PARAINFLUENZA VIRUS TYPE 3 (PIV3) GENE EXPRESSION

副流感病毒 3 型 (PIV3) 基因表达

• 批准号: 3790722
• 负责人: P L COLLINS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/3790722
• 关键词: DNA directed RNA polymerase; Paramyxoviridae disease; Paramyxovirus; chimeric proteins; chloramphenicol acetyltransferase; complementary DNA; gene expression; genetic manipulation; genetic promoter element; genetic regulatory element; genetic transcription; nucleic acid sequence; recombinant DNA; tissue /cell culture; transfection; viral rescue; virus RNA; virus assembly; virus genetics; virus protein; virus replication

A new, simple system was developed for the direct manipulation of sequences of the genomic RNA (vRNA) of human parainfluenza virus type 3 (PIV3). Specifically, cDNA was constructed to encode a truncated synthetic vRNA analog in which all of the viral genes have been removed and replaced by the bacterial chloramphenicol acetyl transferase (CAT) gene. The cDNA is under the control of the promoter for T7 RNA polymerase and, at the distal end, contains an Hga1 site for linearization. Transcription of the cDNA in vitro yields a negative-sense RNA containing the exact, correct vRNA termini. The RNA was transfected into tissue culture cells which were infected with PIV3 to supply complementing proteins. Under these conditions, the RNA was rendered biologically active ("rescued") as evidenced by the intracellular expression of CAT and by the production of a component (presumably infectious particles) that was released into the medium and could be passed productively onto fresh cells in the presence of helper virus. Together with a similar system developed by us for human respiratory syncytial virus (RSV) (accompanying report) and by others for Sendai virus, this represents the first experimental method by which one can synthesize, and thereby manipulate, biologically-active analogs of the vRNA of a nonsegmented negative strand virus. It will now be possible to directly identify and characterize cis-acting signals in vRNA. It should also be possible to modify the system such that rescue is complemented by cDNA-encoded viral proteins in place of PIV3. this would make it possible to characterize the roles of the proteins in replication, transcription and virion morphogenesis. Importantly, the success in rescuing PIV3-CAT supports the idea that it ultimately will be possible to rescue a complete, nondefective synthetic vRNA encoding infectious virus.

开发了一个新的简单系统，用于直接操纵序列 人类帕氏素病毒3型（PIV3）的基因组RNA（VRNA）。 具体而言，构建cDNA以编码截短的合成VRNA 所有病毒基因已被去除并取代的类似物 细菌氯霉素乙酰转移酶（CAT）基因。 cDNA是 在T7 RNA聚合酶的启动子的控制下，在远端 结束，包含用于线性化的HGA1站点。 cDNA的转录 体外产生一个负敏感的RNA，包含精确的，正确的VRNA 终点站。 将RNA转染到组织培养细胞中，该细胞是 感染PIV3以提供补充蛋白质。 在这些 条件，RNA被赋予生物活性（“救出”） 由CAT的细胞内表达和产生来证明 被释放到该的成分（可能是传染性颗粒） 培养基，可以在存在的情况下有效地传递到新鲜细胞上 助手病毒。 与我们为人类开发的类似系统一起 呼吸道合胞病毒（RSV）（随附的报告）和其他人 仙台病毒，这代表了第一个实验方法 可以合成，从而操纵生物活性的类似物 未分段的阴性链病毒的VRNA。 现在可以 直接识别并表征VRNA中的顺式作用信号。 它应该 也可以修改系统，以使救援得到补充 cDNA编码的病毒蛋白代替PIV3。 这将使它成为可能 表征蛋白质在复制，转录和 病毒体形态发生。 重要的是，拯救PIV3-CAT的成功 支持这样的想法，即最终有可能营救一个完整的， 编码传染病的非缺陷合成VRNA。