A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling

Abstract

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X(3)-Trp-X(9)-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

Fig. 1.

Effects of V and P proteins from different genera of the Paramyxovirinae subfamily on TLR7/9-dependent signaling. 293T (A, C, and E), 293XL-mTLR7 (B), or U3A cells (D) (∼1.5 × 10⁵) were transfected with an IFN-α6 promoter-driven reporter plasmid (80 ng), an internal control plasmid (pRL-TK, 10 ng; Promega), and the indicated plasmids (MyD88 [25 ng], TRAF6 [25 ng], IKKα [12.5 ng], IRF7 [15 ng], IPS-1 [10 ng], M [50 ng], P [50 ng], V [50 ng], and NS3/4A [50 ng]). The total mass of transfected DNA was held constant by including the appropriate amount of the empty pCA7 plasmid. (B) 293XL-mTLR7 cells were treated with TLR7 ligand Gardiquimod (100 ng/ml) at 24 h posttransfection. Cells were lysed at 24 (A, C, D, and E) or 48 (B) h posttransfection, and activation of the IFN-α6 promoter was determined by a dual-luciferase reporter assay system (Promega). Data are derived from three independent experiments and are represented by mean values of the relative luciferase activities. Standard deviations are shown as error bars. The IFN-α6 promoter sequence and all the signaling molecule genes on the plasmids are of mouse origin unless otherwise noted. Viral sequences on the plasmids are derived from the SeV Z strain, bPIV3 910N strain, and hPIV2 Toshiba strain. hIRF7, human IRF7; hMyD88, human MyD88; hTRAF6, human TRAF6; hIKKα, human IKKα.

Fig. 2.

Interaction of paramyxoviral V proteins with IRF7. 293T cells (∼7.5 × 10⁵) were transfected with the indicated plasmids. Cells were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1% Triton X-100) at 24 h posttransfection. Flag-tagged proteins (A, C) or V5-tagged proteins (B) were immunoprecipitated (IP) with the anti-Flag antibody or anti-V5 antibody, respectively, and subjected to immunoblot analysis (IB) with the anti-V5 (A, C) or anti-Flag (B) antibody. Whole-cell lysates were also subjected to immunoblot analysis. Asterisks indicate positions of the antibody heavy chain. Plasmids expressing a tagged protein were created by appending each epitope tag to the N-terminal ends of proteins. IRF7, mouse IRF7; hIRF7, human IRF7.

Fig. 3.

V protein binds to the inhibitory domain of IRF7. (A) Schematic diagram of Flag-tagged truncated mutants of mouse IRF7. IRF7 C1, aa 411 to 457; IRF7 C2, aa 238 to 457; IRF7 C3, aa 132 to 457; IRF7 N1, aa 1 to 131; IRF7 N2, aa 1 to 237; IRF7 N3, aa 1 to 410; IRF7 ΔID, N2 fused to C1; IRF7 ID, aa 238 to 410. (B, C, and D) 293T cells (∼7.5 × 10⁵) were transfected with the indicated plasmids and lysed at 24 h posttransfection. Flag-tagged proteins were immunoprecipitated with the anti-Flag antibody and subjected to immunoblot analysis with the anti-V5 (B and D) or anti-Myc (C) antibody. Whole-cell lysates were also subjected to immunoblot analysis. (E) 293T cells were transfected with an IFN-α6 promoter-driven reporter plasmid, pRL-TK, and the indicated plasmids. Cells were lysed at 24 h posttransfection. Activation of the IFN-α6 promoter was determined by dual-luciferase assay. Data are derived from three independent experiments and are represented as mean values. Standard deviations are shown as error bars.

Fig. 4.

The Trp-rich motif in V protein is essential for the interaction with IRF7 as well as the blockade of TLR7/9-dependent signaling. (A) Amino acid sequence alignment of the Cys-rich and Trp-rich C-terminal regions of paramyxovirus V proteins. Conserved His and Cys residues forming the zinc finger-like motif are shaded. Positions (C1 to C6) of the Cys residues are also shown. W1, W2, and W3 indicate positions of Trp residues in the Trp-rich motif. (B, D, and F) 293T cells were transfected with the indicated plasmids. Cells were lysed at 24 h posttransfection. Flag-tagged proteins were immunoprecipitated with anti-Flag antibody and subjected to immunoblot analysis with anti-V5 antibody. Relative intensities of coprecipitated IRF7 bands (%IRF7) are also shown. Whole-cell lysates were also subjected to immunoblot analysis. Asterisks indicate positions of the heavy chain of the anti-Flag antibody (Ab HC). (C, E, and G) 293T cells were transfected with expression plasmids (MyD88, TRAF6, IKKα, and IRF7), an IFN-α6 promoter-driven reporter, pRL-TK, and the indicated plasmid. Cells were lysed at 24 h posttransfection. Activation of the IFN-α6 promoter was determined by dual-luciferase assay. Data are derived from three independent experiments and are represented as mean values. Standard deviations are shown as error bars. V_C12, V(C193A C197A); V_C345, V(C209A C211A C214A); V_C6, V(C221A); V_W123, V(W178H W182E W192A); V_W1, V(W178H); V_W2, V(W182E); V_W3, V(W192A); MeV V_W2, MeV V(W240E); NiV V_W2, NiV V(W416E).