建立嚴重急性呼吸道冠狀病毒第2型3CL蛋白酶體外及體內切割系統

Abstract

嚴重急性呼吸道冠狀病毒2型 ( Severe acute respiratory syndrome coronavirus 2，SARS-CoV-2 )造成嚴重特殊傳染性肺炎( Coronavirus disease 2019，COVID-19 )並造成全球大流行。因此了解病毒與宿主的相互作用有利於抗病毒藥物的研發。其中病毒的3CL蛋白酶是由病毒基因組中的開放閱讀框( ORF1a )轉譯出來的蛋白質，其分子量為33 kDa，具有半胱胺酸( Cysteine )活性。其結構中包含組氨酸( Histidine 41，His 41 )和半胱胺酸( Cysteine 145，Cys145 )形成的催化二元組( catalytic dyad )。在不同冠狀病毒間的3CL序列具有高度保留性因此常作為藥物靶點之一。目前已知3CL蛋白酶除了可藉由切割病毒多蛋白質產生出多個非結構蛋白質來幫助病毒複製。此外3CL蛋白酶也可切割宿主蛋白質影響宿主免疫反應及病毒複製。但目前對於3CL蛋白酶和宿主蛋白質間如何影響病毒複製的詳細機制還有很多是未知的，因此本研究想探討有那些宿主蛋白質可被SARS-CoV-2的3CL蛋白酶切割以及探討其對病毒複製及宿主的影響。論文主軸分成三個部分來建立3CL蛋白酶的切割系統。第一部分是建立體外切割系統：首先建構具有蛋白酶活性的3CL蛋白酶質體和不具蛋白酶活性的3CL-C145A蛋白酶質體且利用大腸桿菌系統純化出3CL蛋白質，並使用酵素活性測試3CL蛋白質活性及已知可被SARS-CoV-2 3CL切割的宿主蛋白質p62、SNX6和DCP1A進行不同體外切割溶液系統的測試。結果顯示以250 ug的Calu-3細胞萃取液和體外切割溶液A和10 ug的SARS-CoV-2-3CL-WT跟SARS-CoV-2-3CL-C145A蛋白質一起於37度下反應12小時為我們最佳體外切割系統條件，這部分也將此反應條件的樣品進行質譜分析以鑑定出可被3CL蛋白酶切割的宿主蛋白質。第二部分是建立在293TACE2細胞中大量表達SARS-CoV-2-3CL和SARS-CoV-2-3CL-C145A質體的細胞模式觀察宿主蛋白質p62、SNX6和DCP1A是否有蛋白質降解現象。結果顯示當大量表達3CL時，DCP1A出現預期的切割產物，而p62和SNX6則無影響。第三部分是建立病毒感染之細胞模式是否有切割現象。結果顯示以0.1 MOI SARS-CoV-2感染CaLu-3細胞24和48小時後，p62、SNX6和DCP1A的蛋白質量減少。同時在0.1 MOI的HCoV-229E病毒感染A549細胞24和48小時後，p62和SNX6的蛋白質量減少，且DCP1A出現預期的切割產物。整合此研究結果我們建立3CL蛋白質在體外及體內的切割系統，未來有利於探討病毒與宿主相互作用機轉。

Severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ) causes Coronavirus disease 2019 ( COVID-19 ) and global pandemic. Therefore, understanding the interaction between viruses and their hosts is beneficial for the development of antiviral drugs. The SARS-CoV-2 3CL protease is a protein translated from the open reading frame (ORF1a) in the viral genome. It has a molecular weight of 33 kDa and possesses Cysteine activity. Its structure contains a catalytic dyad formed by Histidine 41 ( His 41 ) and Cysteine 145 ( Cys145 ). Other research demonstrated that 3CL protease can cleavage viral polyprotein to help viral replication and it is highly conserved in β-coronaviruses. Therefore, it has been recognized as a key target for preventing and treating COVID-19. Increasing evidence demonstrated that 3CL protease can cleavage host proteins and further affect host immune response and viral replication. However, the detailed mechanism of how the SARS-CoV-2 3CL protease and host proteins affect viral replication is still unknown. Therefore, this study wants to identify which host proteins can be cleaved by the SARS-CoV-2 3CL protease and investigate the mechanism of viral replication and host. The main focus of the paper is divided into three parts to establish the cleavage system of the 3CL protease.. The first part involves the establishment of an in vitro cleavage system. Firstly, we constructed a 3CL protease expression plasmid with proteinase activity and a 3CL-C145A protease expression plasmid without proteinase activity. Subsequently, we purified the 3CL protein using the Escherichia coli system. Enzymatic activity assays are performed to evaluate the activity of the 3CL protein, as well as to test different in vitro cleavage buffer systems using known host proteins p62, SNX6 and DCP1A, which can be cleaved by SARS-CoV-2 3CL protease. The results indicate that the optimal in vitro cleavage system for us was to incubate 250 ug of Calu-3 cell lysate with in vitro cleavage buffer A and 10 μg of SARS-CoV-2-3CL-WT and SARS-CoV-2-3CL-C145A proteins together at 37°C for 12 hours. Subsequently, samples from this reaction condition were sent for Mass spectrometry analysis to identify host proteins that can be cleavage by the 3CL protease. The second part involves the establishment of a cell model in 293TACE2 cells to overexpress the SARS-CoV-2-3CL and SARS-CoV-2-3CL-C145A plasmids. This allows the observation whether there is protein degradation of host proteins p62, SNX6, and DCP1A.. The results show that when 3CL is overexpressed, DCP1A exhibits the expected cleavage products, while p62 and SNX6 remain unaffected. The third part investigates the occurrence of cleavage in cell models infected with the virus. It is found that when CaLu-3 cells are infected with 0.1 MOI SARS-CoV-2 for 24 and 48 hours, the protein levels of p62, SNX6 and DCP1A decreased. Similarly, when A549 cells are infected with 0.1 MOI HCoV-229E for 24 and 48 hours also results in decreased protein levels of p62 and SNX6, accompanied by the appearance of cleavage products in DCP1A. Based on the results of this study, we have established an in vitro and in vivo cleavage system for the 3CL protein. This system will be beneficial for investigating the mechanisms underlying the interaction between the virus and the host.