追蹤A型流感病毒在活細胞內的複製動態

Abstract

A型流感病毒（IAV）是造成季節性流感與全球大流行的病原體。由於其具有基因突變率高、傳播速度快、跨物種感染的特性，IAV對於人類的健康造成很大的威脅。因此，目前對於新型抗流感策略的發展有急迫的需求。然而，過去研究流感病毒生活史的實驗方法有它的局限性，不能即時追蹤病毒的基因體RNA（viral RNA, vRNA）在活細胞內與宿主蛋白質交互作用的詳細過程。所以，本研究建立一個以螢光蛋白質為基礎的活細胞影像系統，以偵測流感病毒vRNA在單一細胞層次的即時動態。我們利用MS2噬菌體的殼體蛋白質（MS2 coat protein, MCP）與基因體RNA上的MCP結合位高度專一結合的特性，將24個會形成特殊二級結構的序列（24xMSL）插入至A型流感病毒PB2 vRNA，取代部分PB2基因的編碼序列（coding sequence），形成PB2-24xMSL vRNA。實驗結果顯示，這種攜帶PB2-24xMSL vRNA的流感病毒可以在持續表現PB2蛋白質的MDCK細胞中增殖。另外，為了瞭解不同vRNA是否會在細胞核內相同的區域複製，我們也將18個來自大腸桿菌且會形成特殊二級結構的序列（18xBSL）插入至流感病毒HA vRNA，創造HA-18xBSL vRNA。分析結果顯示PB2和HA在細胞核內不同的位置複製。本研究建立的系統可以突破傳統研究方法的限制，探討流感病毒在宿主細胞內的生理活動。此研究系統可能有助於新型抗流感策略的發展。

Influenza A virus (IAV) is the pathogen causing recurrent influenza epidemics and severe pandemics. IAV has wide disseminations across species and has resulted in many fatal human infections. Therefore, there is an urgent need to develop new anti-influenza strategies to prevent influenza’s spreading. However, there are perceived challenges and limitations in understanding the life cycle of influenza due to the lacks of adequate approaches to investigate the dynamic and kinetic interactions of the virus with its host cells in real time in live cells. In this study, we established a fluorescent protein-based live-cell imaging system to visualize influenza RNA genomes at single-cell level. Accordingly, 24 copies of the stem-loop structures for the binding of bacteriophage MS2 coat protein was inserted into the influenza viral PB2 segment in a reverse genetic system to substitute their coding sequences. Our results have shown that the engineered influenza A virus is likely to be propagated in cells complemented with PB2. This study also engineered influenza virus’ HA vRNA to encode 18 copies of Bgl stem-loops for determining whether IAV’s segments are recruited to specific locations of the nucleus for efficient replication. However, we found that influenza segments replicate independently at their own subnuclear domains rather than replicate together as bundled units. Our reserch aims to investigate the most fundamental aspects of influenza A virus physiology that are difficult to study by using conventional methods. This study will lead to the development of new anti-influenza strategies to prevent its infection.