Figure 4

Two equine tetherin orthologues are similarly sensitive to EIAV Env. (A) The pCMV-EIAV plasmids were transfected into HEK293T cells along with increasing amounts of the indicated tetherin constructs (0, 50, 100 ng). (B) An EIAV GagPol expression construct (2.5 μg) and the tetherin expression vectors as indicated were transfected into HEK 293 T cells in the presence or absence of 1 μg of EIAV Env expression vector. Cells were dissolved at 48 h after transfection and then subjected to SDS-PAGE. Viral proteins were analyzed by Western blotting using anti-EIAV serum. Intracellular expression of tetherin was detected by Western blotting using anti-HA antibody. (C) Tetherin and β-actin mRNA expression in horse and donkey macrophage cultures were quantified by real-time PCR. Histograms represent the averages from three independent experiments (n = 3). (D) Both horse and donkey monocyte-derived macrophages (2 × 10⁶) were infected with EIAV at 1 × 10³ TCID₅₀. Reverse transcriptase activity in supernatants from EIAV-infected horse and donkey macrophages was determined by measuring the reverse transcriptase activity using an RT activity kit (Reverse Transcriptase Assay, Colorimetric kit, Roche, Switzerland) as per the manufacturer’s protocol.