SARS冠狀病毒之PLpro蛋白酶之特性分析

Abstract

SARS-冠狀病毒(SARS-CoV)是造成2003年嚴重急性呼吸道症候群大規模流行的致病源，屬於冠狀病毒屬。冠狀病毒相較一般的RNA病毒，具有較長的基因序列，因此，冠狀病毒的複製以及基因調控的機制比起其他病毒，也顯得更為複雜。一般預期SARS病毒的基因表現，轉譯以及後轉譯調控也非常複雜，詳細的分子作用機轉，需要更進一步研究來加以闡明。來自於SARS-CoV polyprotein ORF1a 的蛋白酶PLpro 被預期會切割在ORF1a N端的三個切點上。ORF1a上另外一個蛋白酶3C-like (3CLpro)則負責作用在其他的11個切點。1a以及1ab經蛋白酶作用所產生的蛋白質產物，一般相信會組合形成依附在細胞內膜上形成replication complex，對SARS病毒的複製，病毒產物的產生，以及致病力扮演重要的角色。 我們利用in vitro以及in vivo酵素活性測定的實驗設計，來檢視蛋白酶PLpro 的活性。首先，將in vitro合成的SARS-CoV PLpro 以及預測具有酵素切點的蛋白質片段混合，來測試酵素的活性。在in vitro實驗中，我們發現PLpro蛋白酶展現出cis-cleavage的活性，但卻沒有trans-cleavage的活性。然而，相同的酵素片段，在in vivo的環境下卻展現出具有相當高的trans-cleavage活性。這樣的實驗結果顯示，某些細胞因子對於PLpro酵素的trans-cleavage活性，扮演著重要且關鍵的角色。此外，透過這次的研究，我們在酵素受質3(substrate 3)的片段，一段含nsp2/nsp3 PLpro 切點的蛋白質片段上，卻發現到一個性質未知的蛋白酶切點。相對於substrate 3所包含的PLpro 切點，這個性質未知的切點，在沒有PLpro 蛋白酶出現的條件下，仍然會被切割，顯示作用在該切點的酵素並非PLpro。我們利用serial deletion的實驗將切點所在位置的範圍，縮小到約莫SARS ORF1a 蛋白質的第950~960個氨基酸之間。這個新切點的酵素作用，不只出現在利用rabbit reticulocyte lysate所合成的蛋白質上，由wheat germ lysate所合成的蛋白質片段，也顯示出相同的結果。然而，如果將合成的蛋白質從lysate中純化出來，類似的酵素作用立刻消失。因此，我們認為，用來合成蛋白質的lysate當中，可能含有某些蛋白酶，可作用在這個新的蛋白酶切點上。

SARS-Coronavirus (SARS-CoV) is the causative agent of the epidemic of the severe acute respiratory syndrome (SARS) in 2003. Coronaviruses are the exceptionally large RNA viruses and employ complex regulatory mechanisms to replicate and express their genomes. Gene expression of SARS-CoV is believed to involve complicated transcriptional, translational and post-translational regulatory mechanisms, the molecular details of which, however, remain to be determined. The PLpro encoded in the SARS-CoV polyprotein ORF1a is predicted to process the N-terminal three sites of ORF1a and 1ab, whereas the 3C-like (3CL) protease is responsible for the cleavage of the rest sites. The products derived from 1a and 1ab are believed to assemble into a membrane-associated viral replication complex, facilitating the replication and downstream viral biogenesis as well as pathogenesis. In this study, we determined the enzyme activity of SARS-CoV PLpro by in vitro and in vivo assay systems. When incubating the in vitro synthesized SARS-CoV PLpro with its predicted substrates, the enzyme exhibited cis-cleavage activity but no detectable trans-cleavage activity in vitro. However, the same enzymatic domain trans-cleaved the substrates efficiently in vivo. The results suggest that some cellular factors of human cells may be important and necessary for the trans-cleavage activity of PLpro. Here, we also identified a cryptic cleavage site within substrate 3, a fragment that contained the predicted nsp2/nsp3 cleavage site for PLpro. The cryptic site was cleaved even in the absence of PLpro, suggesting that it was not a substrate site for PLpro. Serial deletion analysis further established that the cryptic site was around residues 950~960 of SARS-CoV ORF1a. The cryptic processing of substrate 3 was observed not only when the protein was synthesized in rabbit reticulocyte lysate, but also when it was synthesized in wheat germ lysate. However, the cryptic processing was no longer observed when the synthesized substrate 3 was purified from the lysate. Therefore, we concluded that the proteases from the lysate may mediate this cleavage phenomenon.