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

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

Dengue NS3, an RNAi suppressor, modulates the human miRNA pathways through its interacting partner

RNAi acts as a host immune response against non-self molecules, including viruses. Viruses evolved to neutralize this response by expressing suppressor proteins. In the present study, we investigated dengue virus non structural protein 3 (dvNS3), for its RNAi-suppressor activity in human cell lines. Dengue virus (DV) NS3 reverts the GFP expression in GFP-silenced cell lines. Pull-down assays of dvNS3 revealed that it interacts with the host factor human heat shock cognate 70 (hHSC70). Down-regulation of hHSC70 resulted in accumulation of dengue viral genomic RNA. Also, the interaction of dvNS3 with hHSC70 perturbs the formation of RISC (RNA-induced silencing complex)-loading complex (RLC), by displacing TRBP (TAR RNA-binding protein) and possibly impairing the downstream activity of miRNAs. Interestingly, some of these miRNAs have earlier been reported to be down-regulated upon DV infection in Huh7 cells. Further studies on the miRNA-mRNA relationship along with mRNA profiling of samples overexpressing dvNS3 revealed up-regulation of TAZ (tafazzin) and SYNGR1 (synaptogyrin 1), known dengue viral host factors (DVHFs). Importantly, overexpression of dvNS3 in human embryonic kidney (HEK) 293T cells resulted in modulation of both mature and precursor miRNAs in human cell lines. Subsequent analysis suggested that dvNS3 induced stage-specific down-regulation of miRNAs. Taken together, these results suggest that dvNS3 affects biogenesis and function of host miRNAs to regulate DVHFs for favouring DV replication

Underlying image data for the corrected Fig 2D (correct DMSO panel from the original experiment).

Underlying image data for the corrected Fig 2D (correct DMSO panel from the original experiment).</p

Bispidine-Amino Acid Conjugates Act as a Novel Scaffold for the Design of Antivirals That Block Japanese Encephalitis Virus Replication

<div><h3>Background</h3><p>Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties.</p> <h3>Methodology/Principal Findings</h3><p>In this study we have identified 3,7-diazabicyclo[3.3.1]nonane, commonly called bispidine, as a privileged scaffold to synthesize effective antiviral agents. We have synthesized derivatives of bispidine conjugated with amino acids and found that hydrophobic amino acid residues showed antiviral properties against JEV. We identified a tryptophan derivative, Bisp-W, which at 5 µM concentration inhibited JEV infection in neuroblastoma cells by more than 100-fold. Viral inhibition was at a stage post-entry and prior to viral protein translation possibly at viral RNA replication. We show that similar concentration of Bisp-W was capable of inhibiting viral infection of two other encephalitic viruses namely, West Nile virus and Chandipura virus.</p> <h3>Conclusions/Significance</h3><p>We have demonstrated that the amino-acid conjugates of 3,7-diazabicyclo[3.3.1]nonane can serve as a molecular scaffold for development of potent antivirals against encephalitic viruses. Our findings will provide a novel platform to develop effective inhibitors of JEV and perhaps other RNA viruses causing encephalitis.</p> </div

Synthesis of Bisp-W derivatives and its effect on JEV.

<p>(A) Structure of Bisp-W derivatives with tryptophan on one arm of bispidine and benzyl on the other (Bisp-W-Benzyl) and with Boc group deprotected (Bisp-W-NH). (B) Viral titers were determined by plaque assay of N2A cell culture supernatants (22 h pi) infected with JEV and treated with 5 µM derivatives of Bisp-W as indicated. *** P = 0.0003 and 0.0004 and ns: not significant (P = 0.425) as determined by two-tailed, t-test. Error bars represent Mean ± SEM of three replicates. Data are representative of experiments performed twice with three replicates.</p

Bispidine and its derivatives.

<p>(A) Structural relationship of bispidine to natural product sparteine. (B) General structure of bispidine compounds (i) and (ii) represent the conformer equilibrium in diimides of bispidine (iii) Conjugates of bispidine wherein the nitrogen is part of the amino acids. (C) The genesis of design of bispidine. (D) Synthesis of bispidine conjugates i) N-hydroxysuccinimide/DCC/NEt₃/Boc-Leu-OH/dry dichloromethane; ii) N-hydroxysuccinimide/DCC/NEt₃/Z-Leu-OH/dry dichloromethane; iii) Br-CH₂-CO-Leu-Val-OMe; NEt₃, acetonitrile; iv) 25% TFA in dichloromethane.</p

Bisp-W blocks JEV RNA replication.

<p>(A) Viral titers were determined by plaque assay of N2A cell culture supernatants (22 h pi) infected with JEV and treated with DMSO or 5 µM of Bisp-W at the indicated time points. Error bars represent Mean ± SEM. ** P<0.005, **** <0001, * <0.01, ** 0.096 and ** <0.001 for respective time points. (B) Viral titers were determined by plaque assay of N2A cell culture supernatants infected with JEV and treated with PBS or 20 µM minocycline and DMSO or 5 µM of Bisp-W as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002005#s2" target="_blank">materials and methods</a>. Error bars represent Mean ± SEM. *** P = 0.0002. N.D: Not Detected. (C) Western blot analysis of N2A lysates prepared from JEV-infected and DMSO or Bisp-W-treated cells. C- Capsid. β–actin is shown for loading control. (D) Viral titers were determined from supernatants (extracellular) and cell lysates (intracellular) at 22 h pi by plaque assay of samples from N2A cells infected with JEV and treated with 5 µM of Bisp-W at 1 h post-infection. ***P = 0003 and 0.00002 respectively. (E) Total RNA was isolated from N2A cells infected with JEV and treated with 5 µM Bisp-W at 1 h post-infection. JEV genome copy numbers were estimated by quantitative real time PCR normalized to GAPDH mRNA. ** P = 0.0001 by unpaired two-tailed t-test. Error bars represent Mean ± SEM. Data are representative of two or more experiments performed with three replicates.</p

Effect of Bispidine derivatives on JEV infection.

<p>(A) Viral titers were determined by plaque assay of N2A cell culture supernatants (22 h pi) infected with JEV and treated with 100 µM of derivatives of bispidine. * P<0.01 determined by two-tailed, t-test. (B) Cytotoxicity was measured by lactate dehydrogenase (LDH) assay from culture supernatants treated with 100 µM of BLB or DMSO. LDH released from cells incubated with detergent buffer was used as 100% LDH release. (C) Viral titers were determined by plaque assay from Huh7 and C6/36 cell culture supernatants (22 h pi) infected with JEV and treated with 100 µM of BLB. *** P = 0.0002 and **P = 0.0041 determined by two-tailed, t-test. (D) N2A cells were infected as above and at 22 h pi cells were fixed and stained with anti-E antibodies followed by alexa 568-conjugated secondary antibodies. Nuclei were stained by DAPI. (E) Viral titers were determined by plaque assay of N2A cell culture supernatants (22 h pi) infected with JEV and treated with 100 µM of BLB at the indicated time points. UT- Untreated. All the data presented are representative of two or more experiments performed with two or more replicates. *** P = 0.0007, 0.0005 and **P = 0.007 as determined by two-tailed, t-test. Error bars in all figures represent Mean ± SEM.</p

Effect of Bisp-W on other encephalitic viruses.

<p>(A) N2A cells were infected with an MOI of 1 pfu/cell with WNV and treated with DMSO or 5 µM of Bisp-W. Viral titers in cell culture supernatants (22 h pi) were determined by plaque assay. (B) N2A cells were infected with an MOI of 0.1 pfu/cell with CHPV and treated with DMSO or 5 µM of Bisp-W. Viral titers in cell culture supernatants (8 h pi) were determined by plaque assay. Error bars represent Mean ± SEM of three replicates. Data are representative of two experiments performed with triplicate samples. **P = 004 and 0.002 respectively as determined by unpaired, two-tailed t-test.</p

Synthesis of amino acid conjugates of bispidine.

<p>(A) Structure of bispidine conjugated with tryptophan (Bisp-W), lecine+phenylalanine (Bisp-LF) and lysine (Bisp-K). (B) Viral titers were determined by plaque assay of N2A cell culture supernatants (22 h pi) infected with JEV and treated with 5 µM of derivatives of bispidine. *** P = 0.0004 and 0.0004, ** P = 0.0034 and as determined by two-tailed, t-test. Error bars represent Mean ± SEM of three replicates. (C) Cytotoxicity was measured by lactate dehydrogenase (LDH) assay from culture supernatants treated with 5 µM of the indicated bispidine conjugates or DMSO. LDH released from cells incubated with detergent buffer was used as 100% LDH release. (D) IC₅₀ value for Bisp-W in the indicated cell lines was estimated by measuring viral titers in cell culture supernatants (22 h pi) infected with JEV and treated with the indicated concentration of Bisp-W. Error bars represent Mean ± SEM of three replicates. All the data are representative of experiments performed at least twice with three replicates.</p