Parvovirus RNA Processing and Gene Expression Strategies

描述（由申请方提供）：细小病毒亚科是小型无包膜二十面体病毒，是许多动物种属（包括人类）的重要病原体。它们具有5 kb的线性单链DNA（ssDNA）基因组，并且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的转录反式激活过程，替代RNA加工和替代翻译起始，最大限度地提高其编码能力。我们已经确定了三个方面的关键细小病毒复制追求在这个应用程序。 1.与AAV 2不同，AAV 5编码Rep的P19产生的转录物在中央内含子内被多腺苷酸化，因此不剪接，排除了产生Rep 40和Rep 52的AAV 2样机制。令人惊讶的是，AAV 5通过利用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AAV 5的两个AUC之间的150 nt RNA区域作为一种新的正作用信号来编程内部起始。因此，我们在具体目标I中提出：表征AAV小rep基因的替代翻译起始。 2.鹅细小病毒（GPV）剪接的rep基因mRNA的翻译在内部启动，特别是绕过未剪接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GPV内含子内不存在于翻译的mRNA中的元件决定的。rep-基因的选择性翻译起始由剪接过程本身和间插内含子的性质指导，因此GPV RNA的核加工影响这些RNA在细胞质中的翻译命运。因此，我们在特定目标II中提出：描述核RNA加工事件如何控制GPV mRNA的替代翻译。 3.犬细小病毒（MVC）的衣壳基因含有一个有效的内部多聚腺苷酸化位点，我们最近发现，MVC NP 1蛋白对于保留该位点的部分加工的MVC衣壳基因mRNA在细胞质中的积累是必不可少的，因此是生产衣壳蛋白所必需的。这代表了一种新的方式，这种细小病毒调节获得衣壳基因信息。NP 1是第一个被证明直接参与RNA加工的细小病毒蛋白，并且似乎以独特的方式起作用。因此，我们在《特定目标III》中提出：表征NP 1（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，并将在该领域开发新的范例。我们的系统提供高度易处理，严格控制的模型，定位于推进细小病毒学，并提供对基因表达基本方面的重要见解。

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.