Zika Virus Non-structural Protein 4A Blocks the RLR-MAVS Signaling

Abstract

Flaviviruses have evolved complex mechanisms to evade the mammalian host immune systems including the RIG-I (retinoic acid-inducible gene I) like receptor (RLR) signaling. Zika virus (ZIKV) is a re-emerging flavivirus that is associated with severe neonatal microcephaly and adult Guillain-Barre syndrome. However, the molecular mechanisms underlying ZIKV pathogenesis remain poorly defined. Here we report that ZIKV non-structural protein 4A (NS4A) impairs the RLR-mitochondrial antiviral-signaling protein (MAVS) interaction and subsequent induction of antiviral immune responses. In human trophoblasts, both RIG-I and melanoma differentiation-associated protein 5 (MDA5) contribute to type I interferon (IFN) induction and control ZIKV replication. Type I IFN induction by ZIKV is almost completely abolished in MAVS^-/- cells. NS4A represses RLR-, but not Toll-like receptor-mediated immune responses. NS4A specifically binds the N-terminal caspase activation and recruitment domain (CARD) of MAVS and thus blocks its accessibility by RLRs. Our study provides in-depth understanding of the molecular mechanisms of immune evasion by ZIKV and its pathogenesis.

Keywords: NS4A; RIG-I like receptors; RLR; Zika; flavivirus; non-structural protein 4A.

FIGURE 1

The RLR signaling is essential for induction of type I IFN responses to and control of ZIKV infection in human trophoblasts. (A) Immunoblots of RIG-I, MDA5, MAVS and a house keeping gene actin protein expression in wild type (WT) and knockout human trophoblasts. (B) Fluorescent microscopic images of GFP in trophoblasts infected with VSV-GFP at a multiplicity of infection (MOI) of 0.1 for 20 h. Objective: 5×, scale bar: 50 μM. qPCR quantification of cellular (C) viral RNA and (D) IFNB1 mRNA in trophoblasts infected with ZIKV FLR at a MOI of 5 for the indicated time. The bars/data points in C,D are the mean + SEM of the results, n = 3. ^∗p < 0.0.5; ^∗∗p < 0.01 (Student’s t-test). The data shown are representative of three independent experiments.

FIGURE 2

NS4A inhibits polyI:C- induced type I IFN responses. (A) Immunoblots of the FLAG-tagged ZIKV protein expression in HEK293T cells. The proteins were detected by immunoblotting with an anti-FLAG antibody. Actin is a housekeeping protein control. The arrow indicates a non-specific band. The molecular weights in kDa are marked to the right of the blots. (B) Quantification of ISRE-Luc activity in HEK293T cells transfected with either 100 ng of empty vector or the indicated ZIKV gene expressing plasmid for 24 h and then stimulated with 10 μg/ml of heavy molecular weight polyI:C-H for 16 h. Data are expressed as fold induction over unstimulated cells. (C) Quantification of ISRE-Luc activity by a dual luciferase reporter assay similar to B except that an increasing dose of vector and FLAG-NS4A plasmids was applied. (D) Quantification of ISG15 and IFIT1 mRNA by q-PCR in HEK293T cells transfected with either 100 ng of empty vector or NS4A, and then stimulated with polyI:C as in B. The bars/data points in B–D represent the mean + SEM of the results, n = 3. ^∗p < 0.0.5; ^∗∗p < 0.01 (Student’s t-test). The data shown are representative of three independent experiments.

FIGURE 3

NS4A does not interfere with TLR3 or TLR4 signaling. (A) Quantification of ISRE-Luc activity in HEK293T cells transfected with Myc vector + FLAG-TLR3 or Myc-NS4A + FLAG-TLR3 (100 ng each) for 16 h and then stimulated with 20 μg/ml low molecular weight polyI:C-L (no transfection) for 24 h. (B) Quantification of IFNB1 and TNFA mRNA by qPCR. The cells were treated similarly as in A except that polyI:C stimulation was 8 h. (C) Quantification of ISRE-Luc activity in stable TLR4/MD2/CD14-expressing HEK293 cells transfected with 100 ng of Myc vector or Myc-NS4A plasmid for 12 h and stimulated with 100 ng/ml of LPS for 24 h. (D) Quantification of IFNB1 and TNFA mRNA by qPCR. The cells were treated similarly as in C except that LPS stimulation was 8 h. The results are expressed as fold change over unstimulated cells. The bars represent the mean + SEM of the results, n = 3. Three biological replicates were pooled for qPCR. ^∗p < 0.0.5; ^∗∗p < 0.01 (Student’s t-test). The data shown are representative of three independent experiments.

FIGURE 4

NS4A interferes with RIG-I and MDA5-induced type I interferons. Quantification of ISRE-Luc activity in HEK293T cells transfected with 100 ng of FLAG vector or FLAG-NS4A of A ZIKV FS13025 and B FLR strain, together with the indicated individual immune genes of the RLR pathway for 24 h. The immunoblots under the bar graph show the FLAG-NS4A and actin protein expression. (C) Quantification of type I IFN bioavailability in the culture media of A. (D) Immunoblots of FLAG-ΔRIG-I, MDA5, and MAVS proteins in HEK293T cells treated exactly as in A. The FLAG-tagged protein was detected with an anti-FLAG antibody. The bars in A–C represent the mean + SEM of the results, n = 3. ^∗p < 0.0.5; ^∗∗p < 0.01 (Student’s t-test). The data shown are representative of three independent experiments.

FIGURE 5

NS4A interacts with MAVS and blocks RLR binding to MAVS. (A) Co-immunoprecipitation (co-IP) of MAVS with NS4A from HEK293T cells transfected with Myc-NS4A and the indicated FLAG-tagged genes using an anti-FLAG monoclonal antibody, followed by immunoblotting (IB). WCE, whole-cell extract. (B) co-IP of the truncated forms of MAVS with NS4A from HEK293T cells transfected with Myc-NS4A and FLAG-tagged MAVS mutants using an anti-FLAG monoclonal antibody. co-IP of (C) FLAG-MDA5 or (D) FLAG-ΔRIG-I with endogenous MAVS from HEK293T cells transfected with FLAG-MDA5 or FLAG-ΔRIG-I and Myc vector or increasing amounts of Myc-NS4A. The plasmid DNA molar ratio of Myc- NS4A to FLAG-MDA5 is 1:3; to FLAG-ΔRIG-I are 1:6, 1:3, and 1:2. (E) Immunofluorescent staining of endogenous MAVS and MDA5 in human trophoblasts. Trophoblasts were untreated (Mock) or transfected with either a FLAG vector or FLAG-NS4A for 24 h and then stimulated with 20 μg/ml of heavy molecular weight polyI:C-H for 8 h. MDA5/MAVS was stained with a rabbit anti-MDA5 and mouse anti-MAVS antibody followed by secondary antibodies conjugated with Alexa Fluor 594/488. The nuclei were counter-stained with DAPI. The images were acquired using an inverted Nikon Eclipse Ti fluorescence microscope. The arrows indicate colocalizations of MDA5 and MAVS. In (A–C) actin is a housekeeping protein control. The arrow heads point to non-specific bands. The data shown are representative of three independent experiments.