NS5A GENE PRODUCT & MECHANISM OF HEPATITIS C VIRUS INTERFERON RESISTANCE

感染丙型肝炎病毒的患者对干扰素的反应是多种多样的。最近的研究表明，丙型肝炎病毒NS5A基因中存在一个与干扰素耐药相关的区域，称为干扰素敏感性决定区(ISDR)。NS5A也被证明是一种磷蛋白，可能在病毒复制和病毒与宿主的相互作用中发挥重要作用。由于NS5A在功能上的重要性，我们实验室正在进行实验，以确定其功能，并确定作为该丙型肝炎病毒基因产物的功能靶点的细胞因子。利用酵母双杂交系统，已经鉴定了几个与NS5A特异相互作用的独立克隆。其中许多克隆编码带有SH3和/或SH3结合域的蛋白质，其中一些是已知的具有信号转导功能的基因。体外GST融合结合实验和体内免疫共沉淀实验也证实了这些克隆与NS5A的相互作用。值得注意的是，在NS5A的ISDR区域两侧存在几个富含Pro的序列(类似于SH3结合域)。我们的发现与最近的报道一致，即NS5A与信号转导中的SH2/SH3接头分子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。还在努力评估NS5A序列变异的临床意义。此外，最近发现E2蛋白与PKR相互作用并抑制PKR(Science 1999；285：107)。在不同的丙型肝炎病毒中，E2的相互作用序列是不同的，这可能是干扰素反应的基因型差异的原因。我们正在启动临床研究，以解决这种可能性。这种病毒-细胞相互作用的详细特征及其与临床疾病的相关性可能有助于我们理解丙型肝炎病毒的复制和肝细胞损伤的机制。-病毒-宿主相互作用/多重营养效应/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