Human and mouse cyclophilin domains are functionally similar.

<p><b>A</b>) CypA protein levels in Nup358^F/F and Nup358^{−/−[GFP1-1340]} cell lines. Wild type and knockout Jurkat cells were included as positive and negative controls respectively. <b>B</b>) Cyclosporine (Cs) has minimal effect on HIV infection in the presence or absence of the C-terminal domain. Indicated cell lines were transduced with HIV-1 reporter virus in the presence of absence of 5 µM Cs. OMK cells were included in the experiment as a positive control. <b>C</b>) Jurkat PPIA−/− cells were transduced with lentiviral vectors expressing either a HA tagged human or untagged mouse cyclophilin. Cells expressing the indicated constructs were challenged with HIV reporter virus (top panel). Cyclophilin expression was confirmed using antibody to CypA (lower panel). <b>D</b>) TRIM-CHD fusion proteins. The Cyp domain of owl monkey TRIMCyp (OMTC) was replaced with the human or mouse Nup358 CHD (creating proteins OMThNC and OMTmNC respectively). The proteins were expressed stably in CrFK cells, which lack endogenous TRIM5alpha or TRIMCyp restriction. Cells were transduced with dual promoter vectors that express GFP and OMTC, OMThNC or OMTmNC. Expression of appropriate sized fusion proteins was confirmed by western blotting with rat anti-HA. Equivalent GFP expression was also observed. Tubulin was immunoblotted as a loading control. <b>E</b>) Parental and restriction factor-expressing CrFK cells (OMTC, OMThNC, OMTmNC) were challenged with WT HIV-1_luc, N74D HIV-1_luc,, FIV_luc, and NB-MLV_luc. Luciferase activity was measured at 2 days post transduction and normalized to cell number.