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A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity

Nature Immunology volume 17, pages 523–530 (2016)Cite this article

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Abstract

14-3-3 proteins regulate biological processes by binding to phosphorylated serine or phosphorylated threonine motifs of cellular proteins. Among the 14-3-3 proteins, 14-3-3ɛ serves a crucial function in antiviral immunity by mediating the cytosol–to–mitochondrial membrane translocation of the pathogen sensor RIG-I. Here we found that the NS3 protein of dengue virus (DV) bound to 14-3-3ɛ and prevented translocation of RIG-I to the adaptor MAVS and thereby blocked antiviral signaling. Intriguingly, a highly conserved phosphomimetic RxEP motif in NS3 was essential for the binding of 14-3-3ɛ. A recombinant mutant DV deficient in binding to 14-3-3ɛ showed impairment in antagonism of RIG-I and elicited a markedly augmented innate immune response and enhanced T cell activation. Our work reveals a novel phosphomimetic-based mechanism for viral antagonism of 14-3-3-mediated immunity, which might guide the rational design of therapeutics.

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Figure 1: The NS3 protein of DV interacts with 14-3-3ɛ.
Figure 2: NS2B-NS3 of DV inhibits the activation of RIG-I independently of proteolytic activity.
Figure 3: NS3 inhibits the binding of RIG-I to 14-3-3ɛ and thus prevents the translocation of activated RIG-I to mitochondria.
Figure 4: NS3 binds to 14-3-3ɛ via a phosphomimetic RxEP motif.
Figure 5: DV2(KIKP) shows attenuated replication and elicits an enhanced innate immune response.
Figure 6: DV2(KIKP) elicits stronger immune responses in primary human mononuclear phagocytes and T cells than those elicited by DV2(WT).

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Acknowledgements

We thank C. Huang (Center for Disease Control and Prevention) for the DV2 16681 infectious clone pD2/IC-30P; E. Harris (University of California, Berkeley) for antibody E1D8 to NS3; M. Diamond (Washington University in St. Louis) for cDNA encoding NS3 of WNV strain NY99 or Kunjin; Z. Chen (University of Texas Southwestern Medical Center) for cDNA encoding NS3 of HCV strain Con1 and plasmids encoding the HCV NS3-NS4A protease complex and its S139A catalytically-inactive mutant; R. Kuhn (Purdue University) for cDNA encoding NS3 of YFV strain 17D; J. Jung (University of Southern California) for Huh7.5 cells; M. Farzan (Scripps Institute Florida) for Huh7, BHK-21, C6/36 and K562 cells; N. Hacohen (Massachusetts General Hospital) for HEK293T ISRE-luciferase cells, L. Gehrke (Harvard University) for DV4 strain 814699; D. Knipe (Harvard University) for herpes simplex virus type 1; S. Whelan (Harvard University) for VSV-GFP; and A. García-Sastre (Icahn School of Medicine at Mount Sinai) for NDV-GFP. Supported by the US National Institutes of Health (R01 AI087846), the Giovanni Armenise-Harvard Foundation, the William F. Milton Fund, and a John and Virginia Kaneb Fellowship (M.U.G.).

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Authors and Affiliations

  1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Ying Kai Chan & Michaela U Gack

  2. Department of Microbiology, The University of Chicago, Chicago, Illinois, USA

    Michaela U Gack

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  1. Ying Kai Chan
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Contributions

Y.K.C. performed all aspects of this study; and Y.K.C. and M.U.G. designed the study and wrote the manuscript.

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Correspondence to Michaela U Gack.

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Competing interests

The authors are co-inventors on a patent application for use of the RxEP motif and DV2(KIKP) mutant virus.

Integrated supplementary information

Supplementary Figure 1 14-3-3ɛ is critical for the control of DV replication.

