JVI：中科院上海巴斯德研究所在巨細胞病毒晚期基因轉錄調控機制研究方面取得進展

導 讀

人巨細胞病毒(human cytomegalovirus, HCMV)屬於β-皰疹病毒亞群，其感染在人群中廣泛存在，對於免疫力低下的個體感染後果嚴重甚至可危及生命。小鼠巨細胞病毒（murine cytomegalovirus, MCMV）常用於HCMV的替代模型，CMV裂解複製過程中，病毒基因表達受到嚴密調控，但晚期基因轉錄的具體機制尚不清楚。近日，中科院上海巴斯德研究所錢志康研究組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，為研發新一代抗巨細胞病毒的疫苗和治療藥物提供線索。相關研究發表於在國際病毒學雜誌Journal of Virology上，題為「MurineCytomegalovirus Protein pM91 Interacts with pM79 and Is Critical for Viral LateGene Expression」，文章將被雜誌作為亮點文章進行特別推薦。

研究背景

HCMV屬於β-皰疹病毒亞群，其感染在人群中廣泛存在。由於HCMV感染存在嚴格的種屬特異性，MCMV常被作其替代模型。在CMV裂解期複製過程中，病毒基因依次呈現即刻早期基因，早期基因和晚期基因的時序性表達。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，但調控機制尚不完全清楚。

結果速覽

首先，研究者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，其中pM91-pM79之間的相互作用對整個複合物的組成是重要的（圖1）。

之後，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)系統成功得到pM91缺陷突變體，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（圖2）。

最後，研究者發現，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）。

.FIG 1. Six MCMV vTFs form a complex.

FIG 2. Organization of the vTF complex.

FIG 3. Expression pattern and localization of pM91 and pM79 during infection

小 結

本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。本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。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。後續關於MCMV和HCMV晚期基因轉錄調控的研究正在進行中。

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-herpes viruses, but not in α-herpes viruses. Here, we performed coimmuno preciptation 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 indirec tinteractions. 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.

參考文獻：

1.Deng Pana, Tian Hana, Shubing Tanga, Wenjia Xua,Qunchao Baoa, Yamei Suna, Baoqin Xuana# and Zhikang Qiana#..Murine Cytomegalovirus Protein pM91Interacts with pM79 and Is Critical for Viral Late Gene Expression.Journalof Virology,11 July 2018。

doi: 10.1128/JVI.00675-18.

本期編輯：Annabella

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