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. 2013 Aug;87(16):8870-83.
doi: 10.1128/JVI.02774-12. Epub 2013 Jun 5.

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

Pavan Kumar Kakumani  1 , Sanket Singh Ponia, Rajgokul K S, Vikas Sood, Mahendran Chinnappan, Akhil C Banerjea, Guruprasad R Medigeshi, Pawan Malhotra, Sunil K Mukherjee, Raj K Bhatnagar
Affiliations

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

Pavan Kumar Kakumani et al. J Virol. 2013 Aug.

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.

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Figures

Fig 1
Fig 1
Host RNAi regulates dengue virus replication. (Ai) Reduction in dicer, drosha, ago1, and ago2 mRNA levels at 48 h posttransfection in Huh 7 cells transfected with the respective siRNA duplexes (#, P < 0.001). (ii) TaqMan analysis of dengue virus genomic RNA in knocked-down cells at 48 h postinfection (#, P < 0.03). (iii) TaqMan analysis of WNV genomic RNA in knocked-down cells at 48 h postinfection (#, P < 0.02). (B) Downregulation of mRNA levels of dicer, drosha, ago1, and ago2 genes at 24 and 48 h postinfection in dengue virus-infected Huh 7 cells (#, P < 0.05). Data shown are means ± standard deviations from three independent experiments. The # symbols indicate a statistically significant difference in terms of the P value.
Fig 2
Fig 2
Host miRNA response to dengue virus infection. (A) Changes in the host miRNA profile were observed upon dengue virus infection at 48 h in Huh 7 cells. The abundance of downregulated miRNAs (>1.5-fold) are presented in terms of the normalized read counts (i to iv) in both uninfected and infected samples. (B) Validation by stem-loop qRT analysis of some of the miRNAs down- and upregulated upon dengue virus infection at 48 h (#, P < 0.006). (C) Stem-loop qRT analysis of host miRNAs that are affected upon siRNA knockdowns in Huh 7 cells (#, P < 0.005). Data shown in panels B and C are means ± standard deviations from three independent experiments. The # symbols indicate a statistically significant difference in terms of the P value.
Fig 3
Fig 3
Reversion-of-silencing assays show that NS4B is an RNAi suppressor. (Ai) Schematic representation of plasmid constructs used for the generation of the RNAi sensor line (experimental line 1) and the GFP-reverted cells (the RNAi sensor line that overexpressed NS4B and FHVB2) (experimental line 2). (ii) Bar graph representation of the FACS results for FHVB2 and NS4B in the Sf21 sensor cell line, with the percentage of cells expressing GFP represented on the y axis (*, P < 0.008; #, P < 0.05). (Bi) Schematic representation of plasmid construct used for overexpressing NS4B in GFP-silenced leaves of N. xanthi through agroinfiltration. (ii) GFP fluorescence pictures of agroinfiltrated leaves at 8 dpi taken under a UV lamp. The empty vector was used as a mock control, while FHVB2 was used as a positive control in the assay. The tested NS4B protein showed positive reversal of silencing. (Ci) Schematic representation of plasmid constructs used for studying the suppressor activity of NS4B in a mammalian cell line (HEK293T). LTR, long terminal repeat; MSV, mouse sarcoma virus. (ii) Bar graph representation of the FACS results for FHVB2 and NS4B in HEK293T cells, with the percentage of cells expressing GFP represented on the y axis (*, P < 0.001; #, P < 0.003). (Di) Sequence alignment of NS4B proteins from all the four serotypes of dengue virus. (ii) Bar graph representation of the FACS results for NS4B in HEK293T cells, with the percentage of cells expressing GFP represented on the y axis and the dengue virus serotype represented on the x axis. Data shown in panels A, C, and D are means ± standard deviations from three independent experiments. The * and # symbols indicate statistically significant differences at a P value of <0.00004.
Fig 4
Fig 4
