Figure 6.

RecHTLV recombines loxHTLV in a viral context

(A) Scheme of the recombinase expression vector and the HTLV-1 proviral expression plasmids pCS-HTLV-X1MT and pCMV-HT1M. pCS-HTLV-X1MT contains two full-length LTRs while pCMV-HT1M has only one truncated 5′ LTR, therefore lacking one loxHTLV target site. In the bicistronic recombinase expression construct, the self-cleaving 2A peptide (P2A) allows simultaneous expression of the recombinase fused to a 3xFLAG peptide and a nuclear localization signal (NLS-Rec) and a GFP cassette driven by an EF1a. (B and C) Left: detection of Gag p55/p19 protein and FLAG-tagged recombinases 72 h after transient transfection of 293T cells with recombinase expression constructs Puro (control), Cre, and RecHTLV together with proviral expression plasmids (pCS-HTLV-X1MT, pCMV-HT1M). Hsp 90 served as control. Numbers indicate densitometric analysis of Gag (p55 + p19) protein, normalized to Hsp 90. Marked with red arrows are the expected sizes of p55 and p19 products. Blots were cut due to technical reasons. Right: densitometric analysis of Gag protein normalized to Hsp90. The bar shows the mean ± SD of three to four independent experiments (∗p < 0.05; ns, not significant, unpaired two-sided t test using the logarithm of the normalized values).