STRUCTURE-FUNCTION RELATIONS IN HIV REPLICATION

HIV复制中的结构-功能关系

• 批准号: 2067509
• 负责人: Stanley M. Lemon
• 金额: $ 15.74万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067509
• 关键词: Hepatovirus; RNA binding protein; chemical cleavage; gene mutation; genetic mapping; genetic strain; genetic translation; molecular cloning; mutant; nucleic acid sequence; nucleic acid structure; poliovirus; radiotracer; reporter genes; ribosomes; tissue /cell culture; virus RNA; virus genetics; virus protein; virus replication

Hepatitis A virus (HAV) is a unique picornavirus which causes acute hepatitis in man. We are interested in studying the structure and function of the 5' nontranslated region (NTR) of HAV RNA and its role in viral translation and viral replication in cell culture. We developed a model of the secondary structure of the NTR based on identification of covariant nucleotide substitutions, cleavage by double- and single-strand specific RNases, and thermodynamic predictions. We plan to refine this model by (1) phylogenetic analysis of additional HAV strains, (2) RNase and chemical nuclease studies of secondary structure in the less well defined 5' half of the NTR, and (3) Fe(II)-EDTA strand scission mapping of surface features and RNA folding patterns. Our previous work indicates that the 5'NTR contains an internal ribosomal entry site (IRES) extending from 5' of base 354 to the initiator AUG. We plan to map the structural elements of this IRES by examining (1) in vitro translation of bicistonic constructs in which translation of the second cistron is controlled by 5'NTR elements, and (2) in vivo translation of mono- and bicistonic constructs in which expression of a reporter gene (CAT) is under control of 5'NTR elements in permissive cells. These experiments will utilize stably transfected BS-C-1 cells (BT7-6 cell line) which constitutively express the bacteriophage T7 RNA polymerase. We also demonstrated that mutations at several sites within the 5'NTR contribute to a highly host-cell specific enhancement of virus replication in BS-C-1 cells. One (and possibly two) of these sites is within the HAV IRES, suggesting that these mutations may enhance translation in vivo. We will test this hypothesis by examining translation of wt and mutant 5'NTR constructs in BT7-6 cells. Preliminary work indicates that a 39 kDa cytoplasmic protein of BS-C-1 cells (p39) binds specifically to two domains within the 5'NTR. These domains have limited sequence or structural relatedness, yet compete with each other for binding to p39. We aim to (1) carry out a genetic analysis to determine the RNA structural requirements for binding by p39 in both domains, (2) determine whether p39 is part of the eIF-2/2B complex, and (3) assess whether 5'NTR mutations associated with enhanced growth in BS-C-1 cells alter affinity of RNA for p39. Because HAV translation follows binding of the 40S ribosomal subunit to the IRES, we postulate that HAV translation will continue and perhaps be enhanced in the presence of cytoplasmic expression of the poliovirus 2A protein, which should result in cleavage of the p220 component of eIF-4F and shut down translation of capped mRNAs. We will determine whether expression of poliovirus 2A in BT7-6 cells results in continued HAV translation, and influences yields of infectious HAV.

甲型肝炎病毒 (HAV) 是一种独特的小核糖核酸病毒，可引起急性肝炎 男性肝炎。 我们有兴趣研究结构和 HAV RNA 5'非翻译区（NTR）的功能及其在 细胞培养中的病毒翻译和病毒复制。 我们开发了 基于识别的 NTR 二级结构模型 共变核苷酸取代、双链和单链切割 特定的 RNases 和热力学预测。 我们计划完善这个 通过 (1) 其他 HAV 毒株的系统发育分析，(2) RNase 建立模型 和化学核酸酶研究不太好的二级结构 定义了 NTR 的 5' 一半，以及 (3) Fe(II)-EDTA 链断裂图谱 表面特征和 RNA 折叠模式。 我们之前的工作 表明 5'NTR 包含内部核糖体进入位点 (IRES) 从碱基354的5'延伸至引发剂AUG。 我们计划绘制地图 通过检查 (1) 体外翻译来确定此 IRES 的结构要素 双顺结构，其中第二个顺反子的翻译是 由 5'NTR 元件控制，以及（2）单和的体内翻译 双曲结构，其中报告基因（CAT）的表达是 受许可细胞中 5'NTR 元件的控制。 这些实验 将利用稳定转染的 BS-C-1 细胞（BT7-6 细胞系）， 组成型表达噬菌体 T7 RNA 聚合酶。 我们也 证明 5'NTR 内多个位点的突变有助于 BS-C-1 中病毒复制的高度宿主细胞特异性增强 细胞。 其中一个（可能还有两个）位于 HAV IRES 内， 表明这些突变可能增强体内翻译。 我们将 通过检查 wt 和突变体 5'NTR 的翻译来检验这一假设 在 BT7-6 细胞中构建。 初步工作表明 39 kDa BS-C-1 细胞 (p39) 的细胞质蛋白特异性结合两个 5'NTR 内的结构域。 这些结构域的序列有限或 结构相关性，但彼此竞争与 p39 的结合。 我们的目标是 (1) 进行遗传分析以确定 RNA p39 在两个域中结合的结构要求，(2) 确定 p39 是否是 eIF-2/2B 复合体的一部分，以及 (3) 评估 5'NTR 是否 与 BS-C-1 细胞生长增强相关的突变改变亲和力 p39 的 RNA。 因为 HAV 翻译遵循 40S 的绑定 核糖体亚基到 IRES，我们假设 HAV 翻译将 继续并可能在细胞质存在的情况下得到增强 脊髓灰质炎病毒 2A 蛋白的表达，这会导致裂解 eIF-4F 的 p220 组件并关闭 capped 的翻译 mRNA。 我们将确定 BT7-6 中是否表达脊髓灰质炎病毒 2A 细胞导致 HAV 持续翻译，并影响产量 传染性甲型肝炎病毒。