Effect of Pokeweed antiviral protein on hepatitis C virus IRES

描述(申请人提供)：我们建议测试商陆抗病毒蛋白(PAP)对丙型肝炎病毒(丙型肝炎病毒)内部核糖体进入位点(IRES)介导的翻译的影响。丙型肝炎病毒是慢性肝炎的主要病因，可导致危及生命的肝硬变和肝细胞癌。世卫组织1999年的一项全球调查估计，世界上至少有1.7亿人长期感染丙型肝炎病毒。目前干扰素是治疗慢性肝炎的最佳药物。然而，它与相对较差的疗效和不良副作用有关。PAP是从商陆中分离到的一种29 kDa的抗病毒/抗真菌蛋白。它属于一组被称为核糖体失活蛋白(RIPs)的蛋白质。众所周知，RIPs能催化从真核和原核核糖体的大rRNA中高度保守的α-sarcin/ricin环(SRL)中去除特定的腺嘌呤残基，这一过程称为脱嘌呤作用。我们的研究表明，除了先前发现的腺嘌呤(A4324)外，PAP还能从真核大rRNA中去除另一个腺嘌呤(A4321)和一个鸟嘌呤(G4323)，从而抑制翻译。丙型肝炎病毒RNA的翻译是由一个称为内部核糖体进入位点(IRES)的复杂RNA元件介导的，该元件位于病毒RNA的5‘非翻译区。目前的丙型肝炎病毒IRES结构模型表明，存在四个主要的茎环I-IV和一个假结。除了茎环I，所有的都是翻译起始所必需的。在茎环II和Ild上发现了两个SRL基序。我们的初步研究表明，PAP可以影响丙型肝炎病毒IRES，并显著降低报告萤火虫荧光素酶的活性。此外，无毒的PAP突变体可以像野生型PAP一样有效地抑制丙型肝炎病毒IRES介导的翻译。PAP可能通过对丙型肝炎病毒IRES元件上发现的SRL基序进行去嘌呤而发挥其有害作用。因此，本应用的具体目的是：(1)确定无毒的PAP突变体是否抑制丙型肝炎病毒IRES的定向翻译以及它们是否破坏报告基因的稳定；(2)确定PAP是否通过与丙型肝炎病毒IRES结合并提纯SRL基序来抑制丙型肝炎病毒IRES介导的翻译；(3)鉴定丙型肝炎病毒IRES上的PAP结合和去嘌呤位点，并确定这些序列是否对抑制IRES介导的翻译起关键作用。本研究将为设计新型抗丙型肝炎药物治疗慢性肝炎奠定基础。

DESCRIPTION (provided by applicant): We propose to test the effect of pokeweed antiviral protein (PAP) on translation mediated by the internal ribosome entry sites (IRES) of hepatitis C virus (HCV). HCV is the major cause of chronic hepatitis which can result in life threatening cirrhosis and hepatocellular carcinoma. A global survey by WHO in 1999 estimated that at least 170 million people in the world are chronically infected with HCV. Presently interferon is the best treatment for chronic hepatitis. However, it is associated with relatively poor efficacy and unfavorable side effects. PAP is a 29-kDa antiviral/antifungal protein isolated from Phytolacca americana. It belongs to a group of proteins called the ribosome inactivating proteins (RIPs). RIPs are known to catalytically remove a specific adenine residue from the highly conserved alpha-sarcin/ricin loop (SRL) in the large rRNA of eukaryotic and prokaryotic ribosomes, a process called depurination. Our study has indicated that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA resulting in inhibition of translation. HCV RNA translation is mediated by a complex RNA element called the internal ribosome entry site (IRES), located within the 5' untranslated region of the viral RNA. The current structural model of HCV IRES indicates the presence of four major stem-loops I-IV and a pseudoknot. Except for stem-loop I, all are required for translation initiation. Two SRL motifs have been found on stem-loops II and Illd. Our preliminary studies have indicated that PAP can affect the HCV IRES and reduce the reporter firefly luciferase activity significantly. In addition, a non- toxic PAP mutant can inhibit the translation mediated by HCV IRES as efficiently as wild type PAP. PAP may exert its detrimental effect through depurination, possibly on the SRL motifs found on the HCV IRES element. Therefore, specific aims of this application are to: (1) determine if nontoxic PAP mutants inhibit HCV IRES directed translation and whether they destabilize the reporter mRNA, (2) determine if PAP inhibits HCV IRES-mediated translation by binding to the HCV IRES and depurinating the SRL motifs, (3) identify the PAP binding and depurination sites on the HCV IRES and determine if these sequences arp critical for inhibition on IRES-mediated translation. This study will lay the groundwork for designing novel drugs against HCV for the treatment of chronic hepatitis.