腸病毒71型(EV-A71)是正股單股RNA病毒，易感染幼童並引發手足口病和腦膜炎。了解和EV-A71有交互作用的細胞蛋白及其參與感染的生物機制，有助於建立抗病毒策略。實驗室先前結果顯示，細胞內的補體蛋白受體gC1qR與EV-A71鞘蛋白VP4有交互作用，且能在骨骼肌和神經元細胞中對病毒的複製產生負向調控。gC1qR是一種多功能細胞蛋白，更是多種病毒(例如HIV，EBV和HCV)作用的標的。為了了解EV-A71感染時gC1qR-VP4交互作用的功能以及參與反應的相關細胞因子，我們採用免疫沉澱法合併LC-MS/MS技術對EV-A71感染或無感染的HEK293T細胞之內源性gC1qR蛋白質體進行純化和分析。將EV-A71感染(EI)和假性感染(MI)的gC1qR蛋白質體以gC1qR濃度作內部校正，校準後的2042種蛋白質包含503個濃度變化超過±2倍具有顯著意義的蛋白，其中又有332個同時存在於EI和MI相關的gC1qR蛋白質體中。而在濃度變化> 2倍的149個蛋白質中，有61個獨特存在於EI相關的gC1qR蛋白質體中。此結果顯示內源性gC1qR蛋白質體之組成在EV-A71感染過程中的動態變化。我們更使用FunRich生物途徑分析軟體針對內源性gC1qR蛋白質體的2042個蛋白質進行Gene Ontology生物過程和生物途徑的預測。發現在感染期間，503個具有顯著意義的蛋白可能參與在蛋白質代謝和基因轉錄及轉譯的機制中。為了瞭解感染時與gC1qR有較為專一作用的細胞蛋白，我們進一步使用串聯親和純化(Tandem Affinity Purification, TAP)結合LC-MS/MS的蛋白質體分析方法來鑑定外源性gC1qR在細胞內的蛋白質體。gC1qR-SBP-6xHis及SBP-6xHis為兩種含串聯標籤SBP)和6xHis的外源性重組蛋白，我們以SBP-6xHis在感染時的蛋白質體作為控制組，並作為濃度變化的定量基準，發現有3個蛋白獨特存在於校準後的外源性gC1qR蛋白質體中。另外，藉由生物途徑分析內源性和外源性gC1qR蛋白質體，均比對出相同的37種蛋白顯著地參與在轉譯和蛋白質代謝機制中。更重要的是，通過多基因組交互作用的PINA4MS/Cytoscape應用軟體程序，對感染狀態下的gC1qR-interactome和先前已知的VP4-interacrome進行了比較分析，我們得到了一個VP4-gC1qR交互作用網絡，該網絡由40個包含gC1qR的細胞因子組成，並經由預測顯示參與病毒轉譯機制。為了驗證gC1qR在EV-A71的internal ribosome entry site (IRES)轉譯機制中的角色，我們建立了雙基因表達系統。將EV-A71 IRES插入海腎螢光素酶(Renilla luciferase)和螢火蟲螢光素酶(Firefly luciferase)基因之間，來監測宿主cap-dependent translation和EV-A71 IRES引導轉譯的效率。結果顯示降低gC1qR會同時使宿主和病毒的轉譯效率上升，而非單獨影響病毒IRES的轉譯機制，因此gC1qR可能在整體的細胞及病毒轉譯機制中扮演重要角色。此研究提供了gC1qR在EV-A71感染下的交互作用網絡及可能的生物機制，有助於EV-A71感染的關鍵細胞因子和機制的研究和了解。

Enterovirus 71 (EV-A71) is a positive, single-stranded RNA virus of Picornaviridae family, which causes hand, foot and mouth disease (HFMD) and meningitides especially in young children. Understanding viral interacting cellular proteins and their possible interplays during EV-A71 infection is crucial for developing antiviral strategies. Our lab has previously shown that the globular C1q binding protein (gC1qR) is an intracellular interactor of EV-A71 capsid proteinVP4 and negatively modulates viral replication in skeletal muscle and neuron cells. Cellular gC1qR is multifunctional and a critical target of several viruses, such as HIV, EBV and HCV. To better understand the functional aspects of gC1qR-VP4 interaction and associating cellular factors during EV-A71 infection, endogenous gC1qR-interactome(s) in HEK293T cells with or without infection was investigated with a combined technique of Immunoprecipitation and LC-MS/MS. Internal normalization of gC1qR concentration from EV-A71 infection (EI) and mock-infection (MI) related gC1qR interactomes revealed a calibrated interactome of 2042 proteins that contains 503 players with greater than ± 2-fold significant change of concentration. Of the 503 players, 332 co-existed in both EI and MI-related gC1qR interactomes, while 149 had >2-fold concentration change including 61 that were exclusively found in EI-related interactome to account for the dynamic change of endogenous gC1qR interactome during EV-A71 infection. GO biological process and pathways prediction of distinct groups from the 2042 players with FunRich software suggested significant increase and decrease of the 503 interactors in protein metabolism with hypothetical roles in transcription/translational control during EV-A71 infection. To narrow down infection-specific gC1qR-interactors, a combined technique of Tandem-tag affinity purification (TAP) and LC-MS/MS was further conducted to identify an ectopic gC1qR interactome, which was calibrated between interactomes of infected cells that ectopically express a gC1qR-SBP-6xHis and the SBP-6xHis tandem-tag fusion proteins. Three distinct interactors were found after calibration between infection-related ectopic gC1qR-SBP-6xHis and ectopic SBP-6xHis interactomes, while comparison between calibrated endogenous and ectopic gC1qR-interactomes revealed 37 coplayers that all had significant increase of concentration. These 37 critical gC1qR interactors after functional pathway prediction were significantly involved in Translation and Protein metabolism. Furthermore, comparative analysis of critical players within calibrated gC1qR-interactoms and previous identified VP4-interacrome by PINA4MS/Cytoscape, an application for identifying interactions between and within multiple interactomes, revealed a distinctive network of 40 cellular factors, including gC1qR, in viral translation. Hence, a dual-luciferase assay was performed to verify the role of gC1qR on EV-A71 internal ribosome entry site (IRES)-mediated translation. A bicistronic reporter construct was established with EV-A71 IRES inserted within intergenic region between Renilla luciferase (Rluc) and Firefly luciferase (Fluc) genes. The expression of Rluc and Fluc were used to monitor the activities of cellular cap-dependent translation and viral IRES-mediated translation, respectively. Silence of gC1qR affected both host and viral translation rather than IRES-mediated translation of EV-A71 alone, gC1qR may play an important role in both cellular and viral translation machineries. More functional surveys of VP4-gC1qR interactors will be assessed through gene silencing. This study provides detail understanding of gC1qR-interacting cellular networks and paves the way to identify critical cellular factors and machineries involved during EV-A71 infection.

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