Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3’ UTR structures

Abstract

Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines.  The DENV-2 positive-strand RNA genome contains 5’ and 3’ untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins.  In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5’ and 3’ UTRs as “bait”, combined with quantitative mass spectrometry.  We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1, and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs.  Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex.  Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3’ UTR, possibly by formation of a pseudoknot structure.  Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3’ UTR.  These results suggest that the DENV-2 3’ UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3’ UTR is required for DENV replication.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

The authors also would like to thank Shelton Bradrick (Duke University, USA) for the pTNT-FLuc firefly luciferase construct, Matt Marengo (Duke University, USA) for advice on statistical methods, Azlinda Bte Anwar (Duke-NUS Graduate Medical School, Singapore) for critical discussion and proofreading, and Subhash Vasudevan (Duke-NUS Graduate Medical School, Singapore) for technical advice. This research was supported by the Agency for Science, Technology and Research of Singapore and Singapore Ministry of Health Awards R-913-200-002-304 (to M.G.B.), and grants from the Agency for Science, Technology and Research of Singapore (to W.B.).

Figures and Tables

Figure 1 Identification of cellular proteins that interact with the DENV-2 5′ and 3′ UTRs. (A) Schematic of DNA template used to generate RNA for chromatography. The control construct contains the T7 promoter fused to sequences from DENV-2 NGC NS2A coding sequence (nts 3,504–3,674 and nts 3,739–4,221), with the tobramycin aptamer inserted between the NS2A sequences. The 5′ and 3′ UTR construct contains the T7 promoter fused to the DENV-2 NGC 5′ UTR (nts 1–172), tobramycin aptamer sequence and the 3′ UTR from DENV-2 NGC (nts 10,242–10,724). Following in vitro transcription using T7 RNA polymerase, RNA was purified, heated to 95°c, gradually cooled to room temperature and bound to tobramycin-sepharose beads. (B) Scatter plot comparing the results from two independent quantitative mass spectrometric analyses of DENV-2 5′ and 3′ UTR-interacting proteins. In the experiment plotted on the y-axis (forward experiment), the control RNA was incubated with lysate from cells labeled with light arginine and lysine (K0R0) and the DENV-2 5′ and 3′ UTR RNA was incubated with lysate from cells labeled with heavy arginine and lysine (K8R10). In the experiment plotted on the x-axis (reverse experiment), the control RNA was incubated with heavy lysate and the DENV-2 5′ and 3′ UTR RNA was incubated with light lysate. The product of ratio significance in the forward and reverse experiment (p^forward x p^reverse (p) < 1e–10), and minimum log₂ SILAC ratio of 2 for forward experiment and maximum ratio of -2 for reverse experiment were used to select the most confident interacting partners (red dots in upper left quadrant).

Figure 2 DDX6, Caprin1, G3BP1, G3BP2 and USP10 colocalize with sites of DENV replication. HuH-7 cells on coverslips were infected with DENV-2 NGC at an MOI of 1.0 for 24 hours prior to fixation and probing with antibody specific for either SLBP (A and B), DDX6 (C and D), Caprin1 (E and F), G3BP1 (G and H), G3BP2 (I and J) or USP10 (K and L) and dsRNA as a marker for sites of DENV replication as previously shown by Welsch et al. The coverslips probed for SLBP and dsRNA were prepared at a different time, but under similar conditions, than the coverslips used in the rest of the localization studies described here. However, the same preparation of coverslips was also probed for DDX6 and dsRNA with the same colocalization pattern as observed with the other coverslips probed for DDX6 and dsRNA. In the final wash, DAPI was added to visualize cell nuclei and coverslips were sealed prior to visualization using an Olympus IX71 epifluorescent microscope and DP71 digital camera. Images were processed using the ImageJ software package.

