從NP exonuclease抑制劑複合物結構探討半胱氨酸之共價結合酵素活性抑制機制

Abstract

拉薩病毒 ( Lassa virus ) 的 Nucleoprotein exonuclease ( NP exonuclease ) 參與在病毒基因組 RNA 衣殼化、病毒 RNA 合成、以及躲避宿主免疫系統的偵測。NP exonuclease 主要由 N 端結構域 ( N terminal domain ) 與C端結構域 ( C terminal domain ) 所構成，其中 N 端結構域主要功能為截取宿主細胞中mRNA 的5端帽，以幫助進行病毒自身的RNA轉錄與複製；C 端結構域則是屬於 DEDDh 核酸水解外切酶家族成員，能以3端至5端的方向降解感染宿主細胞時所產生的免疫反應誘導病原體 ( Pathogen associated molecular patterns )，例如病毒RNA；使 IRF-3 轉錄因子無法進入細胞核中，避免宿主免疫系統的干擾素合成。若C 端結構域中高度保留性胺基酸突變，會導致拉薩病毒躲避宿主免疫系統的能力下降，換言之，C 端結構域在病毒感染細胞的過程中扮演重要角色；也因此NP exonuclease的 C 端結構域可以做為對抗病毒感染藥物研究中的一個目標蛋白質。本論文中，我們搜尋文獻並利用分子嵌合 ( Molecular docking ) 軟體找出NP exonuclease的抑制劑候選物。透過核酸水解酵素活性測試實驗，篩選出具高抑制效率的抑制劑，如 ATA、PCMPS、PHMB、PV6R 以及 NCI35 等。我們同時也解析出 apo-NP exonuclease 蛋白質晶體結構和 NP exonuclease-PCMPS 複合物晶體結構，其中PCMPS 與 C 端結構域中第409個胺基酸：半胱胺酸 ( Cysteine 409 ) 上的硫氫基共價結合，顯示此共價鍵是抑制 NP exonuclease 酵素活性的一重要因素。我們的生化實驗結果以及兩種蛋白質晶體結構，揭露出PCMPS透過共價鍵與 NP exonuclease 結合之獨特抑制機制，可以應用於對抗病毒藥物開發。

The Nucleoprotein exonuclease ( NP exonuclease ) of Lassa virus is involved in viral genomic RNA encapsidation, viral RNA synthesis and host immune evasion. NP exonuclease is constituted by N-terminal and C-terminal domains, of which the main function of N-terminal domain is to capture the 5' cap of mRNA in the host cell for carrying out transcription and replication of its own viral RNA; the C-terminal domain of NP exonuclease belongs to the DEDDh exonuclease family. The C-terminal domain is used to degrade pathogen associated molecular patterns generated by infecting host cells, like RNA; and thus further make IRF-3 transcription factor unable to enter the nucleus to induce interferon synthesis of immune system. If the highly conserved amino acids in the C-terminal domain are mutated, it will cause a decline in ability for Lassa virus to escape the host immune system, in other words, the C-terminal domain plays an important role in virus infections; hence the C-terminal domain of NP exonuclease can be used as a target for anti-viral drug research. In this study, we found the inhibitor candidates for NP exonuclease by literature reviews and computational molecular docking program. Through the nuclease activity assay, we identified several inhibitors with high inhibition efficiency, such as ATA、PCMPS、PHMB、PV6R and NCI35. We also determined the crystal structures of apo-NP exonuclease protein structure and NP exonuclease-PCMPS complex in which PCMPS was covalently bound to the cysteine ( C409 ) in the C-terminal domain, indicating that the covalent bond is critical to suppress the activation of NP exonuclease. Our biochemical experiments and two crystal structures reveal a unique inhibitory mechanism of PCMPS through covalent linkage to the NP exonuclease, and this work could be applied to the development of antiviral drugs.