dependent enhancement. Interestingly, when cells treated with100IU/ml and200IU/ml of IFNa-2b, luciferase activity of three groups were significant difference (P=0.031and P=0.010). Meanwile, multiple comparisons were performed and showed luciferase activity of NS5A-3group was lowest than those in other groups. This experiment part demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb was sensitive response to IFN therapy.
3. The cell colonies which contain HCV replicon RNA were treated with three schemes of IFN/RBV. After that, we detected the luciferase activity of three groups, when cells treated with IFN50IU/ml plus RBV10μg/ml, luciferase activity of three groups were significant difference (P=0.017). Meanwile, multiple comparisons were performed and showed the luciferase activity of NS5A-1group was highest than those in other groups. It demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb was sensitive response to IFN/RBV therapy. when cells treated with IFN100IU/ml plus RBV10μg/ml, luciferase activity of three groups were significant difference (P=0.005). Meanwile, multiple comparisons were performed and showed significant differences between every two groups. The luciferase activity of NS5A-3group was lowest than those in other groups, and the luciferase activity of NS5A-2group was higher than that in NS5A-1group. This experiment part demonstrated that HCV was more sensitivity in combined IFN/RBV therapy. And the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb, the better response to IFN/RBV therapy.
CONCLUSION:
1. Our results indicated the mutations in NS5A of HCV genotype1b could influence the efficacy of the IFN-a based antivirus therapy in Chinese patients, and the critical regions are ISDR and PKRBD within NS5A protein. We demonstrated that the number of mutations in ISDR was closely associated with response to a more effective PEG-IFN/RBV combination therapy. The mutation number of ISDR as a predictor of SVR was found to be2in our study as compared with4by previous studies. Our experiment part also demonstrated that the more mutation numbers of PKRBD/ISDR within NS5A of HCV-lb, the better response to IFN/RBV therapy than IFN therapy.
2. Our results suggested a special attention should be paid to the potential accuracy mechanism of the effect of different numbers amino acid substitutions in NS5A on the outcome of IFN-a based antivirus therapy.
引文
[1]Williams R. Global challenges in liver disease. Hepatology,2006,44(3):521-6.
[2]中华医学会肝病学分会,中华医学会传染病与寄生虫病学分会.丙型肝炎防治指南.中华肝脏病杂志,2004,12(4)：194-198.
[3]Alberti A, Benvegnu L. Management of hepatitis C. J Hepatol,2003,38 Suppl 1:S104-18.
[4]Seeff LB. Natural history of chronic hepatitis C. Hepatology,2002,36(5 Suppl 1):S35-46.
[5]Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C:a randomised trial. Lancet,2001,358(9286):958-65.
[6]Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med, 2002,347(13):975-82.
[7]Hadziyannis SJ, Sette H Jr, Morgan TR, et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C:a randomized study of treatment duration and ribavirin dose. Ann Intern Med,2004,140(5):346-55.
[8]Weck K. Molecular methods of hepatitis C genotyping. Expert Rev Mol Diagn,2005,5(4):507-20.
[9]丁洋,夏婷婷,窦晓光.慢性丙型肝炎治疗新纪元——口服逆转录酶和蛋白酶抑制剂的临床应用.临床肝胆病杂志,2011,(12)：1266-1269.
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[11]Ghany MG, Nelson DR, Strader DB, et al. An update on treatment of genotype 1 chronic hepatitis C virus infection:2011 practice guideline by the American Association for the Study of Liver Diseases. Hepatology,2011,54(4):1433-44.
[12]黄辉红.HCV基因型和亚型：分型技术、临床意义及其流行病学研 究,2010.
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[2]中华医学会肝病学分会,中华医学会传染病与寄生虫病学分会.丙型肝炎防治指南.中华肝脏病杂志,2004,12(4)：194-198.
[3]Alberti A, Benvegnu L. Management of hepatitis C. J Hepatol,2003,38 Suppl 1:S104-18.
[4]Seeff LB. Natural history of chronic hepatitis C. Hepatology,2002,36(5 Suppl 1):S35-46.
[5]Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C:a randomised trial. Lancet,2001,358(9286):958-65.
[6]Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med, 2002,347(13):975-82.
[7]Hadziyannis SJ, Sette H Jr, Morgan TR, et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C:a randomized study of treatment duration and ribavirin dose. Ann Intern Med,2004,140(5):346-55.
[8]Weck K. Molecular methods of hepatitis C genotyping. Expert Rev Mol Diagn,2005,5(4):507-20.
[9]丁洋,夏婷婷,窦晓光.慢性丙型肝炎治疗新纪元——口服逆转录酶和蛋白酶抑制剂的临床应用.临床肝胆病杂志,2011,(12)：1266-1269.
[10]Ghany MG, Strader DB, Thomas DL, et al. Diagnosis, management, and treatment of hepatitis C:an update. Hepatology,2009,49(4):1335-74.
[11]Ghany MG, Nelson DR, Strader DB, et al. An update on treatment of genotype 1 chronic hepatitis C virus infection:2011 practice guideline by the American Association for the Study of Liver Diseases. Hepatology,2011,54(4):1433-44.
[12]黄辉红.HCV基因型和亚型：分型技术、临床意义及其流行病学研 究,2010.
[13]Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus lb. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest,1995,96(1):224-30.
