運用基因沉默之技術尋找調控甲型流感病毒複製之宿主因子

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题名:	運用基因沉默之技術尋找調控甲型流感病毒複製之宿主因子 To identify host factors that regulate IAV replication through gene silencing
作者:	柯昱羽;Yu-Yu Ke
贡献者:	生物醫學研究所碩士班
关键词:	基因沉默;甲型流感病毒;宿主因子;病毒複製;gene silencing;Influenza A virus;host factor;virus replication;TPCN1
日期:	2018-07-25
上传时间:	2018-12-25 09:30:39 (UTC+8)
出版者:	中國醫藥大學
摘要:	流感病毒為常見之病原體，而其中又以甲型流感病毒更容易造成大流行。現今普遍使用之抗病毒藥物如:克流感、瑞樂沙的藥理機轉主要是針對甲型流感病毒其蛋白質來做治療，但病毒本身的高突變率使藥物治療效果逐漸下降。由於病毒主要仰賴人體細胞來進行複製，因此，找到影響病毒複製之宿主因子可能可以做為減少抗藥性的治療策略。近年已有多篇關於流感病毒宿主因子的研究被發表，然而被篩選出來的宿主因子卻大相逕庭，甚至會因為篩選條件不同而產生偽陽性的可能。我們透過在人類肺部表皮細胞進行篩選實驗，挑選到了數個宿主因子。經由免疫螢光技術進行進階的篩選，我們找到一個名為TPCN1的基因並研究它對甲型流感病毒複製的影響。當TPCN1基因被沉默化，會抑制流感病毒早期的複製階段。透過實驗我們不僅了解病毒與宿主交互作用的分子機制更進一步提供未來抗病毒藥物之潛在標的。 Influenza viruses are a major cause of morbidity and mortality, and Influenza A virus (IAV) in particular has the propensity to cause pandemic outbreaks. Several antiviral drugs such as Oseltamivir, Zanamivir and amantadine are designed to target the viral proteins, while high mutation rate of virus results in the widespread resistance to these drugs. The life cycle of IAV relies on human cellular machinery. Therefore, the investigation of host factors can be an alternative therapeutic strategy that may greatly reduce the crisis of viral resistance. Several studies of screening the host factors have been published, but the criteria used to determine the candidate host factors likely differed among the screens. Each screen might include a number of false positives. Since the CRISPR and RNAi techniques have been widely used for performing genome-wide screening, we conducted a CRISPR screen. Here, CRISPR-cas9 system, coupled with Next Generation Sequencing (NGS), was carried out in human lung epithelial (A549) cells. Based on CRISPR screening, we obtained several candidates. Through further immunofluorescence screening, we identified a gene called TPCN1 and investigated its impact to IAV replication. When TPCN1 was silencing, it would suppress early stage of influenza virus replication. This study can not only inform us the molecular pathways of virus and host cross interaction but also provide potential targets that could be applied to development of antiviral drugs.
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