黏蛋白型瞬時受體電位通道基因變異K370Q對流感病毒感染的影響及胞內運輸機轉分析

Abstract

A型流感病毒（IAV）仍然是全球重要的健康議題，不僅引發季節性流行，還靠能導致大規模疫情。IAV為具包膜的病毒，擁有分節負鏈RNA基因組，主要透過網格蛋白介導的內吞作用（CME）進入宿主細胞。一旦內化，病毒會劫持宿主的內溶體-溶酶體系統，以促進自身的複製與釋放。病毒核糖核蛋白（vRNPs）仰賴內溶體運輸以有效進入細胞核，進可組裝新的病毒顆粒。內溶體-溶酶體系統由早期內溶體、回收內溶體、晚期內溶體及溶酶體組成，負責細胞內物質運輸，並在病毒感染過程中扮演了關鍵角色。該系統主要由Rab蛋白與離子通道所調控，其中包黏黏蛋白型瞬時受體電位通道2（TRPML2）。TRPML2主要而位於早期內體(early endosome)與回收內體(recycling endosome)以協助囊泡運輸。近期研究顯示，TRPML2在A型流感病毒感染中具有重要作用，不僅能促進病毒囊泡運輸，還有助於病毒高效逃離內體區室。值得注意的是，編碼TRPML2的基因MCOLN2的一種罕見等位基因變異被鑑定為功能喪失突變，導致病毒進入受阻。這些發現表明，TRPML2活性在內溶體動態調控中扮演關鍵角色，並可能成為調控感染的潛在標的。

在這項研究中，我們發現TRPML2 K370Q突變在A549細胞中減弱了A型流感病毒的複製，這個結果和不具活性的TRPML2 R310A突變體表現相似，進一步支持了 TRPML2在病毒感染過程中的角色。螢光成像結果顯示，TRPML2 K370Q與早期及晚期內體的共定位增加，但對於回收內體則沒有太大影響。此外，細胞在TRPML2專一性激活劑ML2-SA2的作用下可以逆轉TRPML2 K370Q與早期及晚期內體共而位增加的現象，這表明TRPML2的活性靠能會影響它在細胞中的位置。接著，透過活體即時螢光奈米域鈣成像我們發現K370Q的鈣釋放量低於野生型，暗示它的通道活性靠能受損，進而影響它在病毒運輸和感染過程中的作用。最後，在TRPML2活化劑ML2-SA1作用下，我們在表現K370Q的細胞中觀察到與野生型相當的A型流感病毒表現量，證實了K370Q較弱的活性是導致其無法增強流感病毒感染的主要原因。然而，K370Q如何具體影響流感病毒感染的機制，還需要進一步的研究支解釋。

Influenza A virus (IAV) remains a significant global health concern, causing seasonal epidemics and occasional pandemics. As an enveloped virus with a segmented negative-sense RNA genome, IAV primarily enters host cells via clathrin-mediated endocytosis (CME). Once internalized, the virus hijacks the host endolysosomal system to facilitate its replication and release. The viral ribonucleoproteins (vRNPs) depend on efficient endosomal trafficking for nuclear import and subsequent virion assembly. The endolysosomal system, composed of early endosomes, recycling endosomes, late endosomes, and lysosomes, is essential for intracellular cargo transport and viral infection. This highly dynamic network is regulated by Rab proteins and ion channels, including transient receptor potential mucolipin 2 (TRPML2). TRPML2, a member of the TRPML family, is primarily localized in early and recycling endosomes, where it facilitates vesicular trafficking. Recent studies have highlighted its role in IAV infection, showing that TRPML2 enhances viral vesicular trafficking and promotes efficient viral escape from endosomal compartments. Notably, a rare allelic variant of MCOLN2, the gene encoding TRPML2, has been identified as a loss-of-function mutation, impairing viral entry. These findings suggest that TRPML2 activity plays a crucial role in endosomal dynamics and may serve as a potential target for modulating IAV infection.

In this study, we found that K370Q weakens IAV replication in A549 cells, similar to the inactive R310A mutant, reinforcing the role of TRPML2 in viral infection. Using fluorescence imaging, we observed that K370Q increase its colocalization with early and late endosomes, while having no significant impact on recycling endosomes. Further, ML2-SA2 treatment reversed the increased colocalization between K370Q and endosomes, suggesting that the activity of TRPML2 influence its intracellular localization. Subsequently, calcium imaging revealed less calcium release in cells expressing K370Q, implying a potential defect in channel activity, which may further result in disrupting its enhancement in viral trafficking and infection. Finally, upon treatment with the TRPML2 agonist ML2-SA1, cells expressing the K370Q variant exhibited influenza A virus expression levels comparable to those of wild-type cells. This finding supports that the reduced activity of K370Q is the primary reason for its inability to enhance viral infection. However, it still needs further investigation to clarify the underlying mechanism of how this mutant affects influenza viral infection.