Effect of Pokeweed antiviral protein on hepatitis C virus IRES

美洲商陆抗病毒蛋白对丙型肝炎病毒IRES的影响

• 批准号: 7034327
• 负责人: RONG DI
• 金额: $ 7.7万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7034327
• 关键词: RNA; adenine; binding sites; hepatitis; hepatitis C virus; hepatocellular carcinoma; infection; interferons; model; mutant; proteins; ribosomes

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.

描述（由申请人提供）：我们建议测试美洲商陆抗病毒蛋白（PAP）对丙型肝炎病毒（HCV）内部核糖体进入位点（IRES）介导的翻译的影响。HCV是慢性肝炎的主要病因，慢性肝炎可导致危及生命的肝硬化和肝细胞癌。世卫组织1999年的一项全球调查估计，世界上至少有1.7亿人慢性感染HCV。干扰素是目前治疗慢性肝炎的最佳药物。然而，它与相对较差的疗效和不利的副作用有关。PAP是从美洲商陆中分离的29-kDa抗病毒/抗真菌蛋白。它属于一组称为核糖体失活蛋白（RIP）的蛋白质。已知RIPs可催化去除真核和原核核糖体大rRNA中高度保守的α-八叠球菌素/蓖麻毒素环（SRL）中的特定腺嘌呤残基，该过程称为脱嘌呤。我们的研究表明，除了先前确定的腺嘌呤（A4324），PAP删除另一个腺嘌呤（A4321）和鸟嘌呤（G4323）从真核生物大rRNA导致抑制翻译。HCV RNA的翻译是由一个复杂的RNA元件介导的，称为内部核糖体进入位点（IRES），位于病毒RNA的5'非翻译区。目前的HCV IRES结构模型表明存在四个主要的茎环I-IV和一个假结。除茎环I外，其他都是翻译起始所必需的。在茎环II和IIId上发现了两个SRL基序。我们的初步研究表明，PAP可以影响HCV IRES，并显着降低报告萤火虫荧光素酶活性。此外，无毒PAP突变体可以与野生型PAP一样有效地抑制HCV IRES介导的翻译。PAP可能通过脱嘌呤作用对HCV IRES元件上发现的SRL基序发挥其有害作用。因此，本申请的具体目的是：（1）确定无毒PAP突变体是否抑制HCV IRES指导的翻译以及它们是否使报告mRNA不稳定，（2）确定PAP是否通过结合HCV IRES并使SRL基序脱嘌呤而抑制HCV IRES介导的翻译，（3）鉴定HCV IRES上的PAP结合和脱嘌呤位点，并确定这些序列阿普是否对抑制IRES介导的翻译至关重要。本研究为设计新型抗HCV药物治疗慢性肝炎奠定了基础。