NS5A GENE PRODUCT & MECHANISM OF HEPATITIS C VIRUS INTERFERON RESISTANCE

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。这些克隆中的许多编码具有SH3和/或SH3结合域的蛋白质，其中一些是已知的具有假定信号转导功能的基因。体外gst融合结合实验和体内共免疫沉淀实验也证实了这些克隆与NS5A的相互作用。有趣的是，在NS5A的ISDR区域旁边存在几个富含脯氨酸的序列（类似于SH3结合域）。我们的发现与最近的一篇报道一致，即NS5A在信号转导中与SH2/SH3适配器分子Grb2相互作用（PNAS 1999; 96:5533）。目前正在进行实验，以解决这些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上的相互作用序列在HCV基因型中是可变的，可能是干扰素应答的基因型差异的基础。我们正在启动临床研究来解决这种可能性。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。-病毒-宿主相互作用/多养效应/病毒复制

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. Many of these clones encode proteins with SH3 and/or SH3 binding domains and some of them are known genes with putative signal transduction functions. Interactions of these clones with NS5A were also confirmed by the in vitro GST-fusion binding assay as well as co- immunoprecipitation experiment in vivo. It is interesting to note that several proline-rich sequences (similar to SH3 binding domain) are present and flank the ISDR region in NS5A Our findings are consistent with a recent report that NS5A interacts with Grb2, a SH2/SH3 adaptor molecule in signal transduction (PNAS 1999; 96:5533). Experiments are under way to address the functional significance of these interactions. 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 are variable among the HCV genotypes, possibly underlying the genotypic difference in interferon response. We are initiating clinical studies to address this possibility. 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. - Virus-Host Interaction/Pleiotrophic Effect/Viral Replication