中科院武漢病毒所王華林組揭示桿狀病毒O-糖基化蛋白GP41調控病毒粒子裝配機制

2018年4月12日，國際學術期刊Journal of Virology在線發表了中科院武漢病毒所王華林團隊的最新研究成果，論文題為The functional oligomeric state of tegument protein GP41 is essential for baculovirus BV and ODV assembly（桿狀病毒皮層蛋白GP41的寡聚化對於BV/ODV的裝配是必需的）。

桿狀病毒是一類特異性感染昆蟲的大DNA病毒，在其感染周期中形成兩種不同類型的子代病毒粒子，出芽型病毒粒子BV和包埋型病毒粒子ODV。病毒感染細胞後在細胞核中複製和裝配形成核衣殼，一部分核衣殼經過核膜運輸至細胞膜出芽形成BV，另一部分核衣殼留在細胞核內裝配成ODV，但BV和ODV的裝配機制目前尚不清晰。

O-糖基化修飾是生物界普遍存在的一種蛋白修飾，細胞中的O-糖基化蛋白通常具有重要作用，主要參與調控受體結合，蛋白轉運，信號轉導，核孔複合物形成以及細胞骨架形成等過程。而病毒O-糖基化蛋白的功能現在仍不明確。GP41是桿狀病毒中目前發現的唯一一個O-糖基化蛋白，研究表明其含有豐富的O-糖基化修飾，主要定位於ODV的囊膜和核衣殼之間的皮層結構。前期研究表明GP41參與桿狀病毒核衣殼的出核運輸因而對BV形成非常重要，同時GP41是 ODV的主要成分之一，但其是否參與ODV形成並不清楚。我們通過基因敲除的方法首次闡釋了O-糖基化病毒蛋白GP41在桿狀病毒增殖過程中的作用。我們發現GP41參與BV核衣殼的出核運輸，同時對核衣殼和微囊泡相互作用形成ODV具有重要作用，缺失gp41基因導致BV和ODV都不能形成。同時我們發現，GP41在感染過程中形成寡聚體，其中三聚體被特異性包裝到子代病毒粒子BV和ODV中，推測三聚體是GP41的功能形式。進一步發現，二硫鍵和亮氨酸拉鏈是GP41形成寡聚體的關鍵結構位點（圖1）。該研究為深入解析桿狀病毒O-糖基化蛋白GP41在病毒感染中的作用和病毒的裝配機制提供了重要依據（圖2）。

圖1：GP41的寡聚體形成機制

圖2：GP41在桿狀病毒感染周期中的作用機制

ABSTRACT

gp41, one of the baculovirus core genes, encodes the only recognized tegument (O-glyco) protein of the occlusion-derived virion (ODV) phenotype so far. A previous study using a temperature-sensitive Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) mutant showed that GP41 plays a crucial role in budded virion (BV) formation. However, the precise function of GP41 in the baculovirus replication cycle remains unclear. In this study, AcMNPV GP41 was found to accumulate around the ring zone region (RZ) within the infected nucleus and finally assembled into both BV and ODV. Deletion of gp41 from the AcMNPV genome showed that BVs were no longer formed and ODVs were no longer assembled, suggesting its essential role in baculovirus virion morphogenesis. In infected cells, besides the 42 kDa-monomers, dimers and trimers were detected under non-reducing conditions, whereas only trimeric GP41 forms were selectively incorporated into BVs or ODVs. Mutations of all five cysteines in GP41 individually had minor effect on GP41 oligomer formation, albeit certain mutations impaired infectious BV production, suggesting flexibility in the intermolecular disulfide bonding. Single mutations of key leucines within two predicted leucine zipper-like motifs did not interfere GP41 oligomerization, nor BV and ODV formation, but double leucine mutations completely blocked oligomerization of GP41 and progeny BV production. In the latter case, the usual subcellular localization, especially RZ accumulation of GP41, was abolished. The above findings clearly pointed out a close correlation between GP41 oligomerization and its function, therefore highlighted the oligomeric state as the functional form of GP41 in baculovrius replication cycle.

IMPORTANCE The tegument, which is sandwiched between nucleocapsid and virion envelope, is an important substructure of many enveloped viruses. It is composed of one or more proteins that have important functions during virus entry, replication, assembly and egress. Unlike another large DNA virus, herpesvirus that encodes an extensive set of tegument components, baculoviruses very likely exploit the major tegument protein, GP41, to execute functions in baculovirus virion morphogenesis and assembly. However, the function of this O-glycosylated baculovirus tegument protein remains largely unknown. In this study, we identified trimers as the functional structure of GP41 in baculovirus virion morphogenesis and that both disulfide bridging and protein-protein interactions via the two leucine zipper-like domains are involved in the formation of different oligomeric states. This study advances our understanding of the unique viral tegument protein GP41 participating in the life cycle of baculoviruses.

來源：綜合病毒學國家重點實驗室、JVI

本期編輯：Tony

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