FIG 1.

Overexpression of IFITM3 suppresses PRRSV replication in vitro. (A) Expression of IFITM3 mRNA in PRRSV-infected PAMs. (B) IFA or (C) Western blotting verification of stable expression of IFITM3 in Marc-145 cells. Marc-145-Vector or Marc-145-IFITM3-flag cells were infected with PRRSV (0.1 MOI) at 37°C for 1 h, and cells were subsequently maintained in 3% FBS+DMEM. (D and F) Cells were collected after 24, 36, and 48 hpi for detection of IFITM3 or N protein expression using (D) Western blotting, and supernatant virus titers using (F) TCID₅₀. (E) A portion of cells were fixed using 70% 20°C prechilled alcohol 24 hpi for IFA. (G) PAMs were transduced with recombinant lentivirus expressing IFITM3-flag or control lentivirus for 36 h, and cells were harvested for IFITM3 analysis. (H and I) PAMs transduced with recombinant lentivirus expressing IFITM3-flag or control lentivirus were infected with 0.1 MOI of PRRSV, and cells and supernatants were collected at 24, 36, and 48 hpi to determine (H) IFITM3 and N protein expression and (I) supernatant virus titers. (J) Marc-145 cells were infected with GD-HD-, JXA1-, and VR2332-PRRSV (0.1 MOI). At 48 hpi, cells were harvested and analyzed by Western blotting. IFA, indirect immunofluorescence assay; MOI, multiplicity of infection; hpi, hours postinfection; PAM, porcine alveolar macrophage; IFITM, interferon-induced transmembrane; PRRSV, porcine reproductive and respiratory syndrome virus.