Research ArticleImmunology

LGP2 binds to PACT to regulate RIG-I– and MDA5-mediated antiviral responses

See allHide authors and affiliations

Science Signaling  01 Oct 2019:
Vol. 12, Issue 601, eaar3993
DOI: 10.1126/scisignal.aar3993
  • Fig. 1 ST-LGP2 cells exhibit inhibition of the type I IFN response to RIG-I–specific ligands but amplify the response to MDA5-specific ligands.

    (A) Workflow for generating the ST-LGP2 cell line. (B and C) Measurement of the IFN-β (B) and NF-κB (C) promoter responses by luciferase assay in ST-LGP2 cells (green) and HEK293 cells (black) upon transfection with 100 ng each of the indicated synthetic RLR ligands. HEK293 cells were included as a reference control. Data are means ± SEM of five independent experiments. The asterisk indicates a significant difference. ns, no significant difference. a.u., arbitrary units. (D to F) Measurement of the responsiveness of ST-LGP2 cells to natural RLR ligands. (D) Purification protocol of RLR-specific natural ligands. HeLa cells were infected (at an MOI of 1) with MV, Mengo virus, or Cox virus or were left uninfected (mock). Infection with picornaviruses was performed in the presence of DMSO (as a control) or 1 mM ribavirin (step 1). Total RNAs were then purified and used to transfect ST-LGP2 cells together with the IFN-β reporter plasmid before being subjected to luciferase assays (step 2). (E) Comparative analysis of IFN-β promoter responses in ST-LGP2 cells (green) and HEK293 cells (black) that were transfected with 20 or 100 ng of total RNA isolated from cells infected with the indicated viruses. (F) Comparative analysis of IFN-β promoter responses in the indicated cells after transfection with 100 ng of total RNA isolated from cells infected with the indicated UV-inactivated viruses. Luciferase assays were preformed, and data were analyzed as described for (B). Data are means ± SEM of three (E) and two (F) independent experiments. See table S1 for all details on statistical analysis.

  • Fig. 2 Proteomic analysis of LGP2-specific host protein partners.

    (A) Workflow used to purify LGP2-associated cellular proteins. ST-LGP2 was copurified with interacting cellular partners using STrEP-Tactin beads. Protein complexes were released from the beads and analyzed by direct nano–LC-MS/MS. Nonspecific binding was accounted for by using MS results for ST-CH protein (negative control) and our filtered protocol (fig. S1B). Three biological replicates were obtained and analyzed. (B) LGP2-specific protein cocomplex. Proteins are represented according to their gene symbol and colors depict the corresponding GO term. For the corresponding gene name and protein score for each experimental replicate (see table S2). The P value was obtained with the Fisher exact test, whereas the corrected P value was determined with the Bonferroni test. ncRNA, noncoding RNA.

  • Fig. 3 Validation of the protein-protein interaction between LGP2 and PACT.

    (A) LGP2-specific binding to DICER1, PKR, and PACT. ST-LGP2 cells and ST-CH cells were infected with MV at an MOI of 1 (+) or were left uninfected (−). Twenty-four hours after infection, total cell lysates (INPUT) were subjected to affinity purification with STrEP-Tactin beads (OUTPUT). Both sets of samples were then analyzed by Western blotting with specific antibodies against indicated proteins. Three biological replicates were analyzed. Blots from one representative experiment are shown. (B) The LGP2-specific interaction with PACT persists in the presence of RNase. ST-LGP2 cells and ST-CH cells were infected with MV at an MOI of 1 (+) or were left uninfected (−). Twenty-four hours later, total cell lysates (INPUT) were subjected to affinity purification with STrEP-Tactin beads in the absence or presence of either RNaseA or RNaseIII (OUTPUT). Samples were then analyzed by Western blotting with antibodies against PACT, the STrEP tag, and actin. Two biological replicates were analyzed. Blots from one representative experiment are shown.

  • Fig. 4 Additional validation of the LGP2-PACT interaction.

    (A) Schematic representation of the PCA. Protein A (prey) is fused to the N terminus of the Gaussia luciferase (GL1). Protein B (bait) is fused to the C terminus of the Gaussia luciferase (GL2). If protein A interacts with protein B, the luciferase activity is rescued and the normalized luciferase ratio (NLR) is >3.5. (B) Schematic representation of the organization of the RIG-I, RIG-I CTD (RCTD), LGP2, LGP2 CTD (LCTD), and PACT proteins that were used for the PCA. (C) PACT interacts with the CTD of RIG-I and LGP2. HEK293T cells were cotransfected with the plasmids GL1 and GL2 expressing the indicated proteins, and PCA was performed and luciferase measurements were made. Black columns indicate where protein A was fused to GL1 and protein B was fused to GL2, whereas gray columns indicate where protein A was fused to GL2 and protein B was fused to GL1. Data are means ± SEM of three independent experiments. (D) Binding of PACT protein to RLRs. Left: ELISA plates were coated with PACT and incubated with similar concentrations of MDA5, RIG-I, and LGP2. Cherry (CH) was used as a negative control. Bound RLRs were detected with an antibody against the Strep tag. One representative experiment from a series of four is shown. Nonspecific binding of RLRs to the plates was subtracted. Data are means ± SD of technical duplicates. Right: The indicated samples were analyzed by Western blotting with antibody against the STrEP tag to normalize the amounts of RLRs used for the ELISAs. dsRBD, dsRNA binding domain.

