Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor

Kakumani, Pavan Kumar ; Ponia, Sanket Singh ; Rajgokul, S. ; Sood, Vikas ; Chinnappan, Mahendran ; Banerjea, Akhil C. ; Medigeshi, Guruprasad R. ; Malhotra, Pawan ; Mukherjee, Sunil K. ; Bhatnagar, Raj K. (2013) Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor Journal of Virology, 87 (16). pp. 8870-8883. ISSN 0022-538X

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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.

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Source:Copyright of this article belongs to American Society for Microbiology.
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