(a) Amino acid sequence of 14-3-3ɛ and specific peptides (red) identified by MS upon affinity purification of FLAG-NS3-Pro (DV2, strain NGC) from HEK293T cells. Peptide coverage was ~64.3%. (b) Huh7 cells were transfected with FLAG-14-3-3ɛ and subsequently mock-infected, or infected with DV2 (NGC) at MOI 1 for 24 h. Cells were stained for FLAG (14-3-3ɛ; red), NS3 (green) and NS4A (orange) and imaged by confocal microscopy. Nuclei were stained with DAPI (blue). Scale bar; 5 μm. (c) Huh7 cells were transfected with empty vector or c-myc-tagged 14-3-3ɛ and subsequently infected with DV2 (NGC) at MOI of 0.2 or 0.5. 72 h later, cells were stained for intracellular DV prM and analyzed by flow cytometry. The results are expressed as means ± SD (n = 3). (d) Viral titers at 48 h postinfection in the supernatants from the experiment shown in (c) (MOI 0.5), determined by plaque assay using BHK-21 cells. The results are expressed as means ± SD (n = 3). (e) Huh7 cells were transfected with vector or c-myc-tagged 14-3-3ɛ, and subsequently infected with the indicated DV serotypes (DV1 276 RK1, DV2 16681, DV3 BC188/97 or DV4 814699) at MOI 0.05, or HSV-1 (MOI 0.2). Infected cells were determined by intracellular prM (DV) or ICP8 (HSV-1) staining at 72 and 24 h.p.i, respectively. The infectivity for each virus was normalized to vector-transfected cells. The results are expressed as means ± SD (n = 3). (f) HEK293T ISRE-luciferase reporter cells were transfected with empty vector or FLAG-tagged 14-3-3ɛ and subsequently infected with DV2 (NGC) at MOI 5. 46 h later, cells were subjected to luciferase assay and values normalized to corresponding mock-infected cells. The results are expressed as means ± SD (n = 3). (g) K562 cells were transfected with 14-3-3ɛ-specific siRNA (si.14-3-3ɛ) or non-targeting control siRNA (si.C). 48 h later, cells were infected with DV2 (NGC) at MOI 1. 48 h later, DV prM-positive cells were determined by flow cytometry. The results are expressed as means ± SD (n = 3). Knockdown of 14-3-3ɛ was confirmed by IB. *p< 0.05; **p< 0.005, unpaired t-test. NS; statistically not significant. Data are representative of 1 (a,d), or at least 2 (b, c, e-g) independent experiments.

Supplementary Figure 2 DV NS2B-NS3 does not cleave 14-3-3e, RIG-I or TRIM25.

(a) HEK293T cells were transfected with TRIM25-FLAG, RIG-I-FLAG, FLAG-14-3-3ɛ, or STING-HA together with empty vector or HA-tagged NS2B-NS3. 48 h later, WCLs were subjected to IB with anti-HA, anti-FLAG, and anti-actin antibodies. (b) NS2B-NS3(WT), but not NS2B-NS3(S135A) or NS3 expressed alone, is catalytically active. HEK293T cells were transfected with HA-tagged DV NS2B-NS3(WT), NS2B-NS3(S135A) or NS3 together with HA-tagged STING. WCLs were analyzed by IB with anti-HA and anti-actin antibodies. (c) HEK293T cells were transfected with GST, GST-NS3, or GST-RIG-I(2CARD) (positive control). 48 h later, cells were infected with SeV (50 HAU/ml) for 20 h, or left uninfected. WCLs were subjected to IB with an anti-SeV polyclonal serum, or with anti-GST or anti-actin antibody. P; P protein of SeV; NP; nucleocapsid protein of SeV. Data are representative of 2 (b,c) or 3 (a) independent experiments.

Supplementary Figure 3 DV NS3 has a greatly enhanced 14-3-3ɛ-binding capacity relative to that of RIG-I.

(a) In vitro binding assay was performed by incubating purified TRIM25-FLAG or RIG-I-FLAG with bacterially-purified recombinant (r) human 14-3-3ɛ. Binding was determined by IB with anti-14-3-3ɛ and anti-FLAG antibodies. (b) HEK293T cells were transfected with empty vector, FLAG-RIG-I or FLAG-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 18 h, or left uninfected. WCLs were subjected to FLAG-pulldown (FLAG-PD), followed by IB with anti-14-3-3ɛ or anti-FLAG antibody. Two different exposures of the blot detecting bound 14-3-3ɛ are shown (upper two panels). (c) HEK293T cells were transfected with GST or GST-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 20 h, followed by cytosol-mitochondria fractionation assay and IB analysis. Furthermore, WCLs were analyzed for RIG-I, GST, GST-NS3, and actin protein expressions. (d) Huh7 cells stably expressing empty vector or HA-tagged 14-3-3ɛ were mock-infected or infected with DV2 NGC (MOI 1) for 20 h. Cytosol-mitochondria fractionation assay was performed, followed by IB with anti-RIG-I, anti-MAVS and anti-GAPDH antibodies. Furthermore, WCLs were used for IB with the indicated antibodies. Data are representative of 2 independent experiments (a-d).

Supplementary Figure 4 The NS3(KIKP) protein is impaired in inhibiting the 14-3-3ɛ–RIG-I interaction and antiviral signaling.