Figure 3 DDX6 is required for efficient assembly or release of infectious Dengue virus. (A) Analysis of protein expression of cellular DDX6 and GAPDH proteins at 24 hours post-infection. HuH-7 cells were transfected with 25 nM siRNAs targeting GFP (siGFP) or DDX6 (siDDX6_1, siDDX6_2 and siDDX6_3) and incubated for 48 hours. cells were infected with DENV-2 NGC at an MOI of 0.5 and harvested at 24 hours post-infection. Western blots were probed using rabbit polyclonal antibodies specific for DDX6 and GAPDH. Following incubation with secondary antibody, blots were visualized using the Odyssey Infrared Imaging system. (B) Quantification of western blots for DDX6 protein normalized to GAPDH levels. Quantification was performed using the Odyssey Infrared imaging software package and the level of DDX6 protein in each sample was normalized to GAPDH. DDX6 levels are plotted as a percent relative to cells treated with siGFP. The raw data from quantification was used for one-way ANOVA (p < 0.0001) and Dunnett's Multiple Comparison Test, where p < 0.01 is indicated by an asterisk. All p values were calculated with respect to the siGFP control. (C) Focus forming units (ffu) of tissue culture supernatants 24 hours post-infection. Tissue culture media was diluted and used to infect monolayers of BHK21 cells. Four days post-infection, cells were fixed and analyzed for DENV E protein expression, as described in the Materials and Methods. Virus foci were counted and plotted as ffu/mL. Data was analyzed using a one-way ANOVA (p = 0.0022) and Dunnett's Multiple Comparison Test, where p < 0.01 is indicated by an asterisk. All p values were calculated with respect to the siGFP control. (D) DENV-2 RNA level in the tissue culture media 24 hours post-infection. Total RNA was extracted from tissue culture media of infected cells, reverse transcribed using random primers, and used in Real-time PCR reactions. 25 ng of an in vitro transcript derived form the D. melanogaster Boule mRNA was added to each sample prior to Trizol extraction and used as a normalization control. A standard curve was generated by reverse transcribing 1 ng of in vitro transcribed RNA corresponding to the DENV-2 amplicon. Number of DENV-2 RNA molecules was calculated from the standard curve and plotted. Data was analyzed using a one-way ANOVA (p = 0.0024) and Dunnett's Multiple comparison Test, where p < 0.01 is indicated by an asterisk. All p values were calculated with respect to the siGFP control.

Figure 4 DDX6 interacts with DENV-2 RNA in vivo during infection. (A) RNA immunoprecipitation was performed using either a non-specific IgG or DDX6-specific antibody on uninfected and infected HuH-7 cell lysates. Cells were infected with DENV-2 NGC at an MOI of 0.5 and harvested 48 hours post-infection. Antibodies bound to protein A beads were incubated with uninfected or infected cell lysates and washed 5x with RIPA buffer containing 1 M NaCl. RNA was isolated using Trizol and analyzed by RT-PCR using primers specific for β-actin, c-myc or DENV sequences. (B) RNA immunoprecipitation was repeated in triplicate and processed as in (A), except analysis was performed using Real-time PCR for DENV. To facilitate normalization, an in vitro transcript was added prior to Trizol extraction as a control for recovery. Data was analyzed using a one-way ANOVA (p = 0.0045) and Dunnett's Multiple Comparison Test, where p < 0.01 is indicated by an asterisk.

Figure 5 DDX6 interacts with the DENV-2 3′ UTR via DB2 and DB1 structures. (A–D) Interaction sites for DDX6 were determined using the RNA affinity chromatography method. Deletion mutants were generated from the original 5′ and 3′ UTR construct by PCR and used as templates for T7 in vitro transcription. The left part illustrates the secondary structures present in the DENV-2 3′ UTR based on structural studies and Mfold secondary structure prediction.11,60,61 The deletion mutations used to map 3′ UTR interaction sites are illustrated by black lines under the predicted secondary structures. T7 transcripts were purified, heated to 95°C, cooled and bound to tobramycin-sepharose beads. RNA-bound beads were incubated with cell lysate followed by washing and elution prior to analysis by western blotting for DDX6.

Figure 6 DDX6 interacts with the DENV-2 DB2 and DB1 via pseudoknot structures. (A) Schematic of the DB2 and DB1 structures in the 3′ UTR and sequences predicted to mediate formation of pseudoknot structures (pk2′, pk2, pk1′ and pk1).11,44 (B) RNA affinity chromatography was performed similarly as in Figure 5 except point mutations in the predicted pseudoknot-forming sequences were made in the full-length 3′ UTR. The templates were in vitro transcribed, purified, heated to 95°C, cooled and bound to tobramycin-sepharose beads. RNA-bound beads were incubated with cell lysate followed by washing and elution prior to analysis by western blotting for DDX6.

Figure 7 DDX6, Caprin1, G3BP1, G3BP2 and USP10 bind to different sequences in the 3′ UTR of DENV-2. Interaction sites for DDX6, Caprin1, G3BP1, G3BP2 and USP10 were determined using the RNA affinity chromatography method. Deletion mutants were generated similarly to those described in Figure 5. The left part illustrates the secondary structures present in the DENV-2 3′ UTR based on structural studies and Mfold secondary structure prediction.11,60,61 The deletion mutations used to map 3′ UTR interaction sites are illustrated by black lines under the predicted secondary structures. T7 transcripts were purified, heated to 95°C, cooled and bound to tobramycin-sepharose beads. RNA-bound beads were incubated with cell lysate followed by washing and elution prior to analysis by western blotting for TIAL1 (negative control), DDX6 (positive control), Caprin1, G3BP1, G3BP2 and USP10.

Table 1 List of DENV-2 UTR-interacting proteins identified by mass spectrometry and their respective SILAC ratios with p values from independent forward and reverse experiments