探討日本腦炎病毒NS5蛋白對於第一型干擾素產生的影響

Abstract

當細胞的Pattern Recognition Receptors (PRRs)偵測到病毒入侵時，透過pathogen-associated molecular patterns (PAMPs)與PRRs結合，刺激下游NF-κB，MAPK，以及IRFs等蛋白，開啟第一型干擾素(type I interferons)基因表現的激活途徑，來活化自身與周圍細胞進入抗病毒狀態。目前已知日本腦炎病毒的感染可引發高量的第一型干擾素產生，但日本腦炎病毒誘導第一型干擾素產生的機制仍不是很清楚。先前實驗室曾發現將具有複製酶活性(RNA-dependent RNA polymerase; RdRp)的日本腦炎病毒蛋白NS5送入在細胞中，可以活化帶有第一型干擾素promoter的 p125-Luc reporter質體。因此，在本篇論文中，我們試圖瞭解日本腦炎病毒蛋白NS5誘導第一型干擾素產生的機制。在過度表現正常(wild type)或是不具RdRp活性(RdRp-dead)的突變NS5病毒蛋白的細胞中，只有正常的NS5病毒蛋白能活化第一型干擾素的promoter、與interferon-stimulated基因(ISGs)的表現，而且過度表現正常的NS5病毒蛋白細胞的培養基中具有對抗Sindbis病毒的成分。利用帶有簪子干擾性核醣核酸(shRNAs)的慢病毒(lentivirus)，將細胞中負責誘發干擾素路徑的蛋白進行核酸干擾作用(RNA interference)，結果發現RIG-I，MAVS以及Tom70對於NS5病毒蛋白誘導第一型干擾素的產生是不可或缺的。不過，NS5病毒蛋白引發第一型干擾素產生的量，並不足以保護細胞對抗登革病毒的感染，但能夠增加細胞對外加低劑量干擾素的抗病毒反應。有趣的是，過度表現正常的NS5病毒蛋白的細胞中，有雙股RNA (dsRNA)的產生，並且被粒線體所圍繞著。總結，在本論文研究中，我們發現日本腦炎病毒NS5的複製酶活性可催化雙股RNA，細胞透過偵測雙股RNA，進而誘導第一型干擾素產生。

Induction of type I interferons (IFNs) is triggered after sensing of pathogens by pattern-recognition receptors (PRRs). Binding of PRRs with their ligands, pathogen-associated molecular patterns, stimulates signaling pathways that culminate in the activation of nuclear factors-κB, mitogen-activated protein kinase, and interferon regulatory factors to turn on IFNs expression. Japanese encephalitis virus (JEV) infection can trigger high level of interferon-β (IFN-β) gene expression, but exactly how JEV induces IFN-β production is not clear. Our preliminary data showed that expression of JEV NS5, a RNA-dependent RNA polymerase (RdRp), can activate reporter plasmid p125-Luc containing IFN-β promoter. Thus, this thesis study aims to assess the effect of NS5 on inducing IFN production. We established human A549 cell lines overexpressing wild type (WT) or RdRp-dead mutant (AAG) of NS5. Higher activation of IFN-β gene promoter and IFN-stimulated genes (ISGs) expression were detected in WT NS5-expressing cells when compared to the AAG-mutant. Consequently, antiviral activity against an IFN-sensitive Sindbis reporter virus was detected in the culture supernatant of NS5-expressing cells. By knocking down RNA sensors (RIG-I and MDA5) and adaptors (MAVS and Tom70) in NS5-expressing A549 cells, we found that RIG-I/MAVS and Tom70 are critical in the NS5-induced IFN-β production, whereas MDA5 is dispensable. Furthermore, mitochondria-localized dsRNA signals were detected in NS5-expressing cells. The IFN-induced by JEV NS5 was not sufficient to block the infection of dengue virus in the surrounding non-NS5 expressing cells, but it can sensitize the surrounding cells to exogenously added low dose of IFN. Thus, in this thesis study, we identify RdRp activity of NS5 can catalyze the formation of dsRNA and through RNA sensing that IFN production is turned on.