(a) Schematic representation of the domain structure of DV NS3 as well as GST-fused NS3 truncation mutants. Furthermore, a summary of the results from the 14-3-3ɛ-binding studies shown in Fig. 4a is provided. Numbers indicate amino acids. (b) Ribbon representation of the NS3 (DV4) protease domain (red) with the RxEP motif (green) and the catalytic triad (H51, D75 and S135; cyan). (c) HEK293T cells were transfected with GST, GST-NS3 WT (DV2, harboring RIEP), or the indicated GST-NS3 mutants. 48 h later, WCLs were subjected to GST-PD, followed by IB with anti-14-3-3ɛ and anti-GST antibodies. (d) HEK293T cells were transfected with GST, GST-NS3(WT) or GST-NS3(KIKP). 48 h later, cells were infected with SeV (50 HAU/ml) for 20 h. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-14-3-3ɛ and anti-RIG-I antibodies. (e) HEK293T cells were transfected with GST, GST-NS3(WT) or GST-NS3(KIKP). 48 h later, cells were infected with SeV (50 HAU/ml) for 18 h and then subjected to luciferase assay. Luciferase values were normalized to pGK-β-gal. The results are expressed as means ± SD (n = 3). *p< 0.05, unpaired t-test. NS; statistically not significant. (f) GST, GST-NS3(WT) or GST-NS3(KIKP) was transfected into HEK293T cells. 48 h later, cells were infected with SeV (50 HAU/ml) for 22 h. ISG (ISG54 or RIG-I) protein expression in the WCL was determined by IB with the indicated antibodies. Data are representative of 2 independent experiments (c-f).

Supplementary Figure 5 WNV NS3 binds 14-3-3ɛ via a phosphomimetic RLDP motif.

(a) Binding of endogenous 14-3-3ɛ and GST, or GST-NS3 of YFV (17D), DV (DV2 NGC), or WNV (NY99 or Kunjin) in transfected HEK293T cells, assessed by GST-PD and IB with anti-14-3-3ɛ antibody. (b) IFN-β luciferase activity in HEK293T cells transfected with RIG-I(2CARD) together with GST, or GST-NS3 of DV (DV2 NGC) or WNV (NY99 or Kunjin), normalized to constitutive pGK-β-gal. Viral NS3 expressions were determined by IB with anti-GST antibody. The results are expressed as means ± SD (n = 3). **p< 0.005, unpaired t-test. (c) Binding of endogenous 14-3-3ɛ and GST, or GST-NS3(WT) or GST-NS3(KIKP) from WNV (NY99), assessed in transfected HEK293T cells by GST-PD and IB with anti-14-3-3ɛ antibody. Data are representative of 2 (b, c) or 3 (a) independent experiments.

Supplementary Figure 6 Functional characterization of DV2(KIKP).

(a) The DV NS2B-NS3(KIKP) mutant protein is catalytically active. HEK293T cells were transfected with HA-tagged STING together with empty vector, or HA-tagged NS2B-NS3(WT), NS2B-NS3(S135A), or NS2B-NS3(KIKP). 48 h later, WCLs were analyzed by IB with anti-HA and anti-actin antibodies. (b,c) Vero cells were infected with DV2(WT) or DV2(KIKP) at an MOI of 0.02. Cells were harvested for intracellular prM staining at the indicated time points and analyzed by flow cytometry (b). Viral titers were determined in the supernatants by serial dilution and prM staining of Vero cells (c). IU; infectious unit. The results are expressed as means ± SD (n = 3). (d) Huh7 or Huh7.5 cells were infected with DV2(WT) or DV2(KIKP) at an MOI of 0.01. Cells were harvested for intracellular prM staining and analyzed by flow cytometry. The results are from 2 independent experiments and expressed as means ± SD (n = 6). (e) NHLF cells were infected with DV2(WT) or DV2(KIKP) (both MOI 0.5), NDV (MOI 0.05), or VSV (MOI 0.001) for 48 h and supernatants were analyzed for IFN-β protein by ELISA. The results are expressed as means ± SD (n = 3). (f) Huh7 cells were infected with DV2(WT) or DV2(KIKP) at MOI 0.5 and viral RNA expression was quantified by qRT-PCR for the indicated time points. Transcript levels were normalized to GAPDH and are shown as fold levels compared to DV2(WT)-infected cells of each time point. The results are expressed as means ± SD (n = 3). gRNA; genomic RNA. (g) A549 cells were infected with DV2(WT) or DV2(KIKP) (both MOI 0.2) for 24 h and subjected to immunofluorescence staining of endogenous ISG54 or RIG-I (green), NS3 (red), and DAPI (nuclei, blue). (h) Huh7 cells were infected with DV2(WT) or DV2(KIKP) at MOI 0.5. 48 h later, WCLs were harvested and analyzed by IB with anti-STAT2, anti-NS3 and anti-actin antibodies. (i) Primary moDCs were infected with DV2(WT) or DV2(KIKP) (both MOI 1). 72 h later, cells were harvested and analyzed for CD86 surface expression by FACS analysis. (j) Primary moDCs were infected with DV2(WT) or DV2(KIKP) (both MOI 1), or NDV (MOI 0.5), and then co-cultured with syngeneic naïve pan T cells at 1:1 ratio. 72 h later, T cells were analyzed for intracellular staining of phosphorylated STAT1 (pY701). Representative FACS histograms are shown. *p< 0.05; **p< 0.005, unpaired t-test. NS; statistically not significant. ND; not detectable. Data are representative of 1 (a, f), or at least 2 (b-e and g-j) independent experiments.

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Chan, Y., Gack, M. A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity. Nat Immunol 17, 523–530 (2016). https://doi.org/10.1038/ni.3393

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