G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA

Abstract

Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.

Figure 1. G3BP1, G3BP2 and CAPRIN1 are required for IFN-β mediated antiviral activity against DENV-2.

(A) HuH-7 cells were treated with siGFP or one of two independent sets of siRNAs (siG12C#1 and siG12C#2) targeting G3BP1, G3BP2 and CAPRIN1, and knockdown was confirmed by western blot analysis. (B) Indirect immunofluorescence was used to detect dsRNA-containing replication complexes (red) and CAPRIN1 (green) in HuH-7 cells treated with control siGFP or siRNA targeting G3BP1, G3BP2 and CAPRIN1 (siG12C#1) and pretreated or not with 100 UI/ml IFN-β before infection with DENV-2 at MOI = 1 for 24 h. The percentage of cells with a replication complex (i.e., infected cells) for each condition is indicated in the lower left corner of each image. (C and D) Cells treated with siGFP, siG12C#1 or siG12C#2 were incubated with increasing concentrations of IFN-β for 16 h before DENV-2 infection at MOI = 1. DENV-2 infectivity was determined at 24 h post-infection by indirect immunofluorescence (C) and DENV-2 infectious particles production (D). Asterisks indicate values below detection levels. (E and F) HuH-7 cells treated with control or siG12C#1 siRNAs, pretreated with IFN-β as above and infected with DENV-2 (E) or YFV-17D (F) at MOI = 1. Viral RNAs levels were determined at 24 h post-infection by quantitative real-time RT-PCR and normalized to intracellular GAPDH mRNA levels.

Figure 2. G3BP1, G3BP2 and CAPRIN1 regulate establishment of the antiviral state.

HuH-7 cells treated with siGFP, siG12C#1 or siG12C#2 were stimulated with the indicated concentration of IFN-β for 16 h and ISG mRNA and protein levels were determined. (A–D) Cells were treated with 100 UI/ml IFN-β and PKR, RIG-I, ISG15 and IFITM2 mRNA levels were determined by quantitative real-time RT-PCR, normalized to intracellular GAPDH mRNA and expressed as fold induction compared to control untreated cells. (E) Cells were stimulated with 0, 10, 100 or 1000 UI/ml IFN-β and levels of proteins G3BP1, G3BP2, CAPRIN1 and representative ISGs, PKR (EIF2AK2), RIG-I (DDX58) and ISG15 were analyzed by western blots. Band intensity was determined by densitometry analysis using ImageJ and normalized to ACTINB. Results are presented as one representative experiment. (F) Cells were treated as in (E) and IFITM2 levels quantified in three independent experiments by measuring fluorescence intensity, using the Licor Odyssey system.

Figure 3. G3BP1, G3BP2 and CAPRIN1 are required for association of ISG mRNAs with polyribosomes.

(A) HuH-7 cells treated with control siGFP or two independent sets of siRNAs against G3BP1, G3BP2 and CAPRIN1 and treated or not with 100 UI/ml IFN-β for 4 h were labeled with ³⁵S metabolic for 1 h. Cell lysates were separated on a 4–15% SDS-PAGE gel and radioisotope incorporation assessed using a phosphorimager. (B–G) HuH-7 cells treated with control siGFP or siG12C#1 siRNAs were stimulated with 100 UI/ml of IFN-β for 4 h and cell lysates separated by velocity sedimentation on a 10–50% sucrose gradient. (B) Total RNA from each gradient fraction was separated on a 1% denaturing agarose gel and stained with EtBr. 28S and 18S ribosomal RNAs are indicated. (C–G) Levels of BIP, ELF2, GAPDH, IFITM2, and PKR mRNAs were determined by quantitative real-time RT-PCR and each fraction expressed as percentage of total for the specific mRNA in all fractions. Profiles result from one out of three representative experiments; for comparison, another replicate of these conditions can be found in Figure 4F–4I. (H) HuH-7 cells treated with siGFP or siG12C#1 and infected with DENV-2 (MOI = 1) for 24 h were stimulated with 100 UI/ml and processed for polyribosome fractionation as in (B). The percentage of DENV-2 genomic RNA in each fraction was measured by quantitative real-time RT-PCR.

