Fig 3.

Downregulation of ZAP expression promotes MHV-68 reactivation. (A) S11E cells were transduced with a retrovirus vector expressing myc-tagged M2. The expression of M2 was detected by Western blotting. (B and C) MHV-68 titers in the supernatants of transduced S11E cells were measured at 30 h postransduction (B), and expression of viral lytic replication-associated antigens was detected by Western blotting (C). *, P < 0.05. EV, empty vector. (D) shRNAs against mZAP were cotransfected into HEK293 cells with a construct expressing myc-tagged mZAP along with a construct expressing myc-tagged green fluorescent protein (GFP) to serve as a control. At 48 h posttransfection, mZAP expression levels were measured by Western blotting. (E to I) S11E cells were transduced with retrovirus vectors expressing a scrambled control shRNA or shRNAs against mZAP at an MOI of 1 or were treated with TPA as a positive control. At 30 h postransduction, the supernatants were collected, viral titers were measured (E), and expression of viral replication-associated antigens in the cells was detected by Western blotting (F). The mRNA levels of mZAP (G), M2 (H), and RTA (I) in the S11E cells were measured by real-time PCR. The viral titer data presented are means ± SD of three independent experiments. *, P < 0.05. EV, empty vector; Ctrl, untreated; Scr, scrambled shRNA; TPA, 12-O-tetradecanoylphorbol-13-acetate.