PUTATIVE ENCASIDATION SIGNALS IN PARVOVIRUS LUIII

细小病毒 LUIII 中的推定包壳信号

• 批准号: 3734291
• 负责人: NANETTE DIFFOOT-CARLO
• 金额: --
• 依托单位: UNIVERSITY OF PUERTO RICO MAYAGUEZ
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3734291
• 关键词: DNA binding protein; HeLa cells; Parvoviridae; biological signal transduction; cell growth regulation; cellular oncology; cellular pathology; clone cells; gel mobility shift assay; genetic library; genetic manipulation; minute virus of mice; molecular cloning; neoplastic transformation; nucleic acid hybridization; nucleic acid sequence; nucleocapsid; transfection; virus envelope; virus protein; virus replication

The autonomous parvovirus LuIII, initially isolated from a human cell line, is unique among the mammalian parvoviruses because its genome has non-identical palindromic termini, yet, during replication it encapsidates both strands with equal frequency. With the exception of LuIII, parvoviruses of known nucleotide sequence that encapsidate equal amounts of both DNA strands, have identical 3' and 5' termini. The nucleotide sequence of the Lulll termini shares over 90% sequence identity with that of the rodent parvovirus Minute Virus of Mice (MVM) and an overall genome identity of over 80%. Unlike LuIII, MVM encapsidates 99% minus strand DNA. The minor nucleotide differences observed between the termini of LuIII and MVM very likely do not determine the encapsidation pattern observed for Lulll since similar differences are observed between the sequences of MVM and that of another rodent parvovirus, H-1 which also encapsidates 99% minus strand. Two regions differ significantly between LuIII and MVM. At map unit (m.u.) 92 LuIII has a single copy of a sequence present in tandem in the prototype strain of MVM (MVMp). Although this sequence has been suggested to be involved in replication it most likely is not determining the encapsidation pattern observed for LuIII because a lymphotrophic strain of MVM (MVMi) has a single copy of this sequence and it too encapsidates 99% minus strand. At m.u. 89 LuIII has a unique sequence, rich in A/T residues. Given the location and nature of this sequence and its absence from the genome of MVM this sequence very likely represent a major determinant in the encapsidation pattern observed for LuIII. Deciphering the sequences involved in the encapsidation of LuIII DNA will eventually lead to the characterization of the mechanism resulting in the encapsidation pattern observed for LuIII. The aims of this research include construction of genomic clones of LuIII and MVM (for both MVMi and MVMp) containing a deletion and insert, respectively, of the A-T rich sequence and of minigenomes with and without the A/T rich sequence containing both termini or two copies of either the 3' or 5' terminus. The clones constructed will be transfected into NBE 324K cells and the nature of the progeny viral DNA analyzed. Gel retardation assays of LuIII infected and uninfected NBE 324K cell lysates will be done to identify DNA binding proteins of viral and cellular nature using radiolabelled LuIII termini, A/T rich sequence or a combination of termini including the A/T rich sequence. A cDNA library of HELA cells will also be screened for DNA binding proteins. The long term objective of this project is to elucidate the molecular and cellular changes which lead to the suppression of oncogenic transformation in the host as a result of parvoviral relication and describe a mechanism in which the infection of a parvovirus may be mimicked at a cellular level as to suppress transformation of the cell.

自主细小病毒 LuIII，最初从人体细胞中分离出来 线，在哺乳动物细小病毒中是独一无二的，因为它的基因组具有 不同的回文末端，然而，在复制过程中 以相同的频率包覆两条链。 除了 LuIII，已知核苷酸序列的细小病毒，其衣壳相等 数量的两条 DNA 链具有相同的 3' 和 5' 末端。 这 Lulll 末端的核苷酸序列共享超过 90% 的序列 与啮齿动物细小病毒（MVM）的同一性 整体基因组同一性超过80%。 与 LuIII 不同，MVM 包裹 99% 负链 DNA。 微小的核苷酸差异 在 LuIII 和 MVM 末端之间观察到的情况很可能不是这样 确定 Lulll 观察到的衣壳模式，因为类似 在 MVM 序列和另一个序列之间观察到差异 啮齿动物细小病毒 H-1，也包壳 99% 的负链。 二 LuIII 和 MVM 之间的区域存在显着差异。 以地图单位（m.u.） 92 LuIII 具有串联存在于 MVM 原型应变（MVMp）。 虽然这个顺序已经被 建议参与复制，很可能无法确定 LuIII 观察到的包壳模式是因为淋巴营养性 MVM (MVMi) 菌株也有该序列的单个副本 包壳 99% 负链。 在 m.u. 89 LuIII 具有独特的序列， 富含A/T残留物。 考虑到该序列的位置和性质， 它在 MVM 基因组中的缺失，该序列很可能代表 LuIII 观察到的衣壳模式的主要决定因素。 破译参与 LuIII DNA 衣壳化的序列将 最终导致机制的表征 观察到 LuIII 的衣壳化模式。 本研究的目的 包括构建 LuIII 和 MVM 的基因组克隆（对于 MVMi 和 MVMp) 分别包含富含 A-T 的删除和插入 序列以及具有和不具有 A/T 丰富序列的小基因组 包含两个末端或 3' 或 5' 末端的两个副本。 将构建的克隆转染至NBE 324K细胞中并 分析的子代病毒DNA的性质。 凝胶阻滞测定 LuIII 感染和未感染的 NBE 324K 细胞裂解物将用于 鉴定病毒和细胞性质的 DNA 结合蛋白 放射性标记的 LuIII 末端、富含 A/T 的序列或组合 末端包括富含 A/T 的序列。 HELA 细胞的 cDNA 文库 还将筛选 DNA 结合蛋白。 长期目标 该项目的目的是阐明分子和细胞的变化 导致宿主致癌转化的抑制 细小病毒复制的结果并描述了一种机制，其中 细小病毒的感染可以在细胞水平上进行模拟 抑制细胞的转化。