Herpes Simplex Virus 1 Tegument Protein VP22 Abrogates cGAS/STING-Mediated Antiviral Innate Immunity

Abstract

Cytosolic DNA arising from intracellular pathogens is sensed by cyclic GMP-AMP synthase (cGAS) and triggers a powerful innate immune response. However, herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, has developed multiple mechanisms to attenuate host antiviral machinery and facilitate viral infection and replication. In the present study, we found that HSV-1 tegument protein VP22 acts as an inhibitor of cGAS/stimulator of interferon genes (cGAS/STING)-mediated production of interferon (IFN) and its downstream antiviral genes. Our results showed that ectopic expression of VP22 decreased cGAS/STING-mediated IFN-β promoter activation and IFN-β production. Infection with wild-type (WT) HSV-1, but not VP22-deficient virus (ΔVP22), inhibited immunostimulatory DNA (ISD)-induced activation of the IFN signaling pathway. Further study showed that VP22 interacted with cGAS and inhibited the enzymatic activity of cGAS. In addition, stable knockdown of cGAS facilitated the replication of ΔVP22 virus but not the WT. In summary, our findings indicate that HSV-1 VP22 acts as an antagonist of IFN signaling to persistently evade host innate antiviral responses.IMPORTANCE cGAS is very important for host defense against viral infection, and many viruses have evolved ways to target cGAS and successfully evade the attack by the immune system of their susceptible host. This study demonstrated that HSV-1 tegument protein VP22 counteracts the cGAS/STING-mediated DNA-sensing antiviral innate immunity signaling pathway by inhibiting the enzymatic activity of cGAS. The findings in this study will expand our understanding of the interaction between HSV-1 replication and the host DNA-sensing signaling pathway.

Keywords: DNA sensing; HSV-1; VP22; cGAS.

FIG 1

HSV-1 VP22 inhibits IFN-β activation induced by cGAS/STING. (A) HEK293T cells were cotransfected with IFN-β promoter plasmid (IFN-β-Luc, 200 ng), Renilla luciferase (pRL-TK, 50 ng) reporter plasmid, cGAS-Flag (15 ng), and STING-HA (2.5 ng), along with empty vector (200 ng) or VP22-Flag plasmid (200 ng). Luciferase activity was measured 24 h posttransfection in the cell lysates. (B) HEK293T cells were cotransfected with cGAS-Flag and STING-HA, along with empty vector or VP22-Flag plasmid. At 24 h posttransfection, cells were harvested and subjected to qRT-PCR analysis. (C) Effects of VP22 deficiency on transcription of downstream antiviral genes. HFF cells were infected with WT (MOI = 1) or ΔVP22 (MOI = 1) virus for the indicated times before qRT-PCR analysis. (D and E) HFF cells were infected with WT or ΔVP22 virus at an MOI of 5 for 2 h, cell medium was then replaced by DMEM containing 10% FBS, and ISD (2 μg/ml) was transfected using Lipofectamine LTX according to the manufacturer's recommendations. Cells were harvested and subjected to qRT-PCR analysis at 7 h posttransfection (D) or ELISA analysis at 18 h posttransfection (E). (F and G) HFF cells were infected and transfected as described for panel D, and then cells were harvested at 7 h posttransfection and subjected to WB analysis to detect IRF3 phosphorylation (F) or native PAGE to detect IRF3 dimerization (G). The data represent results from one of triplicate experiments. Error bars represent the standard deviation of results of three independent experiments. Statistical analysis was performed using Student's t test with the GraphPad Prism 5.0 software. (*, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001).

FIG 2

VP22 inhibits the IFN signaling pathway at the level upstream of STING. (A to C) HEK293T cells were cotransfected with IFN-β-Luc reporter, pRL-TK, and STING (A), TBK1 (B), or IRF3/5D (C), along with empty vector or VP22-Flag plasmid. Cells were harvested 24 h after transfection and subjected to a DLR assay. The data represent results from one of triplicate experiments. Error bars represent the standard deviation of results of three independent experiments. Statistical analysis was performed using Student's t test with the GraphPad Prism 5.0 software.

FIG 3

VP22 interacts with cGAS and inhibits the enzymatic activity of cGAS. (A) Colocalization of VP22 with cGAS. HeLa cells were transfected with VP22-EYFP and cGAS-Flag for 20 h, as indicated. Cells were stained with mouse anti-Flag MAb, and TRITC (tetramethyl rhodamine isothiocyanate)-conjugated goat anti-mouse antibody was used as the secondary antibody before confocal microscopy. DAPI, 4′,6-diamidino-2-phenylindole. (B) VP22 interacts with cGAS but not STING. HEK293T cells were transfected with VP22-HA along with cGAS-Flag or STING-Flag for 24 h. HEK293T cells were transfected with UL42-HA and cGAS-Flag as a negative control. The cells were lysed, and the extracts were subjected to IP with anti-Flag MAb or control IgG. Precipitates were analyzed by WB. IB, immunoblotting. (C and D) HFF cells were infected with WT or ΔVP22 virus at an MOI of 1 for 24 h (C) or were infected with WT or ΔVP22 virus at an MOI of 5 for 2 h before ISD transfection (D). Extracts from cells were prepared, DNase treated, heat treated, and incubated with permeabilized HFF cells for 6 h. Cells were then harvested and subjected to qRT-PCR analysis. (E) VP22 inhibits the dimerization of STING. HFF cells were infected with WT or ΔVP22 virus at an MOI of 5 for 2 h, cell medium was then replaced with DMEM containing 10% FBS, and ISD (2 μg/ml) was transfected using Lipofectamine LTX according to the manufacturer's recommendations; cells were then harvested at 7 h posttransfection and subjected to native PAGE to detect STING dimerization. The data represent results from one of triplicate experiments. Error bars represent the standard deviation of results of three independent experiments. Statistical analysis was performed using Student's t test with GraphPad Prism 5.0 software. (**, 0.001 < P < 0.01).

FIG 4

cGAS mediates the defense against replication of ΔVP22 virus. (A to D) The stably transfected HFF-shNC and HFF-shcGAS cells were infected with WT or ΔVP22 virus at an MOI of 2, and then the virus was harvested at the indicated time points postinfection and subjected to WB analysis (A and C) or viral plaque assay on Vero cells (B and D). The data represent results from one of the triplicate experiments. Statistical analysis was performed using Student's t test with GraphPad Prism 5.0 software. (* 0.01 < P < 0.05).