中山大學張萍組揭示新的登革病毒宿主因子調控病毒複製的機制

近期，中山大學張萍教授組在黃病毒與宿主細胞相互作用領域取得新進展，該課題組首次報道了宿主蛋白RNA解旋酶A（RHA）在黃病毒的複製過程中發揮重要的促進作用，並闡明了其分子機制。研究成果在線發表在病毒學國際期刊Journal of Virology上，原文題為「RNA helicase A is an important host factor involved in dengue virus replication」。

登革病毒（Dengue virus，DENV）是引起世界上感染人數最多、威脅最嚴重的蟲媒黃病毒；其感染可引起登革熱、登革出血熱和登革休克綜合症等。目前，臨床上尚沒有有效的疫苗和針對性的藥物可用。近年來，參與病毒複製的宿主蛋白成為抗病毒藥物的作用靶標。

課題組前期篩選得到了一個與固有免疫蛋白PKR結合的蛋白，RNA解旋酶A (RNA helicase A，RHA)。RHA主要位於細胞核中，常在胞核和胞質穿梭；它參與了RNA轉錄、加工與出核、mRNA的翻譯等多個RNA代謝過程。有研究報道了RHA參與了HIV、手足口病毒、流感病毒、粘液瘤病毒等病毒的複製過程，但RHA是否調控黃病毒的複製尚不明確。研究人員發現RHA在登革病毒感染後出核（圖1A）；當在不同組織來源的細胞中（肺癌上皮細胞A549，原代巨噬細胞以及肝癌細胞HepG2等）沉默RHA後，病毒粒子水平均顯著下調（圖1B-F）。此外，RHA可促進多個黃病毒的多輪複製，包括DENV2 NGC株，DENV2 16681株以及日本腦炎病毒（Japanese encephalitis virus，JEV）等，但對寨卡病毒（Zika virus，ZIKV）複製水平沒有顯著影響（圖1G）。

圖一 RHA在黃病毒的複製過程中發揮促進作用

接下來，研究人員明確了RHA參與登革病毒複製的具體階段。他們發現沉默RHA對於病毒吸附和內吞過程沒有影響（圖2A）；但顯著下調了登革病毒的RNA水平（圖2B）和病毒蛋白水平（圖2C-E）。進一步的複製子實驗，證實RHA沒有直接影響病毒蛋白的翻譯過程，而是通過促進病毒RNA的複製間接影響病毒蛋白的表達（圖2F）。

圖二 RHA促登革病毒複製階段的鑒定

更進一步，研究人員揭示了RHA的促病毒效應並不依賴於其解旋酶活性；且RHA與登革病毒RNA、非結構蛋白髮生結合。這些結果提示，登革病毒可招募RHA蛋白進入其複製複合物，RHA通過結合病毒蛋白和RNA，發揮「scaffold」作用，從而促進病毒RNA的複製，最終促進登革病毒的繁殖。該研究發現了一個新的登革病毒宿主因子，並系統闡明了作用機制，為抗病毒藥物研發提供了潛在的靶標。

通訊作者簡介：

張萍教授課題組研究方向是宿主細胞與黃病毒之間的相互作用，包括抗病毒的固有免疫調控機制、參與黃病毒複製的宿主因子研究。近期研究成果發表於Journal of Virology、Journal of Cell Science、Journal of Infection、Immunology、Archives of Virology、Biochemical and Biophysical Research Communications、Virologica Sinica等期刊。

ABSTRACT

Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The siRNA-mediated knockdown of RHA significantly the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multi-step viral growth of DENV2 and Japanese encephalitis virus, but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA binds to the DENV RNA, and associates with three nonstructural proteins including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediate its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents.

IMPORTANCEDengue, caused by dengue virus, is the most rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection, and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

來源：中國病毒學（英文版）

本期編輯：Tony

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