以鄰近標記蛋白質體學探究流感病毒形成時在細胞膜上的病毒—宿主交互作用之動態

Abstract

在A型流感病毒(Influenza A virus, IAV)感染的後期，新合成的病毒結構蛋白與基因組會集合到細胞膜上的脂筏 (lipid raft)處形成子代病毒。與其他包膜病毒不同，IAV不經由常見的內體分選複合體 (endosomal sorting complex required for transport, ESCRT)途徑進行出芽，而是採用一種非ESCRT依賴性的方式進行病毒組裝與出芽。目前關於IAV病毒顆粒形成的模型多著重於病毒蛋白在此過程中的功能，特別是基質蛋白(Matrix proteins)與神經胺酸酶 (Neuraminidase)，而宿主因子(Host Factors)在IAV的組裝與出芽機制中所扮演的角色與作用方式仍不明朗。儘管先前的研究透過許多不同方式提出可能參與IAV生命週期中各個階段的宿主因子，然而在後續進行的驗證中，只有其中少部分的宿主因子被證實對於病毒的組裝或出芽有所貢獻。因此，在這項研究中我們選擇運用以鄰近標記法 (Proximity-Labelling assay, PL assay)為基礎的蛋白質體學方法來辨識特定感染時間點會位在細胞膜周邊、可能與病毒蛋白有相互作用的宿主因子。 鄰近標記法利用特定的催化酶對其周圍半徑大約10奈米內的分子進行標定。而透過將此催化酶與具目標特徵的蛋白質（又稱「誘餌」）融合，就能標定特定亞細胞區域附近的分子。在本研究中，我們將錨定於細胞膜內側的PLCD1作為誘餌，建立了穩定表達其PH結構域與生物素連接酶 (Biotin Ligase) TurboID共同組成的融合蛋白 (PH-PLCD1-GFP-TurboID)的A549穩定細胞株。這個融合蛋白能在添加生物素 (Biotin)後迅速標記活細胞內足夠靠近細胞膜的宿主因子。利用這個方法，我們不僅可以標定本來就位於細胞膜附近的宿主因子，也可以標記那些短暫接近細胞膜或與細胞膜僅有微弱交互作用的因子。在標定後，我們以鏈親和素磁珠 (Streptavidin Magnetic Beads)純化被標定的蛋白質以進行質譜分析。藉此，我們得以鑑定出在IAV感染晚期的細胞膜周圍含量有所改變的宿主因子，進一步揭示其可能的功能，以及病毒與宿主在時間與空間上的交互作用。在未感染與IAV感染的組別中，質譜分析共鑑定出約6000個被生物素標記的蛋白質，我們從中選定了Ras-related proteins Rab-1A (Rab1A)為研究對象。Rab1A在感染後出現不同的分布，且抑制Rab1A表現會導致病毒核蛋白 (Nucleoprotein, NP)在細胞內的基因表現量與病毒產量的改變。我們將進一步研究這些新發現的宿主因子如何影響IAV的生命週期。

During the late stage of influenza A virus (IAV) infection, newly synthesized viral components, including the structural proteins and genomes, are recruited to the lipid raft domains of the cell plasma membrane to produce progeny virions. Unlike many other enveloped viruses, IAV buds from the cell through an endosomal sorting complex required for transport (ESCRT)-independent pathway. Current models of IAV virion formation primarily address the functions and roles of viral proteins, particularly matrix proteins and neuraminidase, in this process. However, the identities and functions of host factors participating in the assembly and budding mechanisms of IAV remain largely unknown. Although various studies had attempted to identify host factors that were involved in individual stages of the IAV life cycle with diverse approaches, only a few candidates were proven to contribute to virus assembly and budding processes. Therefore, in this study, we utilize the proximity-labelling (PL) based proteomic approach to investigate the host-virus interactions occurring near the plasma membrane at the infection times of interest. The PL assay employs specific engineered enzymes that will label biomolecules within its 10-nm radius with certain substrates. By fusing the aforementioned enzyme with the protein possessing features of interest, also known as the “bait”, the labeling can be performed within the vicinity of the subcellular compartment where the bait is located. In this research, we fused the biotin ligase TurboID with the PH domain of PLCD1, which is located at the inner side of plasma membrane, and established a A549 cell line that stably expresses the PH-PLCD1-GFP-TurboID fusion protein. This construct enables prompt labeling of the nearby host factors after the addition of exogenous biotin. With this approach, we can not only label host factors abundant at the plasma membrane, but also those loosely or transiently associated with it. The biotinylated proteins were then purified by the magnetic streptavidin beads and further analyzed via mass spectrometry. This strategy enabled us to identify host proteins with altered abundance at the vicinity of plasma membrane during different stages of IAV infection, implying their potential functional roles in infection events, and study the spatiotemporal-specific virus-host interactome. Using mass spectrometry, we identified 6478 biotinylated proteins across mock and four IAV-infected groups. These were subjected to further data filtering to select high-confidence candidates. Among these candidates, Ras-related proteins Rab-1A (Rab1A) were selected for further examination. Rab1A exhibited altered subcellular distributions upon the infection and the knockdown of Rab1A resulted in changes in viral nucleoprotein (NP) gene expression as well as virus production. How these novel host factors influence the replication cycle of IAV will be further investigated.