Figure 4. G3BP1, G3BP2 and CAPRIN1 depletion specifically inhibits translation of reporters under the control of ISG UTRs.

(A) Schematic representation of IFN-stimulated response element (ISRE)-driven firefly luciferase reporters under the control of ELF2, GAPDH, IFITM2 or PKR UTRs. (B to I) HuH-7 cells stably transfected with the above constructs were treated with control siGFP or siG12C#1 siRNAs, induced with 1000 UI/ml of IFN-β for 10 h and firefly luciferase mRNA determined by quantitative real-time RT-PCR and normalized to GAPDH mRNA levels (B–E). Firefly luciferase protein levels were determined by measuring luciferase activity and normalized to total protein concentration (F–I). Both mRNA and protein activity are expressed as fold induction from control, untreated cells (siGFP, IFN-). Fluc measurements for the GAPDH-Fluc, IFITM2-Fluc and PKR-Fluc constructs were derived from 5 independent experiments in triplicate (n = 15). Fluc measurements for the ELF2-Fluc were derived from 3 independent experiments in triplicate (n = 9). All Fluc mRNA levels were measured in two of these independent experiments (n = 6).

Figure 5. DENV-2 infection interferes with ISG protein expression.

HuH-7 cells were infected with DENV-2 at MOI = 1 and levels of viral products and host IFN response mediators were determined at the indicated times post infection as described above. (A–C) DENV-2 genomic RNA and IFN-β and IFITM2 mRNAs were measured by quantitative real-time RT-PCR and normalized to GAPDH mRNA levels. (D–E) DENV-2 Envelope protein (E) and IFITM2 were detected by western blot and quantified by analysis of fluorescence intensity relative to GAPDH. Open symbols represent mock-infected cells and solid black or grey symbols DENV-2 infected cells for each time-course. (F–I) Control HuH-7 cells (siGFP, black), siG12C-treated (siG12C#1, open) or infected with DENV-2 at MOI = 1 for 24 h (siGFP + DENV-2, grey) and treated with 100 UI/ml IFN-β for 4 h were subjected to polyribosome fractionation. Percentage of ELF2, GAPDH, IFITM2 and PKR mRNAs across fractions was determined by quantitative real-time RT-PCR.

Figure 6. G3BP1, G3BP2 and CAPRIN1 interact with DENV-2 gRNA and sfRNA in infected cells.

(A) The DENV-2 NGC 3′UTR variable region (VR) is predicted to contain five stem-loops (SL-I to SL-V) and two highly conserved pseudoknots PKSL-II and PKSL-IV. Sequences shared by DENV-2 gRNA and sfRNA are highlighted in grey. To relatively quantify these two RNAs species, a differential real-time RT-PCR strategy was designed in which one primer pair, QG, detects DENV-2 gRNA only, while the other primer pair, QGSF, amplifies sequences shared by the gRNA and sfRNA. sfRNA levels are obtained by subtraction of absolute levels of amplicons obtained from both primer pairs calculated against a standard curve (for more details refer to Figure S7). (B–C) HuH-7 cells were infected with DENV-2 at MOI = 1 for 24 h and binding of host RBPs to viral and cellular RNAs lysates was analyzed by RNA immunoprecipitation (IP). (B) A representative western blot shows robust enrichment of G3BP1 and KSRP in the specific IP. (C) Pellet fractions from IP with anti-G3BP1 or anti-KSRP antibodies were analyzed for DENV-2 gRNA and sfRNA as described above. Results are presented as mean ± SEM of the ratio of aforementioned RNAs over GAPDH mRNA in the pellet fraction, normalized to same value for control α-IgG IP. (D–E) RNAs containing a 5′ terminal binding aptamer were used to identify sequences required for G3BP1, G3BP2 and CAPRIN1 binding. RNA matrices DENV-2 3′UTR, the same deleted of SL-II (dSLII), or containing two point mutations in the terminal loop of SLII, which are predicted to disrupt PKSLII (D) were incubated with uninfected cell lysates and bound host RBPs eluted as previously described. The DENV-2 NS2A ORF was used as a negative control . The binding of TIAR (TIAL1), DDX6, G3BP1, G3BP2, or CAPRIN1 was interrogated using specific antibodies. DDX6, which was shown to bind DENV-2 DB region, was used as a positive control . TIAR, which was shown not to interact with DENV-2 positive strand RNA, was used as a negative control . One representative western blot (E) of three done is shown.

