Reconstruction of the cell entry pathway of an extinct virus
Fig 4
Acidic pH is sufficient to trigger HERV-K Env.
(A) HERV-K Env is inactivated by exposure to acidic pH. VSV-HERVK and VSV were incubated in buffer at the indicated pH for 30 minutes at 37°C. Samples were returned to neutral pH before infecting BSRT7 cells. Cells were collected 5 hours post infection and percent GFP-expressing cells quantified by flow cytometry. Values are normalized to the pH 7 condition. Error bars represent SEM from three independent experiments. (B) VSV-HERVK fuses at the plasma membrane when treated with acidic pH. BSRT7 cells were pre-treated with bafilomycinA1 prior to binding virus at 4°C. Cells were treated with buffer at pH 7 or 5. Unbound virus was washed off and cells were collected 6 hours post-infection. Percent infected cells was normalized to cells not treated with bafilomycinA1. Error bars represent SEM for three independent experiments. (C) Endogenous HERV-K Envs are fusogenic at acidic pH. BSRT7 cells were transfected with envs from Phoenix, Xq21.33, and HERV-K 108 and subsequently exposed to buffer at the indicated pH. Syncytia are highlighted with arrows. Data are from a single representative experiment. (D) Endogenous HERV-K Envs bind heparin. 293T cells were transfected with the envs from Phoenix, Xq21.33, HERV-K 108, or VSV G. Cell lysates were incubated with either heparin (H) or protein A (A) beads and bound protein analyzed by Western blot against HERV-K Env and VSV G. T: 10% of total input. Data are from a single representative experiment.