Dysregulation of RIG-I activation by picornavirus 3A protein

Figures

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  Fig 1. Inhibition of IFN-β and ISGs production by the SVA 3A protein. (A–C) HEK-293T cells were transfected with the indicated reporter plasmids (50 ng each): IFN-β (A), ISRE (B), and NF-κB (C), the PRL-TK control reporter (5 ng), and increased doses (0, 100, 200, or 400 ng) of plasmids expressing SVA 3A for 24 h, followed by treating with Sendai virus (SeV) for 12 h. Cell lysates were collected for measuring luciferase activity using a dual luciferase assay. (D and E) HEK-293T cells were transfected with an empty vector or SVA 3A expressing plasmids for 24 h, prior to treatment with SeV for 12 h. Cell lysates were processed to detect IFN-β mRNA levels by qPCR (D), and the supernatants were collected to measure IFN-β protein levels (E). (F) HEK-293T cells were transfected with an empty vector or a plasmid expressing SVA 3A. After 24 h, the transfected cells were treated with SeV for another 12 h. Cell lysate was harvested to evaluate the mRNA levels of ISGs, including ISG15, ISG54, GBP1, TNF-α, IL-6, and IL-1β, using qPCR. (G) PK-15 cells were transfected with an empty vector or a plasmid expressing SVA 3A for 24 h, followed by transfection with poly(I:C) for 12 h. The mRNA levels of…

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  Fig 2. SVA 3A protein interacts with RIG-I. (A) HEK-293T cells were co-transfected with plasmids expressing individual RLR signaling pathway molecules expressing plasmids (RIG-I-CARD, RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7; 100 ng each) and SVA 3A expressing plasmid (100 ng) or empty vector (100 ng), along with ISRE reporter plasmids (50 ng) and PRL-TK control reporter plasmids (5 ng). At 24 h post-transfection, cell lysates were harvested to measure luciferase activity using a dual luciferase assay. (B) HEK-293T cells were transfected with increasing doses of SVA 3A expressing plasmid or empty vector (0, 75, 150, or 300 ng) and RIG-I-CARD expressing plasmid (100 ng), together with ISRE reporter plasmids (50 ng) and PRL-TK control reporter plasmids (5 ng) for 24 h. Cell lysates were harvested, and luciferase activity was assessed by dual luciferase assay. (C) HEK-293 cells were co-transfected with plasmids expressing RIG-I, MAVS, TBK1, IRF3, or IRF7 and SVA 3A for 24 h. Cell lysates were collected to do an immunoprecipitation assay using an anti-Flag antibody. Immunoprecipitation (IP) samples and whole-cell lysates (WCL) were analyzed by western blotting using the indicated antibodies.…

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  Fig 3. RIG-I inhibits SVA replication. (A–C) HEK-293T cells (A), PK-15 cells (B), or IBRS-2 cells (C) were transfected with SVA 3A plasmid (0, 0.25, and 0.5 µg) for 24 h, then inoculated with SVA at 0.1 MOI for 12 h. Cell lysates were collected for quantifying viral mRNA levels by qPCR and for detecting VP2 protein levels by western blotting. Supernatants were harvested to determine viral titers by TCID50. (D) HEK-293T cells were transfected with HA-tagged RIG-I plasmids (0, 0.25, and 0.5 µg) for 24 h, prior to SVA infection (0.1 MOI) for 12 h. Cell lysates were collected to quantify mRNA and protein levels of SVA, and the supernatants were harvested to determine viral titers by TCID50. (E) HEK-293T cells were transfected with a siRNA (5′-GAATTTAAAACCAGAATTATC-3′) targeting RIG-I. After 24 h, the cells were infected with SVA at 0.1 MOI for another 12 h. Cells were then harvested to assess the mRNA and protein levels of the virus, and supernatants were harvested to determine viral titers by TCID50. (F) RIG-I KO and WT PK-15 cells were infected with SVA (0.5 MOI) for 12 h, and the cell lysates were collected. The levels of viral RNA, viral protein, and infectious viral titers were…

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  Fig 4. SVA 3A reduces RIPLET-mediated ubiquitination of RIG-I. (A) HEK-293T cells were transfected with Myc-RIG-I, HA-MAVS, and increasing amounts of SVA 3A expressing plasmids for 24 h, followed with SeV stimulation for 12 h. Cell lysates were harvested to perform immunoprecipitation using an anti-Myc antibody. Immunoprecipitation (IP) samples and whole-cell lysates (WCL) were analyzed by western blotting with the indicated antibodies. (B) HEK-293T cells were transfected with Myc-RIG-I expressing plasmid and HA-tagged K63 ubiquitin plasmids, along with increasing doses of SVA 3A expressing plasmids for 24 h, followed with SeV stimulation for 12 h. Cell lysates were harvested to perform immunoprecipitation using an anti-Myc antibody. IP samples and WCL were analyzed by western blotting to assess the K63-linked ubiquitination of RIG-I. (C) PK-15 cells were mock- or poly (I:C)-treated for 6 h, then cells were infected with mock or SVA (0.5 MOI) for another 12 h to evaluate the ubiquitination status of RIG-I in the context of SVA infection. (D) HEK-293T cells were co-transfected with the indicated plasmid. At 24 h post-transfection, the transfected cells were treated with SeV for an…