Figure 7. DENV-2 3′UTR downregulates ISG protein expression through G3BP1, G3BP2 and CAPRIN1 binding.

(A) Schematics of DENV-2 3′UTR RNAs synthesized to mimic sfRNAs: DENV-2 3′UTR WT, DENV-2 3′UTR YFSLE replaced SL-II/PKSLII with the equivalent structure in YFV-17D (YFV-17D SLE, see panel B) in the DENV-2 3′UTR background, and DENV-2 3′UTR YFSLE-ST4 which contains an additional four point mutations in the middle stem of SL-IV. The corresponding structures are shown in (B). Nucleotides implicated in pseudoknot formation are highlighted in red and substitutions in DENV-2 SL-IV in blue. (C) In vitro transcribed DENV-2 3′UTR RNAs were transfected into HuH-7 cells and their binding to G3BP1 was interrogated by G3BP1 IP. Results are presented as mean ± SEM of three independent experiments, normalized to control IgG IP. (D–G) HuH-7 cells were transfected with 5 ng, 50 ng or 500 ng of in vitro transcribed DENV-2 3′UTR or DENV-2 3′UTR YFSLE, and treated with 100 UI/ml IFN-β for 4 h. (D–E) Levels of viral 3′UTR and IFITM2 RNAs were measured by quantitative real-time PCR and normalized to intracellular GAPDH mRNA levels. (F–G) Levels of IFITM2 protein were determined by western blots and quantified by analysis of fluorescence intensity relative to GAPDH. All results are presented as mean ± SEM of three independent experiments in triplicate. One representative western blot used for IFITM2 protein quantification is shown.

Figure 8. Binding to G3BP1, G3BP2 and CAPRIN1 protects DENV-2 replicons from the antiviral effects of IFN-β.

In vitro transcribed DENV-2 reporter replicons (A) with wild-type 3′UTR or harboring a deletion of SL-II and the four point mutations in SL-IV as described in Figure 6A (D2Rep-WT, thereafter indicated with grey bars, and D2Rep-dSLII/ST4, black bars) were electroporated in HuH-7 cells. (B) Lysates were collected at 72 h post-electroporation and binding of viral RNA to G3BP1 determined by RNA-IP. (C) D2Rep-WT and D2Rep-dSLII/ST4 were electroporated in HuH-7.5 cells, lysates collected at different time-points and analyzed for Renilla luciferase reporter activity. Results are presented as mean ± sem of >2 independent experiments (D–F) HuH-7 cells electroporated with D2Rep-WT or D2Rep-dSLII/ST4 were treated or not with 50 UI/ml IFN-β at 4 h post-electroporation and Renilla luciferase reporter activity measured over time (D). Based on (D), the inhibition of Renilla luciferase accumulation at the 72 h time-point in the presence vs in the absence of exogenously added IFN (E) was calculated for each construct and the rate of increase in Renilla luciferase activity between 48 and 72 h post-electroporation (F, normalized to that of D2Rep-WT in the absence of exogenously added IFN) was calculated for each condition. All results in HuH-7 are derived from three independent experiments, each comprising two to three independent electroporations per reporter.

Figure 9. Model of the DENV-2 sfRNA antagonizing IFN action.

IFNs are produced by infected cells and released in the extracellular space. Binding of IFN to IFNAR on neighboring naïve cells activates a cascade of signaling events leading to selective transcriptional activation of ISGs, which contain an interferon-sensitive response element (ISRE). Host RBPs G3BP1, G3BP2 and CAPRIN1 are required for translation of antiviral ISGs proteins and as a consequence for establishment of the antiviral state. In infected cells, high levels of non-coding sfRNA are produced and act as a RNA sponge, binding to host RNA-binding proteins. G3BP1, G3BP2 and CAPRIN1 bound to DENV-2 sfRNA are prevented to exert their activity in post-transcriptional regulation, leading to downregulation of ISGs, thus protecting DENV-2 replication against IFN antiviral effects.