FIG 7.

PRRSV Nsp1 and Nsp2 interact with LGP2. (A) HEK293T cells were cotransfected with mCherry-tagged Nsp1 and Myc-tagged LGP2 for 24 h. Cell lysates were immunoprecipitated with Myc or IgG antibodies, and the immunoblots are shown with mCherry and Myc antibodies. IgG is a control. (B) HEK293T cells were cotransfected with mCherry-tagged Nsp2 and Myc-tagged LGP2 for 24 h. Cell lysates were immunoprecipitated with Myc or IgG antibodies, and the immunoblots are shown with mCherry and Myc antibodies. IgG is a control. (C) PAMs mock infected or infected with PRRSV (MOI = 1) for 24 h. Cell lysates were immunoprecipitated with LGP2 antibody, and the immunoblots with Nsp2 and LGP2 antibodies are shown. GAPDH is shown as an internal control. (D) Schematic diagrams of the full-length Nsp2 (amino acids 1 to 1196) and Nsp2 fragments (Nsp2-N, amino acids 1 to 405; Nsp2-M, amino acids 323 to 844), with an mCherry tag at the position of N terminus. HV, hypervariable region; TM, transmembrane domain; Nsp2-N, Nsp2 N-terminal; Nsp2-M, Nsp2 middle. (E) Empty vector or plasmids expressing mCherry-tagged Nsp2, Nsp2-N, and Nsp2-M were transfected into HEK293T cells with Myc-tagged LGP2 plasmid, respectively. After transfection, cells were lysed and subjected to Western blotting to analyze the expression of LGP2-myc. GAPDH is included as an internal control. The relative band density was normalized to the loading control GAPDH and then compared to the corresponding control. (F) HEK293T cells were cotransfected with Myc-tagged LGP2 and mCherry-tagged Nsp2 or its truncations. Cell lysates were immunoprecipitated with a Myc antibody, and immunoblots of Myc and mCherry are shown. GAPDH served as an internal control. Asterisks mark the expressed mCherry-fusion proteins of full-length or other truncated Nsp2. (G) GFP-tagged LGP2 and mCherry-tagged Nsp1, Nsp2, Nsp2-N, or Nsp2-M were cotransfected into HEK293T for 24 h, respectively. Immunofluorescence analysis of the colocalization between LGP2 (green) and Nsp1, Nsp2, Nsp2-N, or Nsp2-M (red) was performed. Scale bar, 10 μm.