病毒學國家重點實驗室藍柯組通過合作解析KSHV重要功能蛋白ORF57結構

近日，病毒學國家重點實驗室藍柯組與中科院高能物理所董宇輝研究員及美國國立衛生研究院鄭志明研究員合作，成功解析卡波氏肉瘤病毒（KSHV）重要功能蛋白ORF57 C端（ORF57-CTD）的單體和二聚體結構，並對其二聚化機制進行了詳細的闡釋，研究成果於8月10日在線發表於國際學術期刊PLoS Pathogens上（論文題目為「The crystal structure of KSHV ORF57 revealsdimeric active sites important for protein stability and function」

卡波氏肉瘤病毒 (Kaposi』s sarcoma associated herpesvirus，KSHV)，又名人類皰疹病毒8型，是一種重要的人類腫瘤病毒，能導致卡波氏肉瘤和B細胞淋巴瘤等惡性腫瘤。ORF57是KSHV編碼的早期基因，在病毒複製過程中能穩定病毒轉錄的mRNA從而促進病毒複製，是病毒裂解複製過程中的重要功能蛋白。ORF57蛋白半衰期較短，因此維持其穩定性對其發揮功能至關重要。既往報道證實ORF57蛋白通過二聚化而得以穩定，然而ORF57二聚化的詳細生化機制長期沒有得到充分的闡明。

該研究利用X射線衍射技術成功解析了ORF57-CTD蛋白3.5?解析度的二聚體結構和3?解析度的單體結構，揭示了ORF57-CTD每一個單體球狀核心區域底部都含有一個保守的鋅離子CHCC結合序列並結合一個鋅離子；此外，ORF57-CTD的每個單體N端延伸出一個長臂並包裹住另一個單體核心區域，且兩個單體的核心區域以反向平行的形式相互作用。因此，N端長臂和核心區域的相互作用介導了ORF57的二聚化形成（圖1）。

圖1. ORF57-CTD單體和二聚體三維結構

進一步體外交聯實驗以及免疫熒光實驗表明，刪除N端的長臂（圖2）或者對兩個單體球狀核心區域之間的相互作用氨基酸進行突變均導致ORF57二聚體解離。

圖2. N端長臂區介導ORF57蛋白二聚化

綜上，該研究首次成功解析了ORF57單體及二聚體的結構，詳細揭示了ORF57的穩定性和二聚化的分子機制，為深入理解KSHV生命周期基因表達調控提供了新的線索，也為KSHV相關腫瘤新型治療手段的研發提供了新的潛在靶點。

藍柯教授、董宇輝研究員和鄭志明研究員為論文共同通訊作者，袁菲博士、高增強博士和Vladimir Majerciak博士為論文共同第一作者。該研究得到了國家重點研發計劃項目（2016YFA0502100）、國家傑出青年科學基金（81425017）及美國國立衛生研究院國際項目（1R01AI116442）等的資助。

ABSTRACT

Kaposi』s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus closely associated with Kaposi』s sarcoma, primary effusion lymphoma and multicentric Castleman disease. Open reading frame 57 (ORF57), a viral early protein of KSHV promotes splicing, stability and translation of viral mRNA and is essential for viral lytic replication. Previous studies demonstrated that dimerization of ORF57 stabilizes the protein, which is critical for its function. However, the detailed structural basis of dimerization was not elucidated. In this study, we report the crystal structures of the C-terminal domain (CTD) of ORF57 (ORF57-CTD) in both dimer at 3.5 ? and monomer at 3.0 ?. Both structures reveal that ORF57-CTD binds a single zinc ion through the consensus zinc-binding motif at the bottom of each monomer. In addition, the N-terminal residues 167–222 of ORF57-CTD protrudes a long 「arm」 and holds the globular domains of the neighboring monomer, while the C-terminal residues 445–454 are locked into the globular domain in cis and the globular domains interact in trans. In vitro crosslinking and nuclear translocation assays showed that either deletion of the 「arm」 region or substitution of key residues at the globular interface led to severe dimer dissociation. Introduction of point mutation into the zinc-binding motif also led to sharp degradation of KSHV ORF57 and other herpesvirus homologues. These data indicate that the 「arm」 region, the residues at the globular interface and the zinc-binding motif are all equally important in ORF57 protein dimerization and stability. Consistently, KSHV recombinant virus with the disrupted zinc-binding motif by point mutation exhibited a significant reduction in the RNA level of ORF57 downstream genes ORF59 and K8.1 and infectious virus production. Taken together, this study illustrates the first structure of KSHV ORF57-CTD and provides new insights into the understanding of ORF57 protein dimerization and stability, which would shed light on the potential design of novel therapeutics against KSHV infection and related diseases.

本文來源：武漢大學病毒學國家重點實驗室

本期編輯：rojjer

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