Chikungunya virus antagonizes cGAS-STING mediated type-I interferon responses by degrading cGAS

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus known to cause epidemics resulting in predominantly symptomatic infections, which in rare cases cause long term debilitating arthritis and arthralgia. Significant progress has been made in understanding the roles of canonical RNA sensing pathways in the host recognition of CHIKV; however, less is known regarding antagonism of CHIKV by cytosolic DNA sensing pathways like that of cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING). With the use of cGAS or STING null cells we demonstrate that the pathway restricts CHIKV replication in fibroblasts and immune cells. We show that DNA accumulates in the cytoplasm of infected cells and that CHIKV blocks DNA dependent IFN-β transcription. This antagonism of DNA sensing is via an early autophagy-mediated degradation of cGAS and expression of the CHIKV capsid protein is sufficient to induce cGAS degradation. Furthermore, we identify an interaction of CHIKV nsP1 with STING and map the interaction to 23 residues in the cytosolic loop of the adaptor protein. This interaction stabilizes the viral protein and increases the level of palmitoylated nsP1 in cells. Together, this work supports previous publications highlighting the relevance of the cGAS-STING pathway in the early detection of (+)ssRNA viruses and provides direct evidence that CHIKV interacts with and antagonizes cGAS-STING signaling.

Fig 1. CHIKV inhibits DNA induced type-I Interferon transcription in primary human cells.

(A) HFF-1s were infected with CHIKV 181/25 (MOI = 0.1) and supernatants were plaqued on BHKs at indicated timepoints. (B-D) RT-qPCR from CHIKV growth curve in (A) for specified genes (Ifnb, Isg15, and Tnfa) relative to rps11 at 12 hpi. Data representative of three independent experiments. Data represented as means ± SD (n = 3). Statistical analysis was performed with by a one-way ANOVA with Tukey’s multiple comparisons. (E) NDV viral RNA relative to rps11 from infected HFF-1s (MOI = 0.1) at indicated timepoints. Data represented as means ± SD (n = 3). (F) Diagram of coinfection experiments. Briefly, HFF-1s were infected with either mock, UV-inactivated CHIKV 181/25 (UVC), or CHIKV 181/25 at an MOI of 5.0. Infections were allowed to proceed for 6 hrs in order to allow for viral protein expression. (G & I) 6 hpi, cells were then treated with mock, MVA at an MOI of 2.0, or E. coli DNA (1ug/2.0X10⁵ cells). 6 and 12 hrs postsecondary treatment, cell lysates were collected. Ifnß transcripts were quantified via RT-qPCR for cells stimulated with MVA or E. coli DNA, respectively, while quantification of Isg15 transcripts was performed as previously stated (G & I). Transcripts are represented as “fold over respective mock” E.G. UVC ➔ MVA condition was normalized to UVC ➔ mock condition to determine the relative gene induction resultant from secondary treatment. (H) Quantification of MVA transcripts at 12hrs post infection. (G-I) Data representative of two independent experiments (n = 3). Data are represented by means ± SD (n = 3). Statistical analysis was done with student’s t tests. Statistical significance represented as follows: ns = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.

Fig 2. CHIKV infection results in extra-nuclear DNA in primary cells.

HFF-1s were infected with either mock or different MOIs of CHIKV 181/25 (low = 1.0, high = 10.0). 24 hpi cells were fixed with 4% formaldehyde and permeabilized with 0.01% triton-X before staining with antibodies specific for nsP2 (red), DNA (green), or DAPI (blue). (A) HFF-1s infected at different MOIs of CHIKV were visualized using a Zeiss LSM 880 with Airyscan. Scale bars = 100um. (B) HFF-1s infected with CHIKV (MOI = 10.0) for 24hrs. Imaging performed with a Zeiss LSM 880 with Airyscan and 3D inset image was generated via reconstruction of Z-Stacks with Zen Blue software. Scale bars: 50um (2D), 50 Pixels (3D of inset). Data are representative of three independent experiments.

Fig 3. cGAS and STING restrict CHIKV replication and infectious particle release.

(A) WT or GT MEFs we lysed and analyzed via SDS-PAGE and immunoblotting. (B) WT and GT MEFs infected with CHIKV 181/25 (MOI = 0.1) for 16hr. infectious particles in the supernatant were quantified via plaque assay. (C) RT-qPCR of viral RNA (nsp2) relative to 18S and b-actin. (A-C) representative of three independent experiments. Data are represented by means ± SD (n = 3). Statistical analysis was done with student’s t tests. (D) WT, cGAS KO, or STING KO RAW 264.7 cells were infected with CHIKV 181/25 (MOI = 0.1) and supernatants were collected at indicated timepoints. Infectious particle release in the supernatant was quantified via plaque assay. Data representative of three independent experiments. Data are represented by means ± SD (n = 3). Statistical analysis was done via two-way ANOVA with Tukey’s multiple comparisons. Statistical significance represented as follows: ns = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001.

