Functions Of NS5a & Mechanisms Of Hepatitis C Virus Inte

HCV感染患者对干扰素的反应是可变的。最近的研究表明，在HCV NS 5A基因中存在一个被称为干扰素敏感性决定区（ISDR）的区域，该区域与干扰素耐药相关。NS 5A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NS 5A的功能重要性，我们的实验室正在进行实验，以表征其功能，并确定细胞因子是该HCV基因产物的功能靶点。利用酵母双杂交系统，已经鉴定了几个与NS 5A特异性相互作用的独立克隆。这些克隆中的许多编码具有SH 3和/或SH 3结合结构域的蛋白质，并且其中一些是具有推定的信号转导功能的已知基因。这些克隆与NS 5A的相互作用也通过体外GST融合结合试验以及体内免疫共沉淀试验证实。有趣的是，注意到存在几个富含脯氨酸的序列（类似于SH 3结合结构域），并且位于NS 5A中ISDR区域的侧翼。我们的研究结果与最近的报道一致，即NS 5A与Grb 2，一个SH 2/SH 3衔接分子在信号转导中相互作用。目前正在进行实验，以解决这些相互作用的功能意义。还开始努力评估NS 5A序列变异的临床意义。此外，E2蛋白最近显示与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基因型之间是可变的，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患者治疗前的HCV序列。所有基因型1样本，无论反应有显着的同源性PKR磷酸化位点表明干扰素耐药。基因型2和3的6例患者中的3例以及基因型未知的3例患者中的2例具有与PKR不同的序列，表明对干扰素敏感。在PePHD的第3和第9位氨基酸位置发生变化的突变仅发生在持续应答者中。我们的数据表明，虽然最终的反应可能是多因素的，但PePHD在确定干扰素敏感性方面发挥作用。详细描述这种病毒-细胞相互作用和临床疾病的相关性可能有助于我们理解HCV复制和肝细胞损伤的机制。

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. 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. 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 (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.