2.6. Rev(1.4)-EGFP Nuclear Export Assay

To determine the aa essential for the CAEV Rev protein export, the Rev(1.4)-EGFP nuclear export assay was used [26]. The plasmid construct used in this assay, pRev(1.4)-EGFP, contains the whole HIV-1 Rev protein without the NES. Therefore, a predicted NES sequence can be cloned into the plasmid to promote the nuclear export of the HIV-1-EGFP fusion protein. The NES-deficient Rev(1.4)-EGFP and Rev(1.4)-NES3-EGFP (a construct that contains the intact HIV-1 Rev NES sequence) plasmids were kindly provided by Dr Beric R. Henderson (University of Sydney, Sydney, Australia) [9]. Alanine substitution NES mutant sequences were derived from the predicted CAEV Rev NES sequence by using complementary synthetic oligonucleotides that were ligated into the compatible ends of BamHI- and AgeI-digested Rev(1.4)-EGFP plasmid. After validating all mutant constructs by sequencing, HeLa cells were transfected with Rev(1.4)-EGFP (negative control), Rev(1.4) NES3-EGFP (positive control) or plasmids containing the NES sequence of CAEV Rev WT or each of the CAEV Rev NES mutated sequences. The cells were incubated for 24 h and were left untreated or exposed to both cycloheximide (CHX; 10 μg/mL) and actinomycin D (ActD; 5 μg/mL) 3 h prior to fixation. Finally, cells were fixed, counterstained with DAPI and the coverslips were mounted on glass slides using ProLong Gold antifade reagent (Thermo Fisher). Cells were imaged by CLSM and analyzed as described above. All data shown represent the general expression pattern observed in 30 cells analyzed from three independent experiments (10 analyzed cells per experiment).