  • Fig. 5 The LGP2-PACT interaction is required for modulating RLR signaling activity.

    (A) Structure of the human LGP2 CTD (Protein Data Bank: 2w4r). The domain is colored in yellow. The residues C556, C559, and C612 from the zinc finger domain are colored in blue; C615 is colored in red; the loop between C612 and C615 is colored in cyan; and the zinc atom is in gray. (B) The LGP2-C615A mutant fails to interact with PACT. HEK293T cells were cotransfected with the plasmids GL1 and GL2 expressing the indicated proteins, and PCA was performed and luciferase measurements were made. Black columns indicate where protein A was fused to GL1 and protein B was fused to GL2, whereas gray columns indicate where protein A was fused to GL2 and protein B was fused to GL1. Data are means ± SEM of two independent experiments. (C and D) Characterization of the ST-LGP2–C615A cell line. (C) Analysis of the amounts of LGP2 mRNA relative to that of GAPDH mRNA in the indicated cells. Data are from three biological replicates. (D) Analysis of the binding of LGP2-C615A to PACT. Lysates of the indicated cells (INPUT) were subjected to affinity purification with STrEP-Tactin beads (OUTPUT). The samples were then analyzed by Western blotting with antibodies against STrEP tag, PACT, and β-actin. One representative experiment of three biological replicates is shown. (E and F) Comparative analysis of IFN-β promoter responses to RIG-I ligands. The indicated cells were transfected with 20, 100, and 200 ng of 5′3P-RNA (E) or with RNA isolated from MV-infected HeLa cells (F) before being analyzed by luciferase assay. Data are means ± SEM of three independent experiments. (G and H) Comparative analysis of the relative abundances of IFN-β (G) and IFIT1 (H) mRNAs upon infection of the indicated cell lines with MV. Data are means ± SEM of four independent experiments. See table S1 for all details on statistical analysis. Asterisks indicate statistical significance. ns, not significant.

  • Fig. 6 The LGP2-PACT interaction drives the synergy between LGP2 and MDA5.

    (A) Both WT LGP2 and the LGP2-C615A mutant interact with MDA5. HEK293T cells were cotransfected with the plasmids GL1 and GL2 expressing the indicated proteins, and PCA was performed and luciferase measurements were made. Black columns indicate where protein A was fused to GL1 and protein B was fused to GL2, whereas gray columns indicate where protein A was fused to GL2 and protein B was fused to GL1. Data are means ± SEM of three independent experiments. Samples were analyzed in triplicate. (B) ST-LGP2 cells were transfected with 0, 40, or 100 ng of pCI-neo encoding PACT and 100 ng of total RNA from Mengo- or Cox-infected HeLa cells or 100 ng of HMW poly(I:C) before being analyzed by luciferase assay. Data are means ± SEM of two independent experiments. (C) Comparative analysis of IFN-β promoter responses to MDA5 ligands. The indicated cells were transfected with 20, 100, or 200 ng of HMW poly(I:C) and then analyzed by luciferase assay. Data are means ± SEM of three independent experiments. (D) An ISRE reporter cell line was transfected with pCI-neo plasmids encoding LGP2 (20 ng), RIG-I (20 ng), or MDA5 (20 ng), alone or in the indicated combinations, before promoter activities were measured. Data are means ± SEM of three independent experiments. (E) An ISRE reporter cell line was cotransfected with pCI-neo plasmids encoding MDA5 (25 ng), either WT LGP2 or the LGP2-C615A mutant (0, 5, 25, 50, or 100 ng), and PACT (0, 5, 50, or 100 ng) before promoter activities were measured. Data are means ± SEM of four independent experiments. See table S1 for all details on statistical analysis. Asterisks indicate statistical significance. ns, not significant.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/601/eaar3993/DC1

    Fig. S1. Proteomics approach to study LGP2-specific protein partners.

    Fig. S2. Assessment of RNA integrity within LGP2-protein complexes upon purification in the presence of RNaseA and RNaseIII.

    Fig. S3. PCA analysis of the binding of RIG-I and MDA5 to PACT in the presence of LGP2.

    Fig. S4. PCA analysis of the binding of the LGP2-C615A mutant with a known protein and with RNA binding partners of LGP2.

    Table S1. Statistical analyses.

    Table S2. List of direct and indirect LGP2-specific cellular partners identified by MS.

    Table S3. List of primers.

  • This PDF file includes:

    • Fig. S1. Proteomics approach to study LGP2-specific protein partners.
    • Fig. S2. Assessment of RNA integrity within LGP2-protein complexes upon purification in the presence of RNaseA and RNaseIII.
    • Fig. S3. PCA analysis of the binding of RIG-I and MDA5 to PACT in the presence of LGP2.
    • Fig. S4. PCA analysis of the binding of the LGP2-C615A mutant with a known protein and with RNA binding partners of LGP2.
    • Table S1. Statistical analyses.
    • Table S2. List of direct and indirect LGP2-specific cellular partners identified by MS.
    • Table S3. List of primers.

    [Download PDF]

Stay Connected to Science Signaling

Navigate This Article