The Biology of Influenza A Virus-Generated Small RNAs

描述(申请人提供)：在我们继续努力研究宿主对甲型流感病毒感染的反应时，我们对感染细胞的小RNA进行了深度测序，发现大量甲型流感病毒衍生的小RNA映射到每个病毒RNA片段的5‘端。Northern印迹证实了这种小RNA的表达，需要病毒聚合酶。考虑到病毒基因组的二级结构，我们假设这种小病毒RNA(SvRNA)的产生将导致狭长柄的破坏，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说，我们合成了svRNA，并在转染后观察到mRNA产生减少，vRNA合成增加，流感病毒繁殖普遍丧失。此外，通过竞争性杂交阻断svRNA的可获得性会导致mRNA合成增加和vRNA完全丧失。SvRNA的鉴定回答了一个非常长期存在的问题，即甲型流感病毒如何从转录转换到复制，并展示了一种针对小RNA和病毒感染的新范式。尽管已经确定了svRNA的这一假定功能，但它是如何产生的，其活动的广度在很大程度上仍不清楚。在这里，我们试图更彻底地理解这些细节。AIM 1详细说明了一种策略，以确定每个片段特定的svRNA如何发挥作用。目的2描述建立体外合成svRNA的模型，以确定其合成的分子机制。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，这些靶点可用于产生一类新型的抗流感病毒疗法，并将显著增加我们对流感病毒复制的理解。

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.