探討SARS-CoV2病毒是否經由ORF2蛋白將病毒嵌入宿主DNA中

Abstract

嚴重急性呼吸道症候群冠狀病毒2型 (SARS-CoV-2) 在2020年大量爆發並導致世界各地頻發死亡病例，主要是透過上呼吸道接觸細胞表面的血管收縮素轉化酶2(ACE2)受體感染細胞，導致肺部功能障礙甚至可能導致死亡。然而，在Rudolf Jaenisch, April 19, 2021 的論文中他們用奈米孔定序發現病患身上有人類與冠狀病毒2型的序列嵌合體。這個驚人的發現意味著有某個特殊分子的存在可以幫助冠狀病毒2型的RNA反轉錄成DNA，並嵌入進病患的基因組之中，不過嵌入的DNA都只有部分序列並非全長，也尚未發現是否會對患者造成影響。先前我們的研究發現被冠狀病毒2型感染的細胞會顯著提升長散在核元件 (Long interspersed nuclear element 1, LINE-1)RNA的表現量，藉由直接RNA互作蛋白質鑑定質譜法(iDRiP-MS)發現冠狀病毒2型會與長散在核元件(Long interspersed nuclear element 1, LINE-1)內所編碼的蛋白質(LINE1-ORF2p) 有交互作用。 因此本篇主要想更深入探討LINE-1所編碼的蛋白質（LINE1-ORF2p）是否是幫助冠狀病毒2型基因嵌入的主要因子，我以人胚腎293T細胞(HEK293T)過來表現LINE-1所編碼的蛋白質LINE1-ORF2p並進行純化，過程中我嘗試改變可能的影響因素，包括：實驗的細節步驟、不同來源的抗體、高通量基因克隆(Gateway Cloning)產生的新質體，但是卻始終無法有效產出蛋白。考慮到LINE1-ORF2p在自然界哺乳類動物的細胞中大多是被抑制的狀態，想更換整體的蛋白表現系統，藉由使用桿狀病毒表現系統(Bac-to-Bac expression system)來表現LINE1-ORF2p蛋白於昆蟲細胞(hi five)免於被細胞自身的防禦系統辨識。如果可以順利產出蛋白，我們就可以在體外測試其是否可幫助冠狀病毒2型的RNA進行嵌入作用。

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a massive outbreak in 2020, leading to frequent fatalities worldwide. The virus primarily infects cells through the upper respiratory tract by binding to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface, causing lung dysfunction and potentially leading to death. It was reported by Rudolf Jaenisch ‘s group that chimeric sequences of human and SARS-CoV-2 in patients was observed from long-read nanopore sequencing. This surprising finding suggests the presence of a particular molecule that facilitates the reverse transcription of SARS-CoV-2 RNA into DNA and its integration into the host genome. The integrated DNA consists only of partial sequences, not the full length, and it is not yet known if this integration affects the patients. In our previous research, the expression of Long Interspersed Nuclear Element-1 (LINE-1) was upregulated significantly in Calu3 cells infected with SARS-CoV-2. According to the results from Chia-Yu Guh, using Identification of Direct RNA-Interacting Proteins-Mass Spectrometry (iDRiP-MS), it was shown that LINE-1 open reading frame 2 protein (LINE1-ORF2p) interacts with SARS-CoV-2. To investigate whether LINE1-ORF2p is a key factor in assisting the integration of SARS-CoV-2 genes, I tried to overexpress and purify the ORF2 protein in human embryonic kidney 293T (HEK293T) cells. However, ORF2 protein was unable to be expressed in HEK293 cells. Given that LINE elements are usually repressed but derepressed upon inflammation and infection, I will change the protein expression system by using the Bac-to-Bac expression system to overexpress L1 ORF2p in insect cells (HiFive) after virus infection, and use the synthetic ORFeus-Hs L1 with modified sequences for expressing ORF2 protein. If ORF2 protein is successfully produced, I can test whether it directly facilitates the integration of SARS-CoV-2 RNA in vitro.