中東呼吸道症候群冠狀病毒棘蛋白質透過其受器 DPP4 正調控趨化因子 CXCL10 基因之訊息傳遞

Abstract

中東呼吸道症候群冠狀病毒 (Middle East respiratory syndrome coronavirus，MERS-CoV) 為一種新型的第二型冠狀病毒，具有人畜共通的特性。從 2012 年爆發至今，已由原先集中於中東地區，擴及至歐洲、北非、北美、東南亞以及南韓等地。除了造成嚴重呼吸道感染外，MERS-CoV 還會造成急性腎衰竭，此併發症在 SARS-CoV 感染的個案中不曾出現。相對於具有 9.6% 致死率的 SARS-CoV，MERS-CoV 的致死率高達 36%。此病毒為具有套膜、正向單股的 RNA 病毒，當其感染細胞時，須藉由廣泛分布於各種細胞中之雙基胜肽酶 4 (dipeptidyl peptidase 4，DPP4) 作為受器。在棘蛋白質 (spike，S) 與受器 DPP4 結合時，其構形會發生改變進而引發細胞膜融合促使病毒進入細胞，但至今仍不明瞭在兩者結合時是否會誘導訊息傳遞。本研究根據序列分析發現，DPP4 位於細胞質的羧端具有一個可能被 CDK5 磷酸化之位點。此外，已知 ERK/MAPK 和 PI3K/AKT/mTOR 路徑在 MERS-CoV 感染時扮演重要角色。因此擬探討當 MERS-CoV 的 S 蛋白質與其受器 DPP4 結合時，是否會活化訊息傳遞路徑，進而調控宿主細胞的生理反應，希望藉此有助於闡明 MERS-CoV 致病的原因。首先利用桿狀病毒表現載體系統表現出 MERS-CoV S 蛋白質帶有 receptor binding domain 的次單元 S1，並進一步純化。以可被 MERS-CoV 感染的非洲綠猴腎臟細胞株 Vero E6 作為實驗平台，當 S1 蛋白質加入細胞培養液時，觀察到 DPP4 會被磷酸化、ERK1/2 受到活化，以及 CXCL10 mRNA 的表現量增加。接著以專一性辨認 DPP4 的抗體抑制 S1 蛋白質與受器 DPP4 結合，或是 knockdown CDK5 時，發現皆會影響上述所觀察到之現象。此外，CXCL10 mRNA 的表現量也會受到 ERK1/2 上游 MEK1 抑制劑的影響。綜合以上結果，當 MERS-CoV S1 蛋白質與細胞受器 DPP4 結合時，會促使 CDK5 將 DPP4 磷酸化；此一過程會造成 MEK1 及 ERK1/2 的活化，以及下游 CXCL10 mRNA 的表現量增加。本研究說明了一個新的並具有生理及 MERS-CoV 感染之病理上有意義的 DPP4 訊息傳遞路徑。

Middle East respiratory syndrome coronavirus (MERS-CoV) is a new zoonotic betacoronavirus, that caused an outbreak of severe respiratory disease in 2012 in the Middle East with secondary spread to Europe, North Africa, North America, Southeast Asia, and South Korea. The pathology of patients infected with MERS-CoV included not only respiratory disease but also acute renal failure. To date, there have been 1,368 cases of MERS, with a relatively high mortality rate of 36% in comparism with the 9.6% mortality of SARS-CoV. MERS-CoV is an enveloped, positive single-stranded RNA virus. The virus uses the evolutionarily conserved dipeptidyl peptidase 4 (DPP4) as its functional receptor. The spike (S) protein initiates virus entry by binding to DPP4 followed by conformational changes that lead to membrane fusion. It is not clear whether DPP4 conveys signals from the cell surface to the nucleus upon MERS-CoV infection. Phosphorylation site prediction data in NetPhosK 1.0 Server displayed that there is a putative CDK5 phosphorylation site located in the cytoplasmic side of DPP4. In addition, studies demonstrated that ERK/MAPK and PI3K/AKT/mTOR signaling responses played important roles in MERS-CoV infection. Towards understanding the pathogenesis of MERS-CoV, this study aims to examine whether DPP4 signaling pathway is activated upon binding of viral S protein to the DPP4 receptor. The S1 subunit of S protein which contains the receptor binding domain was produced by baculovirus expression system. MERS-CoV S1 protein secreted into the culture medium was purified and incubated with the virus permissive monkey kidney-derived cell line Vero E6 which expresses DPP4 at a high level. The results showed that the MERS-CoV S1 protein binding to DPP4 receptor caused phosphorylation of DPP4 and activation of ERK1/2. Meanwhile, the mRNA level of CXCL10 was up-regulated. In addition, incubation of Vero E6 cells with antibodies specific to DPP4 or CDK5 knockdown abolished the MERS-CoV S1 protein-induced phosphorylation of DPP4 and ERK1/2 and the up-regulation of CXCL10. Furthermore, the mRNA level of CXCL10 was also reduced by the MEK1 inhibitor PD98059 which blocks ERK1/2 phosphorylation. These data suggest that upon infection of MERS-CoV, DPP4 signaling pathway is activated through the interaction between the viral spike protein and the DPP4 receptor. The DPP4 signaling then activates MEK1-ERK1/2 pathway, resulting in an up-regulation of the downstream gene cxcl10. This novel DPP4 signaling pathway is critical for the cell entry and pathogenesis of MERS-CoV infection.