丙型肝炎病毒(hepatitis C virus, HCV)的分子病毒学—一个基因组，两个非翻译区和十个蛋白质（下）

非结构蛋白4B（NS4B）

    NS4B，跨HCV多蛋白aa1712-aa1972(27kD), 是一个非常疏水的蛋白。目前所知的NS4B的功能只有一个。它和NS3以及NS4A一起，为非结构蛋白NS5A的高度磷酸化所必需；NS5A要从p56形式转变为高度磷酸化的p58形式，要求NS3，NS4A，NS4B和NS5A被编码在一个多蛋白上（Neddermann, et al., 1999; Koch, et al., 1999）。

NS5A

    通过和其他病毒的比较，NS5A（aa1973-aa2420, p58）以及NS4B被预测为复制酶的重要组成成分。NS5A被发现和一个膜结合蛋白hVAP-33（ human vesicle-associated membrane protein-associated protein of 33 kDa）有特异的结合，同时hVAP-33还和HCV的RdRp（NS5B）相结合。NS5A和NS5B分别和hVAP-33的羧端和氨端结合（Tu, et a., 1999）。此结合进一步提示NS5A是复制酶的组成部分并提示了复制酶在膜上定位的机制。

    NS5A的两种形式p56和p58均为磷酸化蛋白，尤其后者为高度磷酸化蛋白。磷酸化暗示了此蛋白具有重要功能，比如在信号传导、转录调控、细胞凋亡等行为中起作用。

    NS5A具有多个脯氨酸富集区域，能和细胞信号分子的SH-3结构花式相结合，免疫印迹的方法显示NS5A和生长因子受体结合蛋白2（Grb2）有特异的结合，Grb2上SH-3的点突变能去除Grb2结合NS5A的能力。同样利用突变方法，证实了和SH-3结合的区域是NS5A的脯氨酸富集区域（Tan, et al., 1999）。NS5A可能通过此途径干扰细胞信号传导（如对ERK1/2磷酸化的抑制，ERK：受细胞外信号调控的激酶），这可能是HCV的致病机制之一。

    有研究表明NS5A可能抑制p21WAF1的转录（Ghosh, et al., 1999）， p21WAF1是细胞周期调控因子。利用酵母双杂交系统，发现NS5A和细胞转录因子SRCAP的羧端有特异结合，此结合为哺乳动物双杂交分析、免疫共沉淀等实验所证实（Ghosh, et al., 2000a）。因此NS5A可能通过SRCAP间接进行转录调控。

    NS5A调控细胞凋亡的证据是它能抑制TNF-α引起的细胞凋亡。NS5A被发现能阻断caspase-3的激活，并抑制死亡底物poly (ADP-ribose) polymerase的酶解（Ghosh, et al., 2000b）。

    在研究基因型对治疗的影响时发现，NS5A在HCV对α干扰素的抗性中起作用，此抗性位于HCV多蛋白aa2209-aa2248 (Enomoto, et al., 1995; Enomoto, et al., 1996)。 病人携带的HCV序列在此区段属1b基因型时，对干扰素治疗几乎无反应。此抗性的分子机制还不清楚。但有证据表明NS5A是PKR的有效的抑制因子（Gale, et al., 1997），而PKR是干扰素诱导的蛋白激酶，它催化真核翻译起始因子2的 α亚单位的磷酸化，造成蛋白合成的终止以及被感染细胞的死亡（图3）。

非结构蛋白5B（NS5B）

    根据和其他病毒的RNA依赖的RNA聚合酶（RdRp）顺序的同源性比较，HCV多蛋白 aa2421-3011被指定为HCV的RdRp，即复制酶复合物最重要的成份，在真核体外表达系统表达产物大小为68kD，即p68（Choo, et al., 1989）。缺失突变分析表明NS5B的N末端负责结合RNA（Ishii, et al., 1999）。杆状病毒表达的NS5B在体外系统中显示了RdRp活性，证实了上述的推测（Behrens, et al., 1996）。进一步的生化和酶动力学分析表明(Lohmann, et al., 1998)：1，NS5B受KCl或高浓度的NaCl抑制；2，NS5B的活性依赖于Mg离子，Mg离子也可被Mn离子所代替；3，NS5B持续性地合成RNA，在22°C的延长速率为150-200nt/min；4，以HCV基因组最后319nt为模板，NS5B对四种核苷酸底物的动力学常数Km值为UTP（约1.0μM）, GTP（约0.5μM）, ATP（约10μM）, 和 CTP（约0.3μM）。

