NP220-沉默逆轉錄病毒染色體外DNA關鍵蛋白

導 讀

近日，美國哥倫比亞大學Stephen P. Goff教授領銜的研究團隊鑒定出沉默逆轉錄病毒染色體外DNA的關鍵蛋白NP220.相關研究成果以「NP220 mediates silencing of unintegrated retroviral DNA」為題，發表在國際著名學術期刊Nature.文章共同第一作者是Yiping Zhu和Gary Z. Wang。

研究背景

逆轉錄病毒（Retrovirus），又稱反轉錄病毒，是RNA病毒的一種。反轉錄病毒DNA的整合是複製病毒RNA的必經階段，反轉錄病毒的最基本特徵是在生命過程活動中，有一個從RNA到DNA的逆轉錄過程，即病毒在逆轉錄酶的作用下，以病毒RNA為模板，合成互補的負鏈DNA後，形成RNA-DNA中間體。中間體的RNA被RNA酶水解，進而在DNA聚合酶的作用下，由DNA複製成雙鏈DNA。

外來DNA進入許多哺乳動物細胞都會觸發先天免疫系統------一套複雜的預防病原體感染的反應。先天免疫包括對細胞內病毒DNA的有效表觀遺傳沉默。與病毒DNA整合到細胞後觀察的穩定表達形成對比，未整合的病毒DNA在所有細胞中轉錄都非常差。Goff教授一直致力於研究病毒和宿主的相互作用以及界定病毒每一個基因產物的功能，他對小鼠白血病病毒和人類免疫缺陷病毒（HIV）這兩組逆轉錄病毒尤其感興趣，他的研究團隊對於導致這種未整合病毒DNA低表達的因素進行了研究，並取得了相關成果。

結果速覽

該研究通過全基因組CRISPR-Cas9篩選的方法，篩選沉默一株整合酶缺陷的MLV-GFP報告病毒所需的基因，以探討抑制人類細胞中未整合病毒DNA的機制。結果篩選鑒定出了DNA結合蛋白NP220、組成HUSH複合物的三個蛋白（MPP8、TASOR和PHLN1）。進一步的染色質免疫沉澱試驗表明，蛋白NP220是募集HUSH複合物SETDB1和組蛋白脫乙醯酶HDAC1和HDAC4來沉默未整合的逆轉錄病毒DNA的關鍵蛋白。N220的敲除加速了逆轉錄病毒的複製。這些實驗鑒定了沉默逆轉錄病毒染色體外DNA的分子機制。

ABSTRACT:The entry of foreign DNA into many mammalian cell types triggers the innate immune system, a complex set of responses to prevent infection by pathogens. One aspect of the response is the potent epigenetic silencing of incoming viral DNAs, including the extra-chromosomal DNAs that are formed imme-diately after infection by retroviruses. These unintegrated viral DNAs are very poorly transcribed in all cells, even in permissive cells, in contrast to the robust expression that is observed after viral integration. The factors that are responsible for this low expression have not yet been identified. Here we performed a genome-wide CRISPR–Cas9 screen for genes that are required for silencing an integrase-deficient MLV–GFP reporter virus to explore the mechanisms responsible for repression of unintegrat-ed viral DNAs in human cells. Our screen identified the DNA-binding protein NP220, the three proteins (MPP8, TASOR and PPHLN1) that comprise the HUSH complex—which silences proviruses in heterochromatin and retrotransposons—the histone methyltransferase SETDB1, and other host factors that are required for silencing. Further tests by chromatin immunoprecipitation showed that NP220 is the key protein that recruits the HUSH complex, SETDB1 and the histone deacetylases HDAC1 and HDAC4 to silence the unintegrated retroviral DNA. Knockout of NP220 accelerates the replication of retroviruses. These experiments identify the molecular machinery that silences extrachromo-somal retroviral DNA.

本期編輯：vetjamie

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