中東呼吸症候群冠狀病毒棘蛋白正調控生長因子FGF-2基因之機轉

Abstract

中東呼吸症候群冠狀病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)係2012年首次出現於沙烏地阿拉伯地區之新興病毒，目前已確認為中東呼吸症候群(Middle East respiratory syndrome, MERS)之病原體。隨著前往中東的旅遊人數增加，此病毒逐漸傳播至其他地區如北美洲、歐洲、非洲及亞洲。截至2017年12月已有2123名確診病例，其中740名病患已經死亡，死亡率約35%，遠高於2003年出現之嚴重急性呼吸症候群(severe acute respiratory syndrome, SARS)的9.6%。MERS-CoV係一正向單股RNA病毒，結構蛋白質包含spike、envelope、membrane及nucleocapsid蛋白質。目前已知MERS-CoV係透過與人類細胞表面之受體dipeptidyl peptidase 4 (hDPP4)結合並入侵宿主細胞，且MERS-CoV感染細胞株及普通狨(common marmoset)時會造成FGF-2及SMAD7的表現量上升，與導致細胞凋亡(apoptosis)有關，然而對於其機轉仍舊不明瞭。故本研究欲探討MERS-CoV係透過何種訊息傳遞路徑誘導FGF-2及SMAD7之表現。利用重組桿狀病毒於昆蟲細胞中表現含受體結合區之MERS-CoV棘蛋白S1次單元，以人胚胎肺纖維母細胞株HFL-1作為實驗平台。結果發現當加入MERS-CoV S1與HFL-1細胞共同培養時，FGF-2的mRNA及蛋白質表現量皆有上升，然而SMAD7之表現則不受影響。此外MERS-CoV S1可促進ERK1/2及JNK1/2之磷酸化，而p38之磷酸化則是下降。若以專一性抑制ERK1/2磷酸化之MEK抑制劑PD98059處理細胞後則MERS-CoVS1蛋白質無法誘導FGF-2之表現，由此可知MERS-CoVS1可能透過ERK1/2之路徑促進FGF-2之表現。另外，預先以DPP4抗體干擾MERS-CoV S1與DPP4之結合時，仍無法抑制MERS-CoV S1誘導FGF-2之能力，故推測MERS-CoV S1可能係透過未知的細胞表面蛋白質誘導ERK1/2所介導之FGF-2蛋白質表現增加。本研究成功找到MERS-CoV透過其棘蛋白S1次單元誘導FGF-2蛋白質表現之可能訊息傳遞路徑。

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emergent zoonotic betacoronavirus which was initially identified in the Kingdom of Saudi Arabia in September 2012. With the increase of travelers to the Middle East, the transmission of MERS-CoV has been reported from countries in North America, Europe, Africa and Asia. To date, MERS-CoV has caused 2123 laboratory confirmed cases with a mortality rate around 35%, which is much higher than the mortality rate of 9.6% caused by severe acute respiratory syndrome coronavirus (SARS-CoV). MERS-CoV is a positive single-strand RNA virus with structural proteins including spike, membrane, envelope and nucleocapsid proteins. The spike (S) protein of MERS-CoV mediates the viral entry into target cells through its specific interaction with human dipeptidyl peptidase 4 (hDPP4) on the cell membrane. MERS-CoV infection can upregulate the expression of FGF-2 and SMAD7 and induce apoptosis in cell models and common marmoset through unknown mechanisms. This study aims to shed light on the mechanism of upregulation of FGF-2 and SMAD7 during MERS-CoV infection. MERS-CoV spike S1 subunit, which contains receptor binding domain was produced by baculovirus expression vector system. Secreted MERS-CoV S1 subunit was purified and incubated with MERS-CoV-susceptible HFL-1 human lung fibroblast cells. Quantitative reverse transcription polymerase chain reaction and Western blot analysis demonstrated increased expression levels of mRNA and protein of FGF-2, respectively, but not SMAD7. Further studies demonstrated that MERS-CoV S1 subunit enhanced phosphorylation of ERK1/2 and JNK1/2, but reduced the phosphorylation of p38 MAPK. In addition, pretreatment of HFL-1 with antibodies specific to DPP4 could not abolish the upregulation of FGF-2 induced by MERS-CoV S1 subunit. These results suggested that MERS-CoV S1 subunit induced upregulation of FGF-2 in an hDPP4-independent manner. In conclusion, this study successfully identified the possible mechanism of MERS-CoV-induced FGF-2 upregulation through the viral spike protein.