近日，中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱獸醫(yī)研究所重要人獸共患病與烈性外來病團(tuán)隊(duì)在乙型腦炎病毒復(fù)制周期機(jī)制研究方面取得新進(jìn)展。相關(guān)研究成果在線發(fā)表在病毒學(xué)國(guó)際期刊《Journal of Virology》上，論文題目為Host factor SPCS1 regulates the replication ofJapanese encephalitis virus 3 through interactions with transmembrane domainsof NS2B。研究首次闡明了宿主因子SPCS1參與乙型腦炎病毒組裝的機(jī)制，進(jìn)一步完善了乙型腦炎等黃病毒復(fù)制生命周期的機(jī)制細(xì)節(jié)，加深了宿主因子與病毒相互作用的理解，為研制新的抗病毒藥物提供了新思路與方向。

BiFC與Co-IP法分析SPCS1與NS2B蛋白相互作用

背 景

乙型腦炎病毒與登革病毒、西尼羅病毒、寨卡病毒等同屬黃病毒科黃病毒屬，由蚊媒傳播，對(duì)動(dòng)物和人類健康構(gòu)成持續(xù)威脅，不斷引發(fā)新的疫情和公共衛(wèi)生危機(jī)。因次，深入研究該類病毒的致病機(jī)制，揭示病毒復(fù)制生命周期中各環(huán)節(jié)的調(diào)控機(jī)制，將為研制新型抗病毒藥物與治療策略提供理論依據(jù)。

結(jié)果速覽

研究團(tuán)隊(duì)以乙型腦炎病毒為模型，利用RNA干擾技術(shù)與基因敲除細(xì)胞系技術(shù)驗(yàn)證了宿主因子SPCS1(Signal peptidase complex subunit 1)參與了病毒的復(fù)制調(diào)控(圖1)。發(fā)現(xiàn)SPCS1分子除影響病毒多聚蛋白的剪切加工外，還影響病毒粒子的組裝(圖2)。進(jìn)一步研究表明SPCS1與乙腦病毒NS2B蛋白發(fā)生相互作用，相互作用發(fā)生在NS2B蛋白的兩個(gè)跨膜結(jié)構(gòu)域與包含跨膜結(jié)構(gòu)域的SPCS1分子的C端片段(圖3)。

Fig 1. Effects of JEV propagation in SPCS1 KO HEK-293 cells following 649 complementation with a plasmid expressing SPCS1

Fig 2. Effects of loss of SPCS1 function on viral entry into cells, genome RNA 692 replication, and protein processing during JEV infection.

Fig 3. Interaction of SPCS1 with the JEV NS2B protein in mammalian cells.

該項(xiàng)研究進(jìn)一步完善了乙型腦炎等黃病毒復(fù)制生命周期的機(jī)制細(xì)節(jié)，加深了宿主因子與病毒相互作用的理解，為研制新的抗病毒藥物提供了新思路與方向。該研究得到國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2016YFD0500403)的資助，該團(tuán)隊(duì)黃病毒研究組組長(zhǎng)華榮虹副研究員和團(tuán)隊(duì)首席步志高研究員為論文通訊作者，碩士研究生馬樂為第一作者。

ABSTRACT

Signal peptidase complex subunit 1 (SPCS1) is a newly identified hostfactor that regulates flavivirus replication, but the molecular mechanism isnot fully understood. Herein, using Japanese encephalitis virus (JEV) as amodel, we investigated the mechanism through which host factor SPCS1 regulatesthe replication of flaviviruses. We first validated the regulatory function ofSPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1.Loss of SPCS1 function markedly reduced intracellular virion assembly andproduction of infectious JEV particles, but did not affect virus cell entry,RNA replication, or translation. SPCS1 was found to interact with NS2B, whichis involved in post-translational protein processing and viral assembly. Serialdeletion mutation of the JEV NS2B protein revealed that two transmembranedomains, NS2B (1-49) and NS2B (84-131), interact with SPCS1. Furthermutagenesis analysis of conserved flavivirus residues in two SPCS1 interactiondomains of NS2B demonstrated that G12A, G37A, and G47A in NS2B (1-49), andP112A in NS2B (84-131), weakened the interaction with SPCS1. Deletion mutationof SPCS1 revealed that SPCS1 (91-169) which containing two transmembranedomains was involved in the interaction with both NS2B (1-49) and NS2B(84-131). Taken together, the results demonstrate that SPCS1 affects viralreplication by interacting with NS2B, thereby influencing post-translationalprocessing of JEV proteins and the assembly of virions.

importANCE

Understanding viral-host interactions is important for elucidating themolecular mechanisms of viral propagation, and identifying potential anti-viraltargets. Previous reports demonstrated that SPCS1 is involved in the flaviviruslife cycle, but the mechanism remains unknown. In this study, we confirmed thatSPCS1 participates in the post-translational protein processing and viralassembly stages of the JEV lifecycle, but not in the cell entry, genome RNAreplication, or translation stages. Furthermore, we found that SPCS1 interactswith two independent transmembrane domains of the Flavivirus NS2B protein. NS2Balso interacts with NS2A, which is proposed to mediate viral assembly.Therefore, we propose a protein-protein interaction model showing how SPCS1participates in the assembly of JEV particles. The findings expand ourunderstanding of how host factors participate in the flavivirus replicationlifecycle, and identify potential anti-viral targets for combatting flavivirusinfection.

文章來源：

1.Le Ma, Fang Li, Jing-Wei Zhang, Wei Li, Dong-Ming Zhao, Han Wang, Rong-HongHua and Zhi-Gao Bu. Host factor SPCS1 regulates the replication of Japaneseencephalitis virus through interactions with transmembrane domains of NS2B.JVI.2018.00197-18.

Doi:10.1128/JVI.00197-18

2.中國(guó)農(nóng)科院哈爾濱獸醫(yī)研究所：哈獸研重要人獸共患病與烈性外來病團(tuán)隊(duì)在乙型腦炎病毒復(fù)制機(jī)制研究方面取得新進(jìn)展。