Epstein-Barr virus latently infected cells are selectively deleted in simulated-microgravity cultures

Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulate d microgravity. Previously, ive showed that the cultivation of lymphoblasto id cells in simulated microgravity results in the suppression of Epstein-Ba rr virus (EBV) reactivation. To determine if the suppression generated by s imulated microgravity could be reversed by changing to static culture condi tions, cells were cultured in an RWV for 5 d, and then switched to static c onditions. Following the switch to static conditions, viral reactivation re mained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine i f chemical treatment could induce viral reactivation in cells from simulate d-microgravity cultures. Cells were cultured in static flask cultures and i n simulated microgravity in RWVs for 4-7 d. The cells were then transferred to 50-cm(3) tubes, and treated with 3 mM n-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for eith er 2 or 48 h, under static conditions. Although EBV was inducible, the cell s from simulated-microgravity cultures treated with n-butyrate displayed si gnificantly lower levels of viral-antigen expression compared with the trea ted cells from static cultures. Also, incubation with TPA for 2-3 h, but no t for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV wa s inducible in cells from static cultures treated for either 2-3 or 48 h wi th TPA. TPA reactivation of EBV following a 2-3-h period of treatment indic ates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the ex posure of B-lymphoblastoid cells from simulated-microgravity cultures to TP A for more than 3-4 h triggered a lyric event (apoptosis or necrosis), whic h prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the absence of any cytotoxic cell s.