Functions Of Ns5a & Mechanisms Of Hepatitis C Virus Inte

丙型肝炎病毒感染患者对干扰素的反应是不同的。最近的研究表明，HCV NS5A基因中有一个被称为干扰素敏感性决定区（ISDR）的区域与干扰素耐药性有关。NS5A也被证明是一种磷蛋白，可能在病毒复制和病毒-宿主相互作用中发挥重要作用。由于NS5A在功能上的重要性，我们的实验室正在进行实验，以表征其功能，并确定作为该HCV基因产物功能靶点的细胞因子。利用酵母双杂交系统，已经鉴定出几个与NS5A特异性相互作用的独立克隆。其中一个NS5A相互作用子是Bin1，它包含一个SH3结构域。在人肝癌细胞中，通过体外结合和体内共免疫沉淀实验证实了NS5A与Bin1之间的蛋白-蛋白相互作用。缺失和突变分析表明SH3和SH3结合域在Bin1与NS5A相互作用中的重要性。Bin1是一种c- myc相互作用的适配器蛋白，具有肿瘤抑制和细胞死亡特性。Bin1的缺失可能通过干扰凋亡通路促进恶性肿瘤的发生。HepG2细胞缺乏Bin1的表达，在腺Bin1感染后，这些细胞发生凋亡，这是通过各种实验确定的。表达野生型NS5A，而不表达sh3结合域突变的突变型NS5A，可抑制bin1诱导的HepG2细胞凋亡。综上所述，我们的研究结果表明NS5A损害了bin1诱导的细胞凋亡，并在细胞生长调节中发挥作用。与许多编码基因产物干扰细胞凋亡的病毒一样，NS5A和Bin1相互作用可能在HCV的生产感染中起重要作用，并有助于丙型肝炎的发病机制。进一步的实验正在进行中，以确定这种相互作用的功能意义。NS5A序列变异的临床意义也正在评估中。此外，E2蛋白最近被证明与PKR相互作用并抑制PKR （Science 1999; 285:107）。E2 （PePHD）上的相互作用序列在HCV基因型中是可变的，这可能是干扰素应答的基因型差异的基础。我们研究了34例接受干扰素单药治疗的HCV预处理序列。所有基因型1的样本，无论反应如何，都与PKR磷酸化位点有显著的同源性，表明对干扰素有耐药性。6名基因型为2和3的患者中有3名以及3名基因型未知的患者中有2名的序列与PKR不同，表明对干扰素敏感。PePHD的第3和第9个氨基酸位置的突变只发生在持续应答者中。我们的数据表明，PePHD在决定干扰素敏感性中起作用，尽管最终的反应可能是多因素的。详细描述这种病毒-细胞相互作用及其与临床疾病的相关性可能有助于我们了解HCV复制和肝细胞损伤机制。

Response to interferon in patients infected with HCV has been variable. Recent studies suggested a region, termed IFN sensitivity determining region (ISDR) in the HCV NS5A gene, that are associated with resistance to interferon. The NS5A has also been shown to be a phosphoprotein, probably playing an important role in viral replication and viral-host interaction. Because of the functional importance of NS5A, our laboratory is conducting experiments to characterize its function and identify cellular factors that are the functional targets of this HCV gene product. Using the yeast two hybrid system, several independent clones that interact specifically with NS5A have been identified. One of the NS5A interactors is Bin1, which contains a SH3 domain. The protein-protein interaction between NS5A and Bin1 was confirmed by in vitro binding and in vivo co-immunoprecipitation assays in human hepatoma cells. Deletion and mutation analyses indicated the importance of the SH3 and SH3 binding domains in the interaction between Bin1 and NS5A. Bin1 is a c-Myc-interacting adapter protein with tumor suppressor and cell death properties. Loss of Bin1 may promote malignancy by interfering with the apoptotic pathways. HepG2 cells lack expression of Bin1 and upon infection with adeno-Bin1, these cells undergo apoptosis, as determined by a variety of assays. Expression of the wild-type NS5A but not the mutant NS5A with mutations in the SH3-binding domain inhibits Bin1-induced apoptosis in HepG2 cells. Together, our results suggest that NS5A impairs Bin1-induced apoptosis and exerts its effect on cell growth regulation. Like many viruses encoding gene products interfering with apoptosis, the NS5A and Bin1 interaction may be important for the productive infection of HCV and contribute to the pathogenesis of hepatitis C. Additional experiments are under way to address the functional significance of this interaction. Effort is also being initiated to evaluate the clinical significance of sequence variations in NS5A. Furthermore, the E2 protein has recently been shown to interact with and inhibit PKR (Science 1999; 285:107). The interacting sequences on E2 (PePHD) are variable among the HCV genotypes, possibly underlying the genotypic difference in interferon response. We studied the pretreatment HCV sequence of 34 patients treated with interferon monotherapy. All genotype 1 samples regardless of response had significant homology with the PKR phosphorylation site suggesting resistance to interferon. Three of the six patients with genotype 2 and 3 as well as two of the three with unknown genotype had sequences which varied from the PKR suggesting sensitivity to interferon. Mutations which varied at the 3rd and 9th amino acid position of the PePHD occurred exclusively in sustained responders. Our data suggest that the PePHD plays a role in determining interferon sensitivity although the final response is probably multifactorial. Detailed characterization of this virus-cell interaction and correlation to clinical disease may contribute to our understanding of HCV replication and mechanisms of hepatocellular injury.