Parvovirus RNA Processing and Gene Expression Strategies

描述（由申请人提供）：细小病毒科是小型非包膜二十面体病毒，是包括人类在内的许多动物物种的重要病原体。它们拥有5kb的线性单链DNA （ssDNA）基因组，它们的复制广泛依赖于宿主核因子。我们最近的工作表明，许多细小病毒的表达谱具有令人印象深刻的复杂转录反激活过程，可选RNA加工和可选翻译起始，这些过程最大化了它们的编码能力。我们已经确定了细小病毒复制在本应用中要追求的三个关键方面。1. 与AAV2不同，AAV5编码Rep- p19生成的转录本在中心内含子内聚腺苷化，因此不剪切，排除了产生Rep 40和Rep52的AAV2样机制。令人惊讶的是，AAV5通过利用一个新的内部翻译起始事件来确保必需的Rep 40样蛋白的产生。位于AAV5的两个aug之间的150 nt RNA区域作为一个新的，积极作用的信号来编程内部起始。因此，我们在特异性目标1中提出：表征AAV小代表基因的替代翻译起始。2. 鹅细小病毒（GPV）剪接代表基因mRNA的翻译在内部启动，特别绕过了未剪接RNA中使用的强启动AUG。虽然内部起始选择在翻译过程中发生在细胞质中，但它是由翻译mRNA中不存在的GPV内含子内的元件决定的。代表基因的替代翻译起始是由剪接过程本身和干预内含子的性质所引导的，因此GPV rna的核加工影响了这些rna在细胞质中的翻译命运。因此，我们在具体目标II中提出：表征核RNA加工事件如何控制GPV mrna的替代翻译。3. 犬细小病毒（MVC）的衣壳基因包含一个有效的内部聚腺苷化位点，我们最近发现MVC NP1蛋白对于保留该位点的部分加工MVC衣壳基因mrna在细胞质中的积累是必不可少的，因此需要衣壳蛋白的产生。这代表了这种细小病毒调节获取衣壳基因信息的一种新方式。NP1是第一个被证明直接参与RNA加工的细小病毒蛋白，并且似乎以一种独特的方式起作用。因此，我们在Specific Aim III中提出：表征NP1（一种独特的病毒RNA加工因子）的功能。这些研究将阐明细小病毒基因表达的关键方面，并将在该领域发展新的范式。我们的系统提供了高度可处理、严格控制的模型，这些模型既可以推进细小病毒学，又可以对基因表达的基本方面提供重要的见解。

DESCRIPTION (provided by applicant): The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. They possess 5kb linear single-stranded DNA (ssDNA) genomes and their replication relies extensively on host nuclear factors. Our recent work has shown how the expression profiles of many of the parvoviruses feature impressively intricate processes of transcriptional transactivation, alternative RNA processing, and alternative translation initiation, which maximize their coding capacity. We have identified three aspects critical for parvovirus replication to pursue in this application. 1. Unlike AAV2, AAV5 Rep-encoding P19-generated transcripts are polyadenylated within the central intron and therefore not spliced, precluding an AAV2-like mechanism for making both Rep 40 and Rep52. Surprisingly, AAV5 ensures the production of the essential Rep 40-like protein by utilizing a new internal translation initiation event. The 150 nt RNA region lying between the two AUGs of AAV5 functions as a novel, positively-acting signal to program internal initiation. Thus, we propose in Specific Aim I: to characterize alternative translation initiation of the AAV small rep gene. 2. Translation of the goose parvovirus (GPV) spliced rep gene mRNA initiates internally, specifically bypassing a strong initiating AUG used in unspliced RNA. Although the internal initiation choice occurs in the cytoplasm during translation, it is dictated by elements within the GPV intron not present in the translated mRNA. Alternative translation initiation of the rep-gene is directed by the splicing process itself, and by the nature of the intervening intron, and thus nuclear processing of GPV RNAs influences the translation fate of these RNAs in the cytoplasm. Therefore, we propose in Specific Aim II: to characterize how nuclear RNA processing events govern alternative translation of GPV mRNAs. 3. The capsid gene of minute virus of canines (MVC) contains a potent internal polyadenylation site, and we have recently discovered that the MVC NP1 protein is essential for the accumulation in the cytoplasm of partially-processed MVC capsid gene mRNAs that retain that site, and thus required for the production of the capsid proteins. This represents a novel way in which this parvovirus regulates access to capsid gene information. NP1 is the first parvovirus protein shown to be directly involved in RNA processing, and appears to act in a unique manner. Thus, we propose in Specific Aim III: to characterize the function of NP1, a unique viral RNA processing factor. These studies will clarify critical aspects of parvovirus gene expression and will develop new paradigms in the field. Our systems afford highly tractable, tightly controlled models that are positioned to both advance parvovirology and to deliver significant insight into fundamental aspects of gene expression.