[14]Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med,1996,334(2):77-81.
[15]Kurosaki M, Enomoto N, Murakami T, et al. Analysis of genotypes and amino acid residues 2209 to 2248 of the NS5A region of hepatitis C virus in relation to the response to interferon-beta therapy. Hepatology,1997,25(3):750-3.
[16]Murashima S, Kumashiro R, Ide T, et al. HCV NS5A 2209-2248 amino acid substitutions affect serum 2,5AS activities. J Hepatol,1999,31(2):382.
[17]Watanabe H, Enomoto N, Nagayama K, et al. Number and position of mutations in the interferon (IFN) sensitivity-determining region of the gene for nonstructural protein 5A correlate with IFN efficacy in hepatitis C virus genotype 1b infection. J Infect Dis,2001,183(8):1195-203.
[18]Gerotto M, Dal Pero F, Sullivan DG, et al. Evidence for sequence selection within the non-structural 5A gene of hepatitis C virus type 1b during unsuccessful treatment with interferon-alpha. J Viral Hepat,1999,6(5):367-72.
[19]Saiz JC, Lopez-Labrador FX, Ampurdanes S, et al. The prognostic relevance of the nonstructural 5A gene interferon sensitivity determining region is different in infections with genotype lb and 3a isolates of hepatitis C virus. J Infect Dis,1998,177(4):839-47.
[20]Puig-Basagoiti F, Saiz JC, Forns X, et al. Influence of the genetic heterogeneity of the ISDR and PePHD regions of hepatitis C virus on the response to interferon therapy in chronic hepatitis C. J Med Virol, 2001,65(1):35-44.
[21]Shen C, Hu T, Shen L, et al. Mutations in ISDR of NS5A gene influence interferon efficacy in Chinese patients with chronic hepatitis C virus genotype 1b infection. J Gastroenterol Hepatol,2007,22(11):1898-903.
[22]Hung CH, Lee CM, Lu SN, et al. Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin. J Viral Hepat, 2003,10(2):87-94.
[23]Yen YH, Hung CH, Hu TH, et al. Mutations in the interferon sensitivity-determining region (nonstructural 5A amino acid 2209-2248) in patients with hepatitis C-1b infection and correlating response to combined therapy of pegylated interferon and ribavirin. Aliment Pharmacol Ther, 2008,27(1):72-9.
[24]Hofgartner WT, Polyak SJ, Sullivan DG, et al. Mutations in the NS5A gene of hepatitis C virus in North American patients infected with HCV genotype 1a or 1b.J Med Virol,1997,53(2):118-26.
[25]Zeuzem S, Lee JH, Roth WK. Mutations in the nonstructural 5A gene of European hepatitis C virus isolates and response to interferon alfa. Hepatology, 1997,25(3):740-4.
[26]Squadrito G, Leone F, Sartori M, et al. Mutations in the nonstructural 5A region of hepatitis C virus and response of chronic hepatitis C to interferon alfa. Gastroenterology,1997,113(2):567-72.
[27]Witherell GW, Beineke P. Statistical analysis of combined substitutions in nonstructural 5A region of hepatitis C virus and interferon response. J Med Virol,2001,63(1):8-16.
[28]Schinkel J, Spaan WJ, Kroes AC. Meta-analysis of mutations in the NS5A gene and hepatitis C virus resistance to interferon therapy:uniting discordant conclusions. Antivir Ther,2004,9(2):275-86.
[29]Pascu M, Martus P, Hohne M, et al. Sustained virological response in hepatitis C virus type 1b infected patients is predicted by the number of mutations within the NS5A-ISDR:a meta-analysis focused on geographical differences. Gut,2004,53(9):1345-51.
[30]Polyak SJ, Paschal DM, McArdle S, et al. Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication. Hepatology,1999,29(4):1262-71.
[31]Song J, Fujii M, Wang F, et al. The NS5A protein of hepatitis C virus partially inhibits the antiviral activity of interferon. J Gen Virol,1999,80 (Pt 4):879-86.
[32]廖继佩,薛小平,尹文,等HeLa细胞中HCV NS5A的表达对干扰素敏感病毒VSV复制的影响.第四军医大学学报,2005,(23)：2122-2124.
[33]Gale MJ Jr, Korth MJ, Tang NM, et al. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology,1997,230(2):217-27.
[34]Gale M Jr, Blakely CM, Kwieciszewski B, et al. Control of PKR protein kinase by hepatitis C virus nonstructural 5 A protein:molecular mechanisms of kinase regulation. Mol Cell Biol,1998,18(9):5208-18.
[35]Berg T, Mas MA, Hohne M, et al. Mutations in the E2-PePHD and NS5A region of hepatitis C virus type 1 and the dynamics of hepatitis C viremia decline during interferon alfa treatment. Hepatology,2000,32(6):1386-95.
[36]Sarrazin C, Herrmann E, Bruch K, et al. Hepatitis C virus nonstructural 5A protein and interferon resistance:a new model for testing the reliability of mutational analyses. J Virol,2002,76(21):11079-90.
[37]Macquillan GC, Niu X, Speers D, et al. Does sequencing the PKRBD of hepatitis C virus NS5A predict therapeutic response to combination therapy in an Australian population. J Gastroenterol Hepatol,2004,19(5):551-7.
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