嚴重急性呼吸道症候群冠狀病毒棘蛋白質透過其受器ACE2訊息傳遞正調控細胞激素CCL2基因之分子機制

Abstract

嚴重急性呼吸道症候群冠狀病毒 (severe acute respiratory syndrome associated coronavirus, SARS-CoV)，是2002年末至2003年之間在全世界造成廣泛感染並有 9.6% 致死率的嚴重急性呼吸道症候群 (severe acute respiratory syndrome, SARS) 的病原體，會引起患者肺部浸潤、纖維化等非典型肺炎症狀。血管收縮素轉化酵素2 (angiotensin-converting enzyme 2, ACE2)，被進一步證實為SARS-CoV感染宿主細胞的重要細胞表面受器，但是ACE2是否會參與在將病毒感染的訊息由細胞表面傳遞到細胞核中，進一步影響到宿主細胞中基因的表現，這些資訊到現在仍不清楚，而且SARS-CoV的致病機制，也還有待進一步的釐清。在本研究中，首先進行SARS-CoV的類病毒顆粒 (virus-like particles, VLPs) 系統的建構。利用會表現SARS-CoV結構蛋白質S、M、E的重組桿狀病毒 (baculovirus) 共同感染昆蟲細胞Sf9，收取細胞培養液進行蔗糖梯度離心，即可收集到純化過的SARS-CoV VLPs，並且純化SARS-CoV spike蛋白質，以模擬病毒感染的生理狀態。為了了解病毒的致病機轉，將SARS-CoV的目標細胞肺癌肺泡上皮細胞A549和SARS-CoV VLPs進行培養後，利用Affymetrix寡核苷酸微陣列系統，大規模地觀察有那些宿主細胞基因的表現受到影響。結果發現這些基因大都參與免疫反應、細胞凋零、細胞週期等反應中。其中一個受影響的基因為C-C motif ligand 2 (CCL2)。利用反轉錄即時聚合酶連鎖反應更進一步確認在A549細胞或是非洲綠猴腎臟細胞株Vero E6中，SARS-CoV VLPs及病毒的spike蛋白質均會正調控CCL2基因的表現，且由酵素免疫分析法也證實了A549細胞培養液中CCL2蛋白質的表現量增加。利用螢光素酶分析法分析CCL2基因啟動子上順式元件在SARS-CoV VLPs處理下扮演的角色，證實了AP-1的重要性，而且c-Fos與c-Jun的表現量也會增加。AP-1的轉錄活性會受到MAPKs的調控，在SARS-CoV VLPs與spike蛋白質培養下，ERK1/2的磷酸化有顯著的增加，其上游的Ras、Raf也參與在CCL2表現正調控的訊息路徑中，而不是透過IkappaBalpha-NFkappaB的路徑。當細胞預先處理專一性辨認SARS-CoV細胞受器ACE2的抗體時，會抑制SARS-CoV VLPs或spike蛋白質引起的CCL2表現量增加與ERK1/2磷酸化，而且利用抑制劑實驗證明casein kinase II會將ACE2胺基酸Ser-787位置進行磷酸化，此一磷酸化對於下游的訊息傳遞是重要的。所以本實驗結果指出，當SARS-CoV感染時，病毒spike蛋白質和其受器ACE2的交互作用，會促使ACE2磷酸化，而引起ACE2訊息路徑的活化，進而活化Ras-ERK-AP-1路徑，導致下游CCL2表現量的增加，可能是導致SARS病人肺部纖維化的原因之一。

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was identified to be the causative agent of SARS, an atypical pneumonia that occurred during late 2002 and the first half of 2003. SARS-CoV infected more than 8,000 people, with a worldwide mortality rate of 9.6%. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV. It is not clear whether ACE2 conveys signals from the cell surface to the nucleus and regulates expression of cellular genes upon SARS-CoV infection. To understand the pathogenesis of SARS-CoV, SARS-CoV virus-like particles (SARS-CoV VLPs) were first generated in this study by co-infecting Sf9 cells with recombinant baculoviruses expressing the viral structural proteins S, E, and M. SARS-CoV VLPs secreted into the culture medium were harvested and used to mimic the infection of SARS-CoV following a sucrose gradient centrifugation. In addition, the SARS-CoV spike protein was purified with an Ni2+ SepharoseTM purification system. Human type II pneumocyte A549 cells were incubated with SARS-CoV VLPs and gene expression profiles were examined by Affymetrix oligonucleotide microarray system. The analysis revealed differentially expressed genes that are involved in humoral immune responses, apoptosis, and cell cycle. The up-regulation of the fibrosis-assocaited chemokine (c-c motif) ligand 2 (ccl2) mRNA in SARS-CoV VLPs-treated and the spike protein-treated A549 cells and Vero E6 cells was confirmed by real-time reverse transcription polymerase chain reaction. In addition, enzyme-linked immunosorbent assay showed elevated levels of CCL2 protein were increased in culture media of both the SARS-CoV VLPs-treated and the spike protein-pretreated A549 cells. Luciferase reporter assay demonstrated a role of activation protein 1 (AP-1) in trans-activating CCL2 promoter upon pretreatment with SARS-CoV VLPs. Western blot analysis further demonstrated SARS-CoV VLP-induced up-regulation of both c-Fos and c-Jun, which constitute AP-1. The up-regulation of CCL2 in A549 cells was mainly mediated by extracellular signal-regulated kinase 1 and 2 (ERK1/2) and AP-1, but not IkappaBalpha-NFkappaB signaling pathway. Further studies demonstrated that Ras and Raf upstream of the ERK1/2 signaling pathway were involved in the up-regulation of CCL2. In addition, the anti-ACE2 antibodies diminished the SARS-CoV VLP- and spike protein-induced phosphorylation of ERK1/2 and the up-regulation of CCL2. An inhibitor assay indicated that ACE2 receptor was activated by casein kinase II–mediated phosphorylation at Ser-787 in cells pretreated with SARS-CoV VLPs. These data suggest that upon infection of SARS-CoV, ACE2 signaling pathway is activated through the interaction between the viral spike protein and the ACE2 receptor. The ACE2 signaling then activates Ras-ERK-AP-1 pathway and results in an up-regulation of the downstream gene ccl2, leading to pulmonary fibrosis.