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  Fig 5. SVA 3A interacts with the helicase and CARD domains of RIG-I. (A) Schematic of full-length RIG-I and its truncated mutants. CARD: Caspase Activation and Recruitment Domain; Hel: Helicase Domain; CTD: C-terminal Domain. (B) HEK-293T cells were transfected with plasmids expressing HA-tagged full-length RIG-I or its mutants and Flag-tagged SVA 3A expressing plasmids for 24 h. Cell lysates were collected for immunoprecipitation using an anti-Flag antibody. Immunoprecipitation (IP) samples and whole-cell lysates (WCL) were analyzed by western blotting using the indicated antibodies. (C) Schematic of full-length SVA 3A and its truncated mutants. (D) HEK-293T cells were transfected with the IFN-β reporter plasmids (50 ng), PRL-TK control reporter plasmids (5 ng), and plasmids expressing full-length SVA 3A or indicated truncated mutants for 24 h, followed by treatment with SeV for 12 h. Cell lysates were collected for measuring luciferase activity using dual luciferase assay. (E) HEK-293T cells were co-transfected with the ISRE reporter plasmids (50 ng), PRL-TK control reporter plasmids (5 ng), and plasmids expressing full-length SVA 3A or mutant, along with RIG-I CARD expressed plasmids…

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  Fig 6. The 3A proteins of EMCV, CA16, EV71, and FMDV suppress the RLR pathway transduction. (A–C) HEK-293T cells were transfected with the indicated reporter plasmids (50 ng each): IFN-β (A), ISRE (B), and NF-κB (C), PRL-TK control reporter (5 ng), and increased doses of plasmids expressing the indicated picornaviral 3A for 24 h, followed by treatment with SeV for 12 h. Cell lysates were collected for measuring luciferase activity using a dual luciferase assay. (E–G) HEK-293T cells were transfected with the indicated picornaviral 3A: EV71 (D), EMCV (E), FMDV (F), and CA16 (G) for 24 h, followed by SeV stimulation for 12 h. Cell lysates were collected to evaluate the mRNA levels of the indicated ISGs. All experiments were repeated at least three times. Statistical analysis: two-way ANOVA (A–C) and two-tailed, unpaired t-test (D–G).

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  Fig 7. The impacts of picornaviral 3A on RIG-I activity. (A) HEK-293T cells were transfected with an HA-RIG-I expressing plasmid and the indicated picornaviral 3A expressing plasmids. After 24 h, cell lysates were subjected to immunoprecipitation using anti-HA antibody and then analyzed by western blotting to evaluate the interaction. (B) HEK-293T cells were transfected with HA-RIG-I expressing the plasmid and the indicated picornaviral 3A expressing plasmids. After 24 h, cell lysates were analyzed by western blotting to evaluate the protein expression. (C) HEK-293T cells were transfected with a plasmid expressing Myc-RIG-I and HA-MAVS, together with increased amounts of the indicated picornaviral 3A expressing plasmids for 24 h, followed with SeV stimulation for another 12 h. Cell lysates were subjected to immunoprecipitation using anti-Myc antibody and then analyzed by western blotting to evaluate the interaction between RIG-I and MAVS. (D) HEK-293T cells were transfected with Myc-RIG-I expressing plasmid and HA-tagged K63 ubiquitin plasmids, along with the indicated picornaviral 3A expressing plasmids for 24 h. Then, the transfected cells were treated with SeV for an additional 12 h,…

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  Fig 8. Proposed models of the picornavirus 3A protein antagonizing the RLR signaling pathway by dysregulating RIG-I. When picornaviruses infect host cells, its genomic RNA is translated into a single polyprotein that is subsequently cleaved by viral proteases into nonstructural and structural proteins. Among these, the 3A protein contributes to the remodeling of host intracellular membranes to form replication organelles for viral RNA synthesis. At the same time, 3A also acts as an interferon antagonist by dysregulating RIG-I activity and impairing its ability to sense viral RNA, thereby suppressing activation of the host antiviral immune response. Picornaviral 3A employs similar yet distinct mechanisms to dysregulate RIG-I, including direct interaction, downregulation of its expression, and reduction of its ubiquitination.