title: Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor.
abstract: 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.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Pavan Kumar
authors.initials: PK
authors.lastname: Kakumani
authors.forename: Sanket Singh
authors.initials: SS
authors.lastname: Ponia
authors.forename: Rajgokul K
authors.initials: RK
authors.lastname: S
authors.forename: Vikas
authors.initials: V
authors.lastname: Sood
authors.forename: Mahendran
authors.initials: M
authors.lastname: Chinnappan
authors.forename: Akhil C
authors.initials: AC
authors.lastname: Banerjea
authors.forename: Guruprasad R
authors.initials: GR
authors.lastname: Medigeshi
authors.forename: Pawan
authors.initials: P
authors.lastname: Malhotra
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Mukherjee
authors.forename: Raj K
authors.initials: RK
authors.lastname: Bhatnagar
mesh_terms: D002460:Cell Line; D004252:DNA Mutational Analysis; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology* / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D016297:Mutagenesis, Site-Directed; D034622:RNA Interference; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: C080107:NS4B protein, flavivirus; D017361:Viral Nonstructural Proteins