Fig 4

(A) HFF-1s were infected with either mock or CHIKV 181/25 at an MOI of 0.1 and cell lysates were collected 4, 12, and 24 hpi. Analysis of protein from mock or CHIKV infected cells was performed via SDS-PAGE followed by immunoblotting for indicated proteins (data representative of three independent experiments). (B) RT-qPCR for cgas transcripts from HFF-1s infected with CHIKV (MOI 0.1), NDVB1 (MOI 0.1), or mock at 4hpi (data representative of three independent experiments. Data are represented by means ± SD (n = 3). Statistical analysis was done with student’s t tests. (C) HFF-1s treated with mock or CHX at 200 μg/ml for 5hrs then treated with mock or puromycin at 10 μg/ml for 15 min. After 15 min puromycin pulse, cells were washed with dPBS and re-fed complete media. Lysates collected and visualized via SDS-PAGE and immunoblotting (data representative of two independent experiments). (D) HFF-1s were infected with mock or CHIKV at an MOI of 1.0. 45 min prior to indicated timepoints, cells were pulsed with puromycin as described in (C). Lysates were collected and proteins were detected as previously stated (data representative of two independent experiments). (E) HEK-293Ts were transfected with indicated constructs at indicated plasmid amounts. Cells were allowed to rest for 24 hr post transfection before lysis and SDS-PAGE/immunoblotting analysis (data are representative of three independent experiments). (F) 293T-IFNb-FFluc cells were transfected with empty vector (Vec), cGAS and STING in conjunction with Vec, or cGAS and STING with the capsid of CHIKV 181/25. Cells were allowed to rest for 36hrs before lysis for collection of protein or quantification of luminescence. data are representative of six independent experiments. Data represented as fold induction over Vector alone. Data are represented by means ± SD (n = 3). Statistical analysis was done with student’s t tests (** = p<0.01)). (G) Protein input for IFN reporter assay in (F).

Fig 5

(A) 293Ts transfected with 200 ng cGAS for 16hrs then infected with CHIKV at an MOI of 10.0. Infections were allowed to proceed for 10 hrs before treatment with 3-MA at 5 mM. Cells were allowed to rest for 8 hrs after drug treatment and were lysed. Lysates were visualized via SDS-PAGE followed by immunoblotting with indicated antibodies. (Data representative of three independent experiments). (B) U2OS cells were transfected with 10 nm siRNA (control or ATG7) for 48 hrs and infected 24 hpt with CHIKV (MOI = 10) or mock infection. Cells were collected and lysed 24 hpi. Analysis by western blot using indicated antibodies. (C) Fold change in cGAS expression relative to respective non-targeting siRNA controls and ATG7 expression via densitometry analysis of western blot bands in figure (n = 2) (B). (D) Quantification of ATG7 knockdown in U2OS cells from western shown in figure (B) (n = 2). (E) Plaque assay of supernatants collected from infected U2OS cells at 24hrs post infection (n = 3) Data are represented as means ± SD. (B-E) Data representative of two independent experiments. Statistical analysis was done with student’s t test.

Fig 6. CHIKV nsP1 interacts with and is stabilized by STING.

(A) Flag-tagged STING was co-expressed in HEK-293T cells, individually with nsPs 1–4 of CHIKV-RT. 24 hpt, cells were lysed and an immunoprecipitation preformed against the Flag-epitope. Protein interactions were visualized via SDS-PAGE followed by immunoblotting (data representative of two independent experiments). (B) HEK-293T cells were transfected with indicated constructs and allowed to rest for 16 hrs. After resting, cells were infected with either mock or CHIKV 181/25 (MOI = 5.0). 12 hpi cells were lysed and an immunoprecipitation preformed against a Flag epitope. Protein interaction was analyzed via SDS-PAGE followed by immunoblotting (data representative of two independent experiments). (C) Diagram of STING deletion constructs used for mapping nsP1-STING interactions. (D) HEK-293Ts were transfected with CHIKV-RT nsP1 and the different STING mutants indicated in (C) and cells were lysed 24 hrs post transfection. An immunoprecipitation was performed against the Flag epitope and protein samples were then analyzed via SDS-PAGE and immunoblotting performed as described previously (data representative of two independent experiments). (E) Schematic of STING inserted in the ER membrane highlighting regions deleted which are located in cytosolic facing domains (schematic representative of poor artistic skill). (F) 293T cells were transfected with internal deletion STING constructs and nsP1. Cells were lysed 24 hpt and an anti-flag IP was performed followed by SDS-PAGE and immunoblotting (data representative of three independent experiments). (G) HEK-293T cells were transfected cells with indicated constructs overnight followed by treatment with 100 uM 2-BP for 24 h. Cells were lysed and analyzed via Western blotting. (H) HEK-293T cells were transfected with indicated constructs overnight and treated for 1 h with 20 uM alk-16 palmitoylation chemical reporter reagent prior to cell lysis. Immunoprecipitation was performed against the HA epitope followed by click chemistry reaction with azido-rhodamine for visualization of protein palmitoylation via fluorescence gel scanning. (G & H) data representative of two independent experiments.