    单链RNA的复制过程要求RdRp具有引物酶活性或者在复制起始时它能利用某个特异的引物。in vitro 实验表明NS5B能使用模板的3’末端羟基作为引物；而如果模板3’末端被封闭或者模板是一个单调的多聚核苷酸，NS5B起始复制时需要特定的寡聚核苷酸引物。这表明NS5B没有内在的引物酶活性（Behrens, et al., 1996; Lohmann et al., 1997）。

3’UTR

    3’UTR的5’端约30nt的片段是一个基因型特异的多变区，不同基因型之间有核苷酸序列的差异。紧接着是一个多聚U区域，不同基因型的HCV的多聚U有不同的长度。然后是一个多聚嘧啶C（U）区域，最后是一段高度保守的98碱基的区域（Kolykhalov, et al., 1996; Tanaka, et al., 1996），被称为X区域。高度保守的3’末端暗示了3’UTR的重要功能。通过和其他RNA病毒相比较，推测这个高度保守的98碱基序列可能和病毒基因组的包装有关系，也可能参与RNA合成起始，或对两者都很重要。从具有感染力的cDNA克隆及其3'UTR的缺失突变株制备RNA转录本，用猩猩来测试感染性，发现多聚U、多聚C（U）或X区域的部分或全部缺失都使RNA转录本丧失了感染性；而破坏了5'端多变区2个茎环结构的缺失却对RNA的感染性没有影响（Ynangi, et al., 1999）。

    3'端多聚A能增强真核mRNA的翻译。HCV的RNA没有多聚A尾巴，但它的3'端X区域被发现具有类似的作用。X-区域具高度保守的茎环结构，能结合一种多聚嘧啶结合蛋白（PTB）。PTB恰能与5'UTR的IRES相结合。缺失突变分析表明，含有X区域的RNA的翻译水平比没有X区域的RNA要高2到4倍，使X区域丧失结合PTB能力的突变降低但没有完全丧失X区域对RNA翻译的增强。X区域对翻译的增强被发现为顺式作用，且能增强另一依赖IRES的翻译，即脑心肌炎病毒RNA的翻译，而对5'加帽的mRNA的翻译没有增强（Ito, et al., 1998）。

结语

    HCV的RNA基因组编码了一个ORF和两个具重要功能的UTR。5’UTR编码IRES，3’UTR可能含有RNA包装或/和复制所需的重要序列。ORF翻译产生的一个多蛋白被蛋白酶切割为十个蛋白质。N末端是三个结构蛋白：核心蛋白和包膜蛋白E1，E2，由宿主编码的信号肽酶从多蛋白上切下。现已知随后的非结构蛋白区编码了四种酶活性：1，自切割蛋白酶NS2-3和NS3氨端丝氨酸蛋白酶，后者切割3/4A，4A/4B，4B/5A和5A/5B位点。NS4A是NS3重要的辅助因子。2，NS3的RNA解旋酶。3，NS3的NTP酶。4，NS5B编码的RdRp。目前在开发抗病毒药物是都是以这些重要的功能蛋白以及UTR的保守序列为目标。在HCV的生长周期中，p7、NS4B和NS5A的功能尚不明确，其它蛋白的功能也需在HCV复制系统中得到进一步证实。

    HCV感染是慢性肝炎的最主要的致病因子，并且引起肝硬化和肝癌。一般认为HCV的核心蛋白能转化细胞引起癌症，其致病机理可能和核心蛋白调控转录的能力有关。NS3也被发现有转化能力，此转化能力可能来源于NS3的DNA解旋酶活性。磷酸化蛋白NS5A对信号传导的干扰、对基因转录、细胞凋亡的调控也可能对HCV的致病性有贡献。阐明它们转化细胞、影响细胞生命活动的机制将有助于抗HCV药物的开发，并增进我们对生命现象中信号传导、转录调控、细胞分化和发育的认识。

    有证据显示HCV编码的核心蛋白、E2和NS5A能抑制宿主的免疫反应。可能免疫抑制和HCV的高突变率都是HCV慢性化以及HCV免疫逃避的重要原因。感染HCV的病人大约有25%能自动清除体内病毒（Paliago, et al., 1999）。有证据表明这是机体产生了较强的体液或细胞免疫反应所致（Choo, et al., 1994; Cooper, et al., 1999）。因此，在疫苗开发和抗病毒药物开发的同时，研究HCV对宿主免疫抑制的机理，以期采取措施避免宿主免疫系统被抑制，激活、提高宿主本身对HCV的抵抗力，也许能为HCV的治疗和预防开辟一条新路。

文献

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