Analysis of deletion mutants for RNAi suppressor activity of NS4B. (A) Schematic representation of predicted transmembrane domains of NS4B with and without its signal peptide 2K. (B) Bar graph representation of the FACS results for NS4B and 2K-NS4B in HEK293T cells, represented on the x axis, with the percentage of cells expressing GFP represented on the y axis (#, P < 0.0004). (C) Schematic representation of deletion mutants of NS4B. (D) Bar graph representation of the FACS results for different deletion mutants of NS4B in HEK293T cells, represented on the x axis, with the percentage of cells expressing GFP represented on the y axis (*, P < 0.0004; #, P < 0.005). Data shown in panels B and D are means ± standard deviations from three independent experiments. The * and # symbols indicate statistically significant differences in terms of the P value.
Fig 5
Fig 5
Localization of amino acid residues responsible for RNAi suppression activity of NS4B. (A) Positions of the DV2 NS4B mutations generated in this study. Mutation sites are described as the amino acid positions in DV2 NS4B. pTMD refers to the predicted transmembrane domain. (B) Bar graph representation of the FACS results for different mutants studied in the pTMD3 region of NS4B in HEK293T cells, represented on the x axis, with the percentage of cells expressing GFP represented on the y axis. Data shown in panel B are means ± standard deviations from three independent experiments. The # symbols indicate a statistically significant difference at a P value of <0.02.
Fig 6
Fig 6
Delineation of domains responsible for IFN antagonism and RNAi suppressor activity of NS4B. (A) Schematic representation of structural features of pISRE-Luc. Upon transfection into mammalian cells (HeLa), the luciferase gene was expressed from a spliced transcript. The change in luciferase expression was observed upon poly(I·C) treatment in the 2K-NS4B-, wt NS4B-, and BC/NS4B-transfected lines. (B) HeLa cells were cotransfected with expression vectors encoding firefly luciferase under the control of an IFN-β-inducible promoter (pISRE-Luc) (100 ng), Renilla luciferase, and 2K-NS4B, wt NS4B, and BC/NS4B at 500 ng each in either the presence or absence of poly(I·C). Luciferase expression was measured at 31 h posttransfection. The relative luciferase expression level corrected for the internal Renilla control (firefly/Renilla) is shown. Data shown in panel B are statistically significant at P values of <0.02 (*) and <0.05 (#).
Fig 7
Fig 7
NS4B inhibits the processing of shRNA/dsRNA into siRNAs both in vivo and in vitro. (i) Histogram showing levels of gfp-specific siRNA in HEK293T cells transfected for a GFP reversion assay and Northern blot analysis of GFP siRNAs in transfected cell lines (HEK293T). Lane 1, GFP-expressing cells; lane 2, GFP-silenced cells; lane 3, GFP-reverted cells expressing wt NS4B; lane 4, GFP-reverted cells expressing the NS4B F112A protein with rRNA loading controls. (ii) Purification of recombinant NS4B F112A proteins run on 12% SDS-PAGE gels in increasing concentrations. (iii) EMSAs using labeled dsRNA probes with purified NS4B protein. Lane 1, free probe; lane 2, dengue virus NS3 (1.0 μg); lanes 3 to 6, NS4B protein (0.1, 0.5, 1.0, and 2.0 μg, respectively). (iv) In vitro dicer assay in the presence of recombinant NS4B F112A proteins in various concentrations. The purified dsRNA could be a mixture of dsRNAs of various sizes, but the majority of the dsRNA molecules were ∼900 bp. Lane 1, ladder; lane 2, dsRNA substrate; lane 3, control dicing reaction; lanes 4 to 11, dicing in the presence of recombinant proteins at various concentrations (lanes 4 to 8, NS4B protein [1.0, 0.5, 0.2, 0.1, and 0.01 μg, respectively]; lane 9, glutathione S-transferase [GST] [1 μg]; lane 10, NS4B [1 μg]; lane 11, F112A mutant [3 μg]). The average size of the diced siRNAs was 22 bp. (v) Western analysis for expression check of NS4B and its F112A mutant in reversal-of-silencing assays in HEK293T cells.
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