探討細胞eEF1A因子與病毒核蛋白間交互作用及干擾

Abstract

真核延伸因子1α（eEF1A）是在哺乳類動物細胞中被分為兩種亞型的重要轉譯因子，且先前的藥物篩選研究指出抑制轉譯作用，例如：eEF1A的抑制劑，具有潛在降低新型冠狀病毒（SARS-CoV-2）複製的能力。由於新冠肺炎（COVID-19）的快速肆虐全球，比起發展新療法，舊藥新用能夠緩解人類面臨的緊急情況且更能縮短研發的時間。宿主導向的抗病毒藥物（host-directed antivirals, HDA）因有著低風險的抗藥性而脫穎而出，所以本論文研究的eEF1A的抑制劑Aplidin（Plitidepsin）正符合標準且具有抑制SARS-CoV-2的抗病毒能力。我們研究發現病毒的核殼蛋白（nucleocapsid, N）及細胞eEF1A亞型因子間的交互作用具有在分子病毒學及細胞激素誘發上的研究與治療價值。一開始透過在Calu-3細胞中進行基因表現RNA干擾方法，我們發現eEF1A的兩種亞型，eEF1A1及eEF1A2，都參與了病毒增殖。但有趣的是，我們透過免疫共沉澱的實驗發現N蛋白傾向於與eEF1A1亞型結合，並且更深入確認N蛋白片段（胺基酸第355至363位點，N355-363）是與eEF1A1的重要結合區域（結構域III）。而且，Aplidin在細胞培養中能干預並抑制此結合。此外Aplidin在Vero E6及Calu-3細胞株的病毒感染實驗中證明具有優異地抑制多株代表性SARS-CoV-2變異株的能力。最後，我們也發現到Aplidin能夠有效降低致命性細胞激素表現，例如IL-6。總結來說，我們發現病毒N蛋白會傾向性的與eEF1A1結合，此結合不僅在病毒的複製及生長上扮演重要的角色，並且可能是Aplidin所具有的抗病毒能力及致命性細胞激素的抑制藥用機制。

Eukaryotic elongation factor 1 alpha (eEF1A) is a crucial translation factor with two isoforms that had been identified in mammalian cells for protein synthesis. Previous screening studies have revealed that eEF1A inhibitors have the potential to reduce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Due to the Coronavirus disease 2019 (COVID-19) pandemic has rapidly ravaged the world, in comparison to new regimens and/or drug development, repurposing clinically used drugs can relieve urgency and shorten the period of development time. Notably, host-directed anti-viral drugs (HDA) have stood out with good advantages of low-risk drug resistance and higher safety. Therefore, this dissertation focused on (i) eEF1A inhibitors, specifically Aplidin (Plitidepsin), which meet the above criteria well and also exhibit a surpassing anti-viral activity against SARS-CoV-2; (ii)The interactions between viral nucleocapsid (N) protein and cellular eEF1A isoforms are characterized by molecular virology and cytokine induction; (iii) the mechanism of action of Aplidin, especially in relation with N protein and eEF1A isoform preference. In these above regards, we have found that two eEF1A isoforms, eEF1A1 and eEF1A2, are critically involved in virus reproduction revealed by shRNA knockdown approach using the Calu-3 cell system. Further, co-immunoprecipitation (co-IP) experiments showed that N protein preferentially binds to eEF1A1. Detailed domain-mapping analysis then revealed that the binding motif of N protein located at C-terminus amino acid (a.a.) from 355 to 363 (N355-363) and the N-interacting domain of eEF1A1 was mapped at C-terminus Domain III. The peptide N355-363 was synthesized and, similar to Aplidin, showed an intervention potential in vitro using cellular extracts. Notably, Aplidin showed great antiviral activity against the original strain and prevalent variants of SARS-CoV-2 (such as variant of concern/interest) using both Vero E6 and Calu-3 cellular infection systems. We also found that Aplidin effectively reduced the expression of lethal cytokines, such as IL-6. To sum, we found out a preferential interaction of the eEF1A1 isoform with viral N protein and this interaction not only plays an important role in the viral reproduction but also underlies anti-viral activity and the relieving effect of lethal cytokine expression by Aplidin.