嚴重急性呼吸道症候群冠狀病毒7a蛋白質與血紅素氧化酶結合參與病毒之致病機轉

Abstract

嚴重急性呼吸道症候群相關之冠狀病毒 (severe acute respiratory syndrome associated coronavirus，SARS-CoV) 被證實與引起 SARS 之非典型肺炎有關，其致死率約為 10%。SARS-CoV 為一含有套膜之新型冠狀病毒，基因體為一正向的 RNA，長約 30,000 個核苷酸，具有 14 個 ORFs，其中包含8個組別特異性蛋白質(group-specific proteins)，包括ORF 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b，這些蛋白質在不同組別的冠狀病毒中具有顯著的序列差異性，一般認為在致病機轉中扮演重要的角色。本實驗的研究對象是 SARS-CoV 7a 蛋白質，探討其與宿主細胞蛋白質的交互作用及其功能。 本實驗室欲探討 7a 致病的分子機制，利用高效率酵母菌功能性基因模組從 200 個跟細胞凋亡相關之蛋白質篩選出一個細胞因子heme oxygenase-1 (HO-1)，其會與 7a 和 8a 產生交互作用。Heme oxygenase-1會催化 heme 代謝的速率決定步驟，最終產物為鐵離子、一氧化碳 (CO) 、以及膽紅素 (bilirubin) 。目前研究指出， HO-1在細胞當中主要是透過其產物 CO 扮演具有細胞保護的角色。為進一步確認 7a 和 8a 與 HO-1 的交互作用，首先利用免疫共沉澱的方式證明，在哺乳類動物細胞中 7a 的確會與 HO-1有交互作用，並利用雷射掃描共軛焦顯微鏡觀察到7a與HO-1在細胞中有colocalization的情形。此外，文獻指出7a會阻礙細胞週期的進行，使其停留在G0/G1階段，並且是透過抑制cyclin D3的表現而造成;為探討 7a 與 HO-1 的交互作用之生理意義，我們進行cyclin D3的 luciferase reporter assay，發現受到 HO-1 活化的 cyclin D3 promoter 的活性可以被 SARS-7a 所抑制。 經由 SARS-CoV 7a 與 8a 的序列比對發現，7a 與 8a 的 21-35胺基酸序列具有高度相似性，因此推測7a和8a可能是透過這段序列與 HO-1 結合。未來的工作將會釐清 7a、8a 對於 HO-1 酵素活性的影響。

Severe acute respiratory syndrome (SARS) is an atypical pneumonia caused by SARS-coronavirus (SARS-CoV). The overall mortality rate was about 10 %. SARS-CoV is an enveloped, positive single-stranded RNA virus with an RNA genome about 30,000 nucleotides in length, which encodes 14 open reading frames (ORFs). The genome contains eight group-specific open reading frames (ORFs)：ORF 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b, which encode group-specific proteins with no known homologues. These accessory proteins are highly associated with the virulence and pathogenesis of SARS-CoV. The specific aim of this study is to understand the function and pathogenesis of SARS-CoV 7a. By performing high throughput yeast functional array analysis, our laboratory has identified heme oxygenase-1 (HO-1) as a SARS-CoV 7a-, and 8a-interacting protein. HO-1 is an enzyme which can catalyze heme metabolism and generally acts as a cytoprotective protein through its by-products, CO, Fe2+, and bilirubin. The interaction between SARS-CoV 7a and HO-1 protein was confirmed by co-immunoprecipitation. In addition, confocal microscopy demonstrated the colocalization of SARS-CoV 7a and HO-1. Furthermore, it is reported that SARS-CoV 7a blocks cell cycle at G0/G1 phase by inhibiting the expression of cyclin D3. In this study, luciferase reporter assay demonstrated that SARS-CoV 7a inhibited the up-regulation of cyclin D3 promoter activity mediated by HO-1. Alignment of SARS-CoV 7a and 8a amino acid sequences showed that the N-terminal 21-35 amino acid resideus of 7a and 8a have high similarity. Thus, it is possible that 7a and 8a interact with HO-1 through amino acid residues 21-35.