透過適應性演化研究牛痘病毒A56/K2 蛋白複合體如何抑制病毒進入細胞

Abstract

在牛痘病毒進入宿主過程中，牛痘病毒需要通過11種蛋白質的病毒融合複合物與宿主膜融合，但詳細機制仍不清楚。先前研究顯示，病毒蛋白A56和K2會在感染的細胞上表達，並透過與病毒融合複合物的兩個分子（G9和A16）結合以防止細胞外痘苗病毒的過度感染，從而抑制膜融合。為了研究A56 / K2複合物如何抑制膜融合，我們透過在HeLa 細胞表面表達A56和K2蛋白並用三種痘苗病毒WR，WRΔA26和WRΔB5進行病毒體外演化實驗，以分離出適應性突變病毒。適應性突變病毒的全基因組定序結果顯示，三種病毒基因對於痘苗病毒克服A56 / K2表達的融合抑制作用很重要。我們確定的有一個三種病毒都共同擁有的突變發生在G9R ORF中，它在經過多代的篩選後出現了一個單一鹼基突變C130T，會導致胺基酸His44變成Tyr。G9H44Y突變並不影響病毒融合蛋白複合物的形成，也不影響其與A56 / K2蛋白複合物的相互作用。這些結果說明G9H44Y突變蛋白模擬了酸誘導的中間構象，使膜融合更容易發生。由於此突變共同出現在三個傳代病毒庫中，可能表明該突變是發生病毒適應的主要驅動力。除了G9H44Y突變之外，有兩個突變只專一性的存在於WR傳代病毒中，A28 ORF中的P79Q和A26 ORF中發生的多個插入或缺失的適應性突變。A28P79Q突變僅短暫累積，隨後在之後的傳代過程中被A26 ORF中發生的多個插入或缺失突變所取代，這也說明了這些基因之間存在著遺傳上相互作用的關係。 實際上，我們發現A26和A28蛋白在免疫共沈澱實驗結果中顯現有相互結合的關係，然而A28P79Q突變並不影響其與A26蛋白的結合。最後，我們發現雙重突變病毒WR-G9H44Y＋A28P79Q具有比WR-G9H44Y好的病毒傳播能力，這結果可能暗示著A28P79Q控制著mature virus (MV)到extracellular virus (EV)的運輸或EV釋放的步驟。

For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, I performed experimental evolutionary analyses by repeatedly passaging three vaccinia viruses—WR, WRΔA26 and WRΔB5—in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that three viral genes are important for vaccinia virus to overcome fusion inhibition by A56/K2 expression. The first viral open reading frame (ORF) I identified is G9R, which harbors a specific mutation (C130T) from adaptive selection, resulting in a His44Tyr amino acid change. G9H44Y did not affect viral fusion protein complex formation nor its interaction with A56/K2 protein complex. These results suggest that G9H44Y mutant protein mimics an acid-induced intermediate conformation more prone to membrane fusion. Since this mutation is shared by all three passaged virus pools, this mutation appears to be the major driving force for virus adaptation. In addition to G9H44Y, I also identified two viral mutations specific to adaptive mutants derived from WR passaging, i.e., P79Q of A28 ORF and multiple insertions/deletions of A26 ORF. A28P79Q only accumulated transiently and was subsequently replaced by insertions/deletions of A26 ORF in subsequent passaging events, indicating genetic interplay between these two genes. Indeed, a co-immunoprecipitation experiment revealed that A26 and A28 protein bind to each other and that the P79Q mutation of A28 does not affect its binding to A26 protein. Finally, I observed that the double mutant virus WR-G9H44Y+A28P79Q exhibits enhanced virus spreading relative to WR-G9H44Y, indicating that A28P79Q controls the transition of mature virus (MV) to extracellular virus (EV) or the EV release step.