Overexpression of hZAP inhibits XMRV infection.

(A) Schematic structure of XMRV-luc vector. The coding sequences of Gag-Pol and part of Envelope were replaced with luciferase-coding sequence to generate pXMRV-luc. (B) Overexpression of hZAP inhibits XMRV-luc infection. 293TRex cells expressing hZAP-v1-myc and hZAP-v2-myc upon tetracycline induction were infected with VSV-G pseudotyped XMRV-luc. Cells were equally divided into two dishes at 6 h postinfection, with one mock treated and the other treated with tetracycline. Cells were lysed and luciferase activity was measured at 48 h postinfection (upper panel). The luciferase activity in the absence of ZAP was set as 100. Data presented are means ± SD of three independent experiments. The expression of hZAP was confirmed by Western blotting (lower panel). (C) ZAP inhibits XMRV-luc in an expression-level-dependent manner. 293TREx-hZAP-v2 cells were infected with XMRV-luc. At 6 h postinfection the cells were equally split and tetracycline was added to the concentrations indicated. Cells were lysed and luciferase activity was measured at 48 h postinfection. Fold inhibition was calculated as the luciferase activity in mock treated cells divided by the luciferase activity in the tetracycline treated cells (upper panel). Data presented are means ± SD of three independent experiments. The expression levels of hZAP-v2 were measured by Western blotting (lower panel). (D) ZAP inhibits XMRV replication. 293Trex-hZAP-v2 Cells were infected with XMRV produced in 293T cells. At 8 h postinfection, cells were mock treated or treated with doxycycline to induce hZAP-v2 expression. Samples were taken every day and subjected to RT assays.