Figure 1.

Evolution of the dox-inducible SIV-rtTA variant. (A) In the SIVmac239-based SIV-rtTA variant, the Tat-TAR regulatory mechanism was inactivated through mutation of TAR (TAR^m), and functionally replaced by the dox-inducible Tet-On regulatory system through the introduction of the gene encoding the rtTA transcriptional activator protein at the site of the nef gene and two dox-responsive tet operator (tetO) elements between the NFκB and Sp1 sites in the U3 promoter region [25]. The TAR mutations and tetO elements were introduced in both the 5' and 3' LTR. (B) The TAR RNA element of SIV-rtTA can fold a branched hairpin structure with three stem-loop domains (SL1-3). The mutations that had been introduced in SL1 and SL2 to inactivate TAR, are encircled in gray (SL1: +27^U-A, +28^U-A, +34^C-A, +36^G-U; SL2: +62^U-A, +68^C-A, +70^G-U). Upon long-term culturing of SIV-rtTA in PM1 cells, additional nucleotide substitutions are observed in TAR. The number of the culture in which the mutation is observed is shown (#), with the asterisk (*) indicating the transient presence of the mutation. (C) Alternative folding of the SL1 domain can result in a 6-bp spacer between the bulge and loop sequences. However, this spacer extension slightly reduces TAR stability (ΔG_{5 bp} = -67.5 kcal/mole; ΔG_{6 bp} = -67.2 kcal/mole). Alternative folding of the +63^A-G mutated TAR RNA results in a 6-bp bulge-loop spacer in SL2 but does not affect TAR stability (ΔG_{5 bp} = ΔG_{6 bp} = -67.5 kcal/mole). Formation of an A⁺⁶³-U⁺⁷⁸ base pair in the +78^C-U mutant results in a similar 6-bp bulge-loop spacer in SL2 and increases the stability of this TAR variant (ΔG_{5 bp} = -65.2 kcal/mole; ΔG_{6 bp} = -65.8 kcal/mole). (D) TAR can fold an alternative extended hairpin structure in which the SL1 and SL2 sequences fold a large stem-loop structure. The introduced and acquired mutations are shown as in B.