Novel Inhibitors of Poxvirus Replication

描述(申请人提供)：痘病毒是在受感染细胞的细胞质中复制的双链DNA病毒。痘病毒编码复制DNA、转录病毒基因和处理病毒信使核糖核酸所需的酶。病毒基因可分为三类，它们的表达时间、启动子利用和转录起始因子不同。早期基因在感染后立即在病毒核心中转录。早期基因是唯一一类对序列依赖转录终止作出反应的基因。早期的基因转录终止需要RNA中的信号UUUUUNU，NPH I，一个依赖于SSDNA的ATPase，VTF，病毒RNA聚合酶中的病毒终止因子，它有一个独特的亚基Rap94，H4L基因的产物。最近，我们发现，在体外和分离的病毒核心中，加入具有UUUUUUUUUU序列的RNA寡核苷酸可刺激提前转录终止。当加入具有突变或化学修饰的终止信号的RNA时，没有观察到过早终止，显示出特异性。重要的是，提前终止的刺激与模板DNA序列无关。因此，所有早期基因转录终止都对添加含有寡核苷酸的UUUUUNU敏感。我们建议利用这一观察结果，并确定通过刺激提前转录终止来抑制痘病毒复制的嵌合RNA寡核苷酸。我们将在体外和分离的病毒核心中合成和评估选定的化学修饰的寡核苷酸刺激提前终止的能力。活性寡核苷酸将在组织培养中测试其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是否具有抑制鼠痘病毒在BALB/c小鼠体内复制的能力。这些研究将使我们能够确定是否可以基于通过抑制早期mRNA合成来抑制病毒复制的能力来合成安全有效的抗痘病毒药物。

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.