上海巴斯德研究所錢志康組在巨細胞病毒晚期基因轉錄調控機制研究方面取得進展

近日，中科院上海巴斯德研究所錢志康研究組在國際病毒學雜誌Journal of Virology上在線發表了題為「Murine Cytomegalovirus Protein pM91 Interacts with pM79 and Is Critical for Viral Late Gene Expression」的論文，報道了在巨細胞病毒晚期基因轉錄調控機制研究方面取得的重要進展。該文章將被雜誌作為亮點文章進行特別推薦。文章鏈接：http://jvi.asm.org/content/early/2018/07/06/JVI.00675-18.long

人巨細胞病毒(human cytomegalovirus, HCMV)是β-皰疹病毒亞群的一個典型代表，其感染在人群中廣泛存在。對於免疫力低下的個體，例如新生兒、艾滋病患者、器官移植術受者和癌症患者，HCMV感染後果嚴重甚至可危及患者生命。HCMV感染存在嚴格的種屬特異性，所以常採用小鼠巨細胞病毒(murine cytomegalovirus, MCMV)作為替代模型。在CMV裂解期複製過程中，病毒基因呈時序性表達，分為即刻早期基因，早期基因和晚期基因。研究發現CMV晚期基因的表達受病毒DNA複製的調控，同時也需要一些反式激活因子的參與(1)。在β-和γ-皰疹病毒中有6個保守的病毒反式激活因子(viral transactivation factors, vTFs)參與了晚期基因的表達調控(2)，但具體的調控機制尚不完全清楚。

本研究中，研究者們通過免疫共沉澱技術證明MCMV 的6個vTFs通過一定的相互作用組成了一個複合物（圖1），其中pM91-pM79之間的相互作用對整個複合物的組成是重要的。另外，研究者們通過基於內含肽剪接的蛋白質穩定性調控(intein-mediated modulation of protein stability, imPS)系統(3)成功得到pM91缺陷突變體（圖2），發現pM91缺失會抑制病毒晚期基因的表達及病毒的生長，但幾乎不影響病毒DNA的複製。最後，研究者們發現pM91的4個氨基酸殘基（E61、D62、D89和D96）對pM91-pM79相互作用是重要的，在病毒基因組中引入雙突變E61A/D62A或D89A/D96A可阻止病毒擴增，由此推測pM91-pM79相互作用可能是調控病毒晚期基因的轉錄所必需的（圖3）。

圖1. MCMV 病毒晚期基因轉錄複合物的模型。

圖2. 利用imPS系統構建pM91缺陷病毒SMdd91。

圖3. pM91-pM79相互作用對MCMV病毒生長是必需的。

該研究初步闡明了巨細胞病毒晚期基因轉錄調控複合物的組成方式，證明了病毒蛋白pM91和pM79之間的相互作用對複合物的形成及病毒正常生長是重要的，並發現了pM91中參與pM91-pM79相互作用的重要氨基酸殘基，為研發新一代抗巨細胞病毒的疫苗和治療藥物提供線索。

中科院上海巴斯德研究所錢志康研究員和宣寶琴副研究員為共同通訊作者，博士研究生潘登為第一作者。本研究得到了國家自然科學基金以及科技部重點研發項目的資助。後續關於MCMV和HCMV晚期基因轉錄調控的研究正在進行中。

參考文獻：

1. Isomura H, Stinski MF. 2013. Coordination of late gene transcription of human cytomegalovirus with viral DNA synthesis: recombinant viruses as potential therapeutic vaccine candidates. Expert Opin Ther Targets 17:157-66.

2. Gruffat H, Marchione R, Manet E. 2016. Herpesvirus Late Gene Expression: A Viral-Specific Pre-initiation Complex Is Key. Front Microbiol 7:869.

3. Pan D, Xuan B, Sun Y, Huang S, Xie M, Bai Y, Xu W, Qian Z. 2016. An intein-mediated modulation of protein stability system and its application to study human cytomegalovirus essential gene function. Sci Rep 6:26167.

ABSTRACT

Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors, [vTFs]) supporting late gene expression were conserved in β- and γ-herpesviruses, but not in α-herpesviruses. Here, we performed coimmunopreciptation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics, and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex, and the pM91-pM79 interaction is essential for viral late gene expression.

IMPORTANCECytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex; (ii) demonstrated that pM91 interacts with pM79, and that they colocalize in the nuclear viral replication compartments; (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication; and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation how CMV regulates late gene expression, and have important implications for the design of antiviral strategies.

來源：中國病毒學（英文版）、JVI

本期編輯：Tony

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