Novel Inhibitors of Poxvirus Replication

痘病毒复制的新型抑制剂

• 批准号: 6818481
• 负责人: Edward G. Niles
• 金额: $ 31.4万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-15 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6818481
• 关键词: Poxviridae; RNA biosynthesis; adenosine; enzyme linked immunosorbent assay; gene expression; genetic transcription; high performance liquid chromatography; inhibitor /antagonist; laboratory mouse; oligonucleotides; tissue /cell culture; uridine; vaccinia virus; virus replication

DESCRIPTION (provided by applicant): Poxviruses are double stranded DNA viruses that replicate in the cytoplasm of infected cells. Poxviruses encode enzymes needed to replicate their DNA, transcribe viral genes and process viral mRNA. Viral genes can be sorted into three classes that differ in their time of expression, promoter utilization and transcription initiation factors. Early genes are transcribed in the virus core immediately after infection. Early genes are the only class that responds to sequence dependent transcription termination. Early gene transcription termination requires the signal UUUUUNU in the mRNA, NPH I, a ssDNA dependent ATPase, VTF, the viral termination factor in the virion RNA polymerase that has a unique subunit, Rap94, the product of gene H4L. Recently, we showed that addition of an RNA oligonucleotide that has the sequence UUUUUUUUU stimulated premature transcription termination in vitro and in isolated virus cores. Premature termination was not observed when RNA possessing a mutant or chemically modified termination signal was added, demonstrating specificity. Importantly, stimulation of premature termination was independent of the template DNA sequence. Thus all early gene transcription termination is sensitive to the addition of a UUUUUNU containing oligonucleotide. We propose to exploit this observation and identify chimeric RNA oligonucleotides that inhibit poxvirus replication through the stimulation of premature transcription termination. We will synthesize and evaluate the ability of selected chemically modified oligonucleotides to stimulate premature termination in vitro and in isolated virus cores. Active oligonucleotides will be tested for their ability to inhibit virus gene expression and replication in tissue culture. Finally, the most active chimeric oligonucleotides will be tested for their ability to inhibit ectromelia virus (mousepox) replication in BALB/c mice. These studies will permit us to determine whether safe effective anti-poxvirus drugs can be synthesized based on their ability to inhibit virus replication through inhibition of early mRNA synthesis.

描述（由申请人提供）：痘病毒是在感染细胞的细胞质中复制的双链DNA病毒。痘病毒编码复制其DNA、转录病毒基因和处理病毒mRNA所需的酶。病毒基因可分为三类，它们在表达时间、启动子利用和转录起始因子方面存在差异。早期基因在感染后立即在病毒核心转录。早期基因是唯一一类对序列依赖性转录终止有反应的基因。基因早期转录终止需要mRNA中的UUUUUNU信号，NPH I，一种依赖于ssDNA的atp酶，VTF，病毒粒子RNA聚合酶中的病毒终止因子，具有独特的亚基，Rap94，基因H4L的产物。最近，我们在体外和分离的病毒核中发现，添加一个序列为UUUUUUUUU的RNA寡核苷酸刺激了转录的过早终止。当加入具有突变或化学修饰的终止信号的RNA时，未观察到过早终止，证明了特异性。重要的是，过早终止的刺激与模板DNA序列无关。因此，所有早期基因转录终止都对添加含有UUUUUNU的寡核苷酸敏感。我们建议利用这一观察结果，鉴定嵌合RNA寡核苷酸，通过刺激过早转录终止抑制痘病毒复制。我们将合成并评估选定的化学修饰寡核苷酸在体外和分离的病毒核中刺激过早终止的能力。将测试活性寡核苷酸在组织培养中抑制病毒基因表达和复制的能力。最后，将测试最活跃的嵌合寡核苷酸在BALB/c小鼠中抑制鼠痘病毒复制的能力。这些研究将使我们能够确定是否可以合成安全有效的抗痘病毒药物，基于它们通过抑制早期mRNA合成来抑制病毒复制的能力。