MECHANISMS OF GENOMIC RNA PACKAGING IN INFLUENZA VIRUS

描述（由申请人提供）：流感病毒是人类最多产的病毒杀手，在典型的一年里杀死数万名美国人，当新的大流行毒株出现时，全世界有多达2000万至1亿人死亡。为了具有传染性，单个病毒粒子必须包含（包装）组成其基因组的八个线性RNA片段中的每个片段的至少一个副本。当一种现有菌株在双重感染的宿主中包装另一菌株的一个或多个片段，产生具有新的免疫和致病特性的基因重组时，大流行就会出现。因此，了解流感RNA包装对于理解和控制这种致命的重新出现的病毒至关重要。然而，迄今为止，包装所需的顺式作用决定因素（即包装信号）尚未完全阐明任何区段，所涉及的反式作用因素是未知的。我们的实验室在确定HIV对RNA包装的要求方面有丰富的经验。在最近的研究中，我们已经将有效包装所需的信号定位在三个流感RNA片段的一端或两端的离散45至200个碱基区域内。我们现在建议详细绘制活性区域，并确定RNA序列和/或二级结构的哪些特定特征是包装这些和其他五个片段所必需的。我们还将追求我们令人惊讶的观察，即3‘或5’包装区域不是简单地可互换的区段之间，这意味着，迄今未知的组合规则管理他们在包装中的相互作用。此外，我们已经获得了令人信服的证据，表明流感PA蛋白（三聚体病毒RNA聚合酶的一个组成部分）是包装所有八个病毒RNA片段所需的反式作用因子。我们现在将研究PA和其他病毒蛋白识别病毒RNA片段中的包装信号并指导其整合到病毒粒子中的分子途径。我们提出的研究将有助于阐明包装与病毒粒子组装之间的联系，将扩展我们对流感遗传学和重组的理解，并可能揭示抗病毒药物开发的脆弱新靶点。

DESCRIPTION (provided by applicant): The influenza virus is humanity's most prolific viral assassin, killing tens of thousands of Americans in a typical year and as many as 20-100 million people worldwide when new a pandemic strain emerges. In order to be infectious, an individual virion must incorporate (package) at least one copy of each of the eight linear RNA segments that make up its genome. Pandemics are thought to arise when an existing strain packages one or more segments from another strain in a dually-infected host, yielding a genetic reassortant with novel immunologic and pathogenic properties. Understanding influenza RNA packaging is therefore critical for understanding and, perhaps, controlling, this lethal re-emerging virus. To date, however, the cis-acting determinants required for packaging (i.e., the packaging signals) have not been fully elucidated for any segment, and the trans-acting factors involved are unknown. Our laboratory has extensive experience in defining the requirements for RNA packaging by HIV. In recent studies, we have localized the signals required for efficient packaging to within discrete 45- to 200-base regions at one or both ends of three influenza RNA segments. We now propose to map the active regions in detail and to determine which specific features of RNA sequence and/or secondary structure are required for packaging of these and the other five segments. We will also pursue our surprising observation that 3' or 5' packaging regions are not simply interchangeable among segments, which implies that as-yet-unknown combinatorial rules govern their interactions in packaging. In addition, we have obtained compelling evidence implicating the influenza PA protein (a component of the trimeric viral RNA polymerase) as a trans-acting factor required for packaging all eight viral RNA segments. We will now investigate the molecular pathway by which PA and other viral proteins recognize the packaging signals in viral RNA segments and directing their incorporation into virions. The studies we propose will help elucidate the linkage between packaging and virion assembly, will extend our understanding of influenza genetics and reassortment, and may reveal vulnerable new targets for antiviral drug development.