MxA異構mRNA定量分析與第一型單純疱疹病毒對MxA啟動子之調控

Abstract

第一型單純疱疹病毒(Herpes simplex virus 1)具有多種機制可以影響干擾素媒介的抗病毒免疫反應。其中，第一型干擾素誘發表現的MxA蛋白已知具有抗RNA病毒的能力。然而，我們過去的研究發現，人類胚胎肺臟纖維母細胞受到HSV-1感染後，會使細胞的MxA基因經由選擇性剪接的方式轉錄MxA異構mRNA ，簡稱為varMxA，且轉譯成蛋白，協助病毒的複製。本篇論文將探討纖維母細胞經過IFN-α的刺激或HSV-1感染後，MxA與varMxA的表達情況，並且利用螢火蟲冷光報導質體探討HSV-1調控MxA啟動子的可能機制。 我們利用 MxA 與varMxA 專一性引子進行real-time PCR 的實驗，分析經過IFN-α或HSV-1 處裡後的纖維母細胞MxA 與varMxA的轉錄情形。實驗結果發現，給予纖維母細胞IFN-α的刺激後，MxA 與varMxA 的mRNA 表現量都有約100 倍的上升，且穩定表現至刺激後24 小時。另一方面，在HSV-1 感染纖維母細胞的組別中，housekeeping 基因的表現會受到HSV-1 的影響而顯著的下降，例如β-actin、GAPDH。然而，MxA 與varMxA 的mRNA 表現量在HSV-1 感染後，不僅不會受到抑制，反而有2 至3 倍的上升且持續至感染後24 小時才降低至細胞內基本的表現量。這個結果顯示，給予細胞IFN-α與HSV-1 的刺激後，都可以增加MxA 與varMxA mRNA 的表現量。但是在HSV-1 感染後的細胞選擇性地轉錄varMxAmRNA 成為蛋白的機制為何，仍是未知的問題。 我們利用轉染的方式將帶有MxA 啟動子序列的冷光報導質體送入黑色素瘤細胞，探討MxA 啟動子的活性。當MxA 啟動子上的ISRE1 與ISRE2 經過位點突變後，IFN-α不具有增加該啟動子活性的能力。然而，HSV-1 引發的MxA 啟動子活性不僅不會受到影響，反而MxA 啟動子的活性會有顯著的上升。實驗結果顯示，HSV-1 引發MxA 啟動子的活性不需要依賴IFN-α的訊息傳遞。進一步利用序列刪除實驗發現，MxA 啟動子上-533 到-250 的片段可能是HSV-1 媒介MxA 啟動子活性的區域。利用TRANSFAC 資訊軟體分析發現，該區域有一個可能的ICP4/TFIID結合區，進行 ICP4/TFIID 結合區位點突變後，會降低HSV-1 媒介MxA 啟動子一半的活性，進一步利用ICP4 質體DNA 進行轉染實驗，反而不影響MxA 啟動子的活性，顯示ICP4/TFIID 結合區位點突變可能是影響轉錄因子TFIID 的結合，使得病毒蛋白 ICP4 無法與細胞轉錄因子TFIID 交互作用後，降低MxA啟動子的活性。然而，HSV-1 感染細胞後誘發表現的varMxA 是如何受到病毒調控的，仍需要進一步的研究。

Herpes simplex virus 1 (HSV-1) is a member of human alphaherpesviruses which has evolved different strategies to evade type I interferon (IFN) response. Mammalian Mx proteins are dynamin-like GTPases that are induced by interferon (IFN) α/β and have antiviral activity against RNA viruses. Our previous studies have found that HSV-1 induces an alternatively spliced MxA isoform (varMxA) to enhance viral replication in primary human fibroblast in the absence of IFN triggering. In this study experiments have been designed to investigate expression of MxA isoforms in IFN-treated versus HSV-1 infected fibroblasts as well as the effect of HSV-1 on MxA promoter activity in luciferase reporter system. MxA isoform-specific primers were designed and used to measure the expression of MxA and varMxA transcripts in IFN-treated or HSV-1 infected human fibroblasts by real-time PCR analysis. The results showed that IFN-α treatment induced both MxA and varMxA expression at least 100 folds and remained stable over the 24h-course of treatment. Whereas HSV-1 infection resulted in degradation of housekeeping genes such as β-actin and GAPDH, expression of MxA and varMxA transcripts remained stable and increased slightly over an interval of at least 10h and started to decline at 24h after infection. These data suggested that either IFN-α or HSV-1 stimulated the expression of MxA and varMxA mRNA. The importance that MxA isoforms were not degraded but rather selectively remained expression in HSV-1 infected cells is deserved for further investigation. HSV-1 mediated MxA promoter activity was evaluated by transient transfection of melanoma cells with a luciferase reporter plasmid containing MxA promoter region (-533 to -1). HSV-1 infection result in the activation of MxA promoter by 5 folds was not dependent on ISRE (IFN-stimulated response element) binding since disruption of ISRE1 and ISRE2 binding sites did not eliminate MxA promoter activity after HSV-1 infection. However, HSV-1 mediated luciferase activity from MxA promoter mutant constructs with deletion in the frangment from -533 to -250 was significantly reduced to basal level. Site-directed mutagenesis at ICP4/TFIID putative binding site at -317 to -306 also showed a reduced luciferase activity either by HSV-1 infection or by co-transfection with a plasmid expressing ICP4. These experiments have demonstrated that ICP4 was able to trans activate MxA gene. Whether the effect requires the coorperation with cellular transcription factor TFIID remains to be elucidated.