The Biology of Influenza A Virus-Generated Small RNAs

描述（由申请人提供）：在我们继续研究宿主对甲型流感病毒感染的反应的过程中，我们对感染细胞的小RNA进行了深度测序，发现了大量甲型流感病毒衍生的小RNA，这些小RNA位于每个病毒RNA片段的5'端。这种小RNA的表达通过northern blot证实，并且需要病毒聚合酶。鉴于已知的病毒基因组二级结构，我们假设这种小病毒RNA （svRNA）的产生会导致狭长链的破坏，并导致从转录到复制的转换。由于svRNA产生的时间支持这一假设，我们合成了svRNA，并在转染后观察到mRNA产生减少，vRNA合成增加，流感病毒传播普遍丧失。此外，通过竞争杂交阻断svRNA的可用性导致mRNA合成增加和vRNA的完全丧失。svRNA的鉴定回答了一个长期存在的问题，即甲型流感病毒如何从转录转向复制，并举例说明了小rna和病毒感染的新范式。尽管确定了svRNA的这一假定功能，但它是如何产生的及其活性的广度在很大程度上仍然未知。在这里，我们试图更彻底地了解这些细节。目标1详细介绍了确定每个特定段的svRNA如何起作用的策略。目标2描述了开发体外svRNA合成模型以确定其合成的分子机制的努力。目的3研究svRNA如何影响宿主细胞环境。包含这些目标的实验策略将揭示令人兴奋的新分子靶点，可用于产生一类新的抗流感病毒疗法，并将大大增加我们对流感病毒复制的了解。

DESCRIPTION (provided by applicant): In our continued efforts to study the host response to influenza A virus infection, we performed deep sequencing on small RNAs from infected cells and discovered a large population of influenza A virus derived small RNAs mapping to the 5' end of the each of the viral RNA segments. Expression of this small RNA was corroborated by northern blot and required the viral polymerase. Given the known secondary structure of the viral genome, we hypothesized that generation of this small viral RNA (svRNA) would result in disruption of the panhandle and would cause a switch from transcription to replication. As the timing of svRNA production supported this hypothesis, we synthesized svRNA and, upon transfection, observed a decrease in mRNA production, an increase in vRNA synthesis, and a general loss of influenza virus propagation. Furthermore, blocking svRNA availability through competitive hybridization resulted in an increase in mRNA synthesis and complete loss of vRNA. Identification of svRNA answers a very longstanding question as to how influenza A virus switches from transcription to replication and exemplifies a new paradigm for small RNAs and virus infection. Despite determining this putative function for svRNA, how it is generated and the breadth of its activity remains largely unknown. Here we seek to understand these details more thoroughly. Aim 1 details a strategy to ascertain how each segment-specific svRNA functions. Aim 2 describes efforts to develop a model for in vitro svRNA synthesis to determine the molecular mechanism by which they are synthesized. Aim 3 investigates how svRNA affects the host cell environment. The experimental strategy comprising these aims will reveal exciting new molecular targets that can be exploited to generate a novel class of anti-influenza virus therapeutics and will significantly increase our understanding of influenza virus replication.