Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor
Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor
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DOI：
10.1128/jvi.02774-12
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发表时间：
2013-08-01
影响因子：
5.4
通讯作者：
Bhatnagar, Raj K.
中科院分区：
文献类型：
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作者：
Kakumani, Pavan Kumar;Ponia, Sanket Singh;Bhatnagar, Raj K.
RNA干扰（RNAi）是植物和无脊椎动物中重要的抗病毒防御反应，然而，它在哺乳动物抗病毒防御中的作用的证据很少。在本研究中，我们证明了RNAi在哺乳动物细胞中的抗登革病毒作用。登革病毒感染Huh 7细胞降低了宿主RNAi因子（即Dicer、Drosha、Ago 1和Ago 2）的mRNA水平，因此，病毒感染细胞中这些基因的沉默增强了登革病毒的复制。此外，我们观察到许多已知的人类microRNAs（miRNAs）在病毒感染后下调。使用沉默逆转试验，我们进一步表明所有四种登革热病毒血清型的NS 4 B是有效的RNAi抑制剂。我们产生了一系列的缺失突变体，并证明NS 4 B介导的RNAi抑制通过其中间和C-末端结构域，即，跨膜结构域3（TMD 3）和TMD 5。重要的是，NS 4 B的N-末端区域，包括信号序列2K，这已经牵连在干扰素（IFN）拮抗特性，不参与介导RNAi抑制活性。保守残基的定点突变显示，TMD 3区域中的Phe-to-Ala（F112 A）突变导致RNAi抑制活性的显著降低。野生型NS 4 B显著降低了GFP沉默系的绿色荧光蛋白（GFP）-小干扰RNA（siRNA）生物合成，而F112 A突变体消除了这种降低。这些结果进一步证实了在体外dicer测定。总之，我们的研究结果表明miRNA/RNAi途径参与登革病毒的建立，登革病毒NS 4 B蛋白在调节宿主RNAi/miRNA途径以促进登革病毒复制中起重要作用。
RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication.