Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Dec;86(24):13486-500.
doi: 10.1128/JVI.01104-12. Epub 2012 Oct 3.

Noncoding flavivirus RNA displays RNA interference suppressor activity in insect and Mammalian cells

Esther Schnettler  1 , Mark G Sterken, Jason Y Leung, Stefan W Metz, Corinne Geertsema, Rob W Goldbach, Just M Vlak, Alain Kohl, Alexander A Khromykh, Gorben P Pijlman
Affiliations

Noncoding flavivirus RNA displays RNA interference suppressor activity in insect and Mammalian cells

Esther Schnettler et al. J Virol. 2012 Dec.

Abstract

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3'-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.

PubMed Disclaimer

Figures

Fig 1
Fig 1
WNV RNA replication suppresses shRNA-mediated gene silencing in mammalian cells. (A) Schematic representation of the puromycin-selectable WNV replicon, encoding β-galactosidase (Bgal) as a reporter. UTR, untranslated region; PAC, puromycin acetyltransferase; NS, nonstructural protein. (B) WNV replication in Vero cells expressing the WNV replicon (WNVrep) was verified by β-galactosidase detection using X-Gal staining (upper panel). Wild-type Vero cells were stained as a control (lower panel). (C) Suppression of shRNA-induced silencing in Vero WNVrep cells (gray) or normal Vero cells (black). Cells were cotransfected with Firefly luciferase (Fluc), Renilla luciferase (Rluc), and shRNA, either Fluc-specific (shFluc) or off-target (sh-scrambled). Normalized relative luciferase expression (Fluc/Rluc) was determined at 48 hpt, and the means of four independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance determined by an independent two-sample Student t test (P < 0.05).
Fig 2
Fig 2
WNV sfRNA inhibits shRNA-induced silencing in mammalian cells. (A and B) Electromobility gel shift analysis performed by incubating cell lysates of normal Vero, BHK, Ap61, or Vero WNVrep/DENVrep, BHK WNVrep, Ap61 WNVrep cells with radiolabeled siRNA molecules, ath-miRNA171/miRNA171* duplex miRNA molecules, or 114-nt radiolabeled dsRNA molecules for 20 min at room temperature. RNA-protein complexes were separated on a native polyacrylamide gel, dried and exposed overnight to a phosphorimager screen. MBP-NS3 of RHBV, P19 of CymRSV, or influenza virus NS1 were used as positive controls. (C) WNV NS1-5 does not suppress RNA silencing in plants. Agrobacterium tumefaciens harboring vectors encoding mGFP were coinfiltrated in Nicotiana benthamiana leaves with MBP (negative control), Tomato spotted wilt virus (TSWV) NSs (positive control), or different WNV proteins (C107 [capsid], NS12A, NS2B3, NS4AB, and NS5) constructs, respectively. Green fluorescence under UV light was determined in infiltrated leaves 5 days after agroinfiltration. (D) Suppressor activity of WNV proteins and sfRNA on shRNA-induced silencing in Vero cells. Luciferase activity of cells cotransfected with Firefly luciferase (Fluc), Renilla luciferase (Rluc), a specific (shFluc) or off-target (sh-scrambled) shRNA, and different WNV proteins (C107 [capsid], NS12A, NS2B3, NS4AB, and NS5) or WNV sfRNA (gray bars) was measured at 48 hpt. The NS3 of RHBV and VA RNA were used as positive controls (black bars) and a NS3 mutant (NS3m RHBV) as negative control (white bar). The means of two independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance by Tukey's HSD (P < 0.05). (Inset) Schematic representation of the sfRNA structure. SL, stem-loop; DB, dumbbell; RCS, repeated conserved sequence; CS, conserved sequence. (E) To determine the lack of putative RNAi suppressor activity by the added HDVr sequence, the experiments performed for panel D were repeated with MBP, MBP-HDVr, WNV-sfRNA, and VA RNA. The means of two independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance determined by Tukey's HSD (P < 0.05).
Fig 3
Fig 3
sfRNA interferes with Dicer cleavage of dsRNA in vitro and suppresses RNAi in a concentration-dependent manner. (A and B) dsRNA of 700 bp was incubated either in the absence (A) or in the presence (B) of decreasing amounts of in vitro-transcribed WNV sfRNA with human recombinant Dicer and loaded onto an ethidium bromide-stained agarose gel. (C) Renilla luciferase (Rluc), Firefly luciferase (Fluc), either specific (shFluc) or off-target (sh-scrambled) shRNA, and decreasing concentrations of WNV sfRNA were cotransfected into Vero cells. RHBV NS3 or a dysfunctional mutant (RHBV NS3m) were used as positive and negative controls, respectively. Relative luciferase expression (Fluc/Rluc) was determined at 48 hpt and normalized to the cells transfected with off-target shRNA. The means of two independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance determined by Tukey's HSD (P < 0.05). (D) Bypassing of sfRNA-mediated RNAi suppression by siRNA transfection. Renilla luciferase (Rluc), Firefly luciferase (Fluc), either specific (siFluc) or off-target (si-scrambled) siRNA, and plasmids expressing WNV/DENV sfRNA were cotransfected into Vero cells. Tombusvirus P19, which binds siRNA, was used as a positive control. The relative luciferase expression (Fluc/Rluc) was determined at 48 hp siRNA transfection and normalized to the cells transfected with off-target siRNA. The means of four independent experiments performed in triplicate are shown with the standard errors. Asterisks indicate significance determined by Tukey's HSD (P < 0.05).
Fig 4
Fig 4
Suppression of the mammalian miRNA pathway in WNV replicon cells. (A) Vero cells either expressing the WNV replicon (Vero-WNV [▩]) or lacking the replicon (Vero wild type [■]) were transfected with expression vectors encoding pCMV-luc-miR30-AP or pCMV-luc-random alone or in combination with either a specific miRNA (hsa-miRNA30) or off-target (hsa-miRNA21). The results shown are the means of two independent experiments performed in duplicate. Asterisks indicate significance determined by independent two-sample Student t tests (P < 0.05). (B) The luciferase expression was measured 2 days posttransfection, and the relative luciferase expression (Fluc/Rluc) was determined. The luciferase level measured with nonspecific has-miRNA21 was set at 1.0. The results shown are representative of three independent experiments performed in duplicate. Asterisks indicate significance determined by an independent two-sample Student t test (P < 0.05).
Fig 5
Fig 5
Mapping of RNAi suppressor activity within WNV sfRNA. (A to D) 3′SL-derived miRNA sensor assay using Firefly luciferase-based sensor constructs. (A) Schematic representation of miRNA sensor constructs for expression in mammalian cells. Reverse complement (rc) of A1A2 and C1C2 tandem repeats are indicated in black and gray, respectively. Sequences used for shRNA cloning into pSuper plasmids are indicated in boldface. Fluc, Firely luciferase; SV40, simian virus 40 promoter; pA, polyadenylation signal; Xb, XbaI restriction site; Xh, XhoI restriction site. (B) Schematic representation of the WNV 3′SL. A1A2 and C1C2 sequences for tandem repeat cloning into miRNA sensor constructs are indicated in black and gray, respectively. (C) Functionality of miRNA sensor constructs. Cells were cotransfected with pRL-TK, pGL3-(sensor constructs), and either pSuper-A1A2 or pSuper-C1C2 or control plasmid. The relative luciferase expression (Firefly/Renilla) was determined at 24 hpt. The means of two independent experiments performed in triplicate are shown with the standard errors. Significance was tested by an independent two-sample Student t test (P < 0.05). (D) Silencing of miRNA sensor constructs by sfRNA expression in Vero cells. Cells were cotransfected with pRL-TK, pGL3-(sensor constructs), and either pDEST40-sfRNA or control plasmid. The relative luciferase expression (Firefly/Renilla) was determined at 24 hpt. The means of three independent experiments performed in duplicate are shown with the standard errors. Significance was tested by an independent two-sample Student t test (P < 0.05). (E) Schematic representation of WNV sfRNA with abbreviations as in Fig. 2. The numbers are nucleotide positions from the 3′ terminus of the WNV 3′UTR. Deletions within sfRNA are indicated. (F) Suppressor activity of WNV sfRNA truncations on shRNA-induced silencing in Vero cells. The luciferase activity of cells cotransfected with Firefly luciferase (Fluc), Renilla luciferase (Rluc), a specific (shFluc) or off-target (sh-scrambled) shRNA, and WNV sfRNA variants was measured at 48 hpt. The NS3 of RHBV was used as a positive control, and MBP was used as a negative control. The means of two independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance determined by Tukey's HSD (P < 0.05).
Fig 6
Fig 6
WNV sfRNA interferes with siRNA-induced silencing in insect cells. (A) Schematic representation of SFV constructs. The Renilla luciferase (Rluc) gene is inserted upstream of nsP4. sfRNA or the EMCV IRES sequence is inserted downstream of the 26S subgenomic promoter. Schematic RNA structures of sfRNA and the EMCV IRES are shown. (B) Capped, in vitro-transcribed SFV RNA was transfected in Aedes albopictus U4.4 mosquito cells. Rluc expression was measured at 24 hpt. The means of four independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance determined by independent two-sample Student t test (P < 0.05). (C) Aedes albopictus U4.4 mosquito cells were cotransfected with Firefly luciferase (Fluc), Renilla luciferase (Rluc), and MBP or WNV sfRNA constructs. After 24 h, silencing was induced by transfection of either specific (dsFluc) or off-target (ds-scrambled) in vitro-transcribed dsRNA. The luciferase activity was measured at 48 hpt, and the normalized relative luciferase activity (Fluc/Rluc) is shown with the standard errors (means of two independent experiments performed in duplicate). Asterisks indicate significance determined by Tukey's HSD (P < 0.05). (D) Concentration-dependent effect of sfRNA on shRNA-induced silencing determined in D. melanogaster S2 cells by transfection of decreasing concentrations of WNV-sfRNA (140, 50, and 20 ng) construct in concert with Firefly luciferase (Fluc), Renilla luciferase (Rluc), and either specific (shFluc) or off-target (sh-scrambled) shRNA. RHBV NS3 was used as a positive control. The luciferase activity was measured at 48 hpt, and the normalized relative luciferase activity (Fluc/Rluc) is shown with the standard errors (means of three independent experiments performed in duplicate). Asterisks indicate significance determined by Tukey's HSD (P < 0.05).
Fig 7
Fig 7
WNV sfRNA interferes with Ago1-dependent miRNA-induced silencing in insect cells. (A) Suppression of dme-miRNA1-induced silencing in S2 cells by cotransfection of a pMT-Renilla luciferase (Rluc), pMT-Firefly luciferase (Fluc)-dme-miRNA1 sensor construct, either specific (dme-miRNA1) or off-target (dme-miRNA12) primary miRNA and either MBP, CIRV P19 (as a positive control), or WNV sfRNA (180 or 50 ng). After induction at 48 hpt, the relative luciferase expression (Firefly/Renilla) was determined at 72 hpt, and the means of three independent experiments in duplicate are shown with the standard errors. Asterisks indicate significance by Tukey's HSD (P < 0.05). (B) Silencing of the pMT-Firefly luciferase (Fluc)-dme-miRNA1 sensor construct is Ago1 dependent. S2 cells were soaked with 200 ng of dsRNA either Ago1, Ago2, or EGFP (unspecific) during seeding. dme-miRNA1-induced silencing was induced by cotransfection of pMT-Renilla luciferase (Rluc), pMT-Firefly luciferase (Fluc)-dme-miRNA1 sensor construct, either specific (dme-miRNA1) or off-target (dme-miRNA12) primary miRNA. The means of three independent experiments in duplicate are shown with the standard errors. Asterisks indicate significance determined by an independent two-sample Student t test (P < 0.05). (C) Confirmation of successful Ago1/2 depletion in S2 cells. To check the knockdown level of Ago1/2, RT-PCR was carried out on total RNA purified from dsRNA-silenced cells. Western blot analysis of Drosophila melanogaster Ago1/2 was performed to confirm successful depletion of Ago1/2 in S2 cells.
Fig 8
Fig 8
Suppression of RNA silencing in mammalian and mosquito cells by DENV sfRNA. (A) Viral RNA replication in Vero cells stably expressing the DENV1 replicon was verified by immunofluorescence using a primary J2 anti-dsRNA antibody and a corresponding rhodamine-labeled secondary antibody (upper panel). Wild-type Vero cells were stained as a control (lower panel). (B) The effect of DENV RNA replication on shRNA-induced silencing was investigated in normal Vero cells (black) and in Vero cells stably expressing the DENV replicon (gray). The cells were cotransfected with Firefly luciferase (Fluc), Renilla luciferase (Rluc), and shRNA, either Fluc-specific (shFluc) or off-target (sh-scrambled). The normalized relative luciferase expression (Fluc/Rluc) was determined at 48 hpt. The means of three independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance determined by an independent two-sample Student t test (P < 0.05). (C) RNA silencing suppression by the DENV sfRNA was investigated in Vero cells by cotransfection of pFluc, Rluc, either specific (shFluc) or off-target (sh-scrambled) shRNA, and expression constructs of MBP, MBP-HDVr, DENV, sfRNA, or adenoviral VA RNA. The luciferase expression was measured at 48 hpt. The means of two independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance by Tukey's HSD (P < 0.05). (D) Effect of DENV sfRNA was determined by cotransfection of Aedes albopictus-derived U4.4 cells with Firefly luciferase (Fluc), Renilla luciferase (Rluc) and MBP, DENV sfRNA, or WNV sfRNA (as positive control) constructs. After 24 h, silencing was induced by transfection of either specific (dsFluc) or off-target (ds-scrambled) in vitro-transcribed dsRNA. The luciferase activity was determined 48 h after primary transfection and normalized to the cells transfected with off-target dsRNA. The means of three independent experiments performed in duplicate are shown with the standard errors. Asterisks indicate significance determined by Tukey's HSD (P < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aliyari R, Ding SW. 2009. RNA-based viral immunity initiated by the Dicer family of host immune receptors. Immunol. Rev. 227:176–188 - PMC - PubMed
    1. Ambros V. 2004. The functions of animal microRNAs. Nature 431:350–355 - PubMed
    1. Andersson MG, et al. 2005. Suppression of RNA interference by adenovirus virus-associated RNA. J. Virol. 79:9556–9565 - PMC - PubMed
    1. Aparicio O, Razquin N, Zaratiegui M, Narvaiza I, Fortes P. 2006. Adenovirus virus-associated RNA is processed to functional interfering RNAs involved in virus production. J. Virol. 80:1376–1384 - PMC - PubMed
    1. Asgari S. 2011. Role of microRNAs in insect host-microorganism interactions. Front. Physiol. 2:48. - PMC - PubMed

Publication types