研究 SARS-CoV-2 ORF3a 的錯義突變對溶小體鈣穩態的影響

Abstract

鈣離子在細胞內體系統中扮演關鍵角色。它們不僅透過內溶小體通道驅使內體融合、幫助溶小體重構，也參與鈣信號傳遞以及膜蛋白運輸。這些功能對維持細胞的調控、功能穩定與平衡非常重要。而當病毒感染細胞時，鈣離子訊號傳導成為病毒侵入到釋出的生命週期中的關鍵因子。 病毒通道蛋白會破壞膜電位梯度及細胞穩態，並影響胞內囊泡運輸及訊號傳導，達到大量複製繁衍的目的。透過微觀顯像工具的進步以及全球基因組即時比對的監測，現今已然能推測病毒蛋白質的功能性，並瞭解結構中錯義胺基酸取代對病毒感染致死力的影響。 SARS-CoV-2 ORF3a，依據 cryo-EM 結構和序列比對的相似性，被描述為一種多功能的病毒通道蛋白。ORF3a 蛋白在內溶小體系統的作用日益受到關注。Two pore segment channel 2 (TPC2) 則是表達在內溶小體膜上的的非選擇性陽離子通道，藉由調控內溶小體奈米域鈣離子的釋放，調節內溶小體的胞內運輸及生理功能。近期研究則指出利用 TPC2 拮抗劑能降低 SARS-CoV-2 病毒進入細胞的程度，然而其詳細機制仍不明。 在這裡，我們假設SARS-CoV-2 ORF3a會直接或間接影響內溶小體系統的奈米域鈣離子訊號。我們利用 SARS-CoV-2 ORF3a 錯義突變可能造成病毒膜蛋白功能的增益或喪失，觀察是否影響 TPC2 所介導內溶小體奈米域鈣離子訊號。透過活細胞影像，我們觀察到 SARS-CoV-2 ORF3a 與溶小體陽離子通道 TPC2 共表達，能夠影響細胞內奈米域鈣訊號的釋放。透過共軛焦顯微鏡和分子動力學模擬，來檢查 SARS-CoV-2 ORF3a 的細胞內分佈和蛋白結構的穩定性我們觀察到SARS-CoV-2 ORF3a 與TPC2 高度共定位，且Q57E突變導致的功能喪失與ORF3a蛋白構型的崩潰無關。 我們的結果闡明 SARS-CoV-2 ORF3a 極有可能造成內溶小體中鈣離子梯度的變化，從而影響內溶小體的通道對鈣離子的調控機制，間接揭示SARS-CoV-2 ORF3a 為一個鈣離子通道的可能性。因此，開發針對 ORF3a 的拮抗劑來影響病毒感染的調節機制可能是一種有效的治療策略。

Intracellular calcium ions play a crucial role in the intracellular system. They drive the process of endolysosomal fusion through endolysosomal channels, assist in the reconstruction of compartments, participate in calcium signalling pathways, and transport membrane proteins. These functions are vital for maintaining cellular regulation, functional stability, and balance. During viral infection, Ca2+ signalling becomes a critical factor in the viral life cycle, from entry to release. Viroporins disrupt membrane potential gradients and cellular homeostasis, affecting intracellular vesicle transport and signalling to replicate themselves extensively. With advancements in microscopic imaging tools and real-time global genome alignment monitoring, it is now possible to infer the functional properties of viral protein subunits and understand the impact of missence amino acid substitutions on viral pathogenicity. SARS-CoV-2 ORF3a, based on cryo-EM structure and sequence alignment, is described as a multifunctional viral pore protein. The role of the ORF3a protein in the endolysosomal system has garnered increasing attention. Two pore segment channel 2 (TPC2) is a non-selective cation channel expressed on the endolysosomal membrane, regulating intracellular transport and physiological functions of endolysosomes by controlling the release of calcium ions within endolysosomal nanodomains. Recent studies have indicated that using TPC2 inhibitors can reduce the extent of SARS-CoV-2 viral entry into cells, though the detailed mechanism remains unclear. Here, we hypothesise that SARS-CoV-2 ORF3a directly or indirectly affects the calcium ion signalling within endolysosomal nanodomains. First, we investigate whether missence mutations in SARS-CoV-2 ORF3a, which may result in gain or loss of function in viral membrane proteins, influence TPC2-mediated endolysosomal calcium ion signalling. Through live-cell imaging, we observed that co-expression of SARS-CoV-2 ORF3a and the lysosomal cation channel TPC2 affects intracellular nanodomain calcium signalling. Using confocal microscopy and molecular dynamics simulations to examine the intracellular distribution and structural stability of SARS-CoV-2 ORF3a, we observed that SARS-CoV-2 ORF3a is highly co-localized with TPC2. Additionally, the loss of function caused by the Q57E mutation is not related to the collapse of the ORF3a protein conformation. Our results elucidate that SARS-CoV-2 ORF3a likely causes changes in calcium ion gradients within endolysosomes, thereby affecting the regulatory mechanisms of calcium ions in endolysosomal channels. This indirectly reveals the potential of SARS-CoV-2 ORF3a as a calcium ion channel. Therefore, developing antagonists targeting SARS-CoV-2 ORF3a to modulate viral infection mechanisms may be an effective therapeutic strategy.