The effects of combining multiple mutations in the <i>pol</i> and <i>env</i>-<i>nef</i> silencing regions.

<p>(A) The <i>pol</i> and <i>env</i>-<i>nef</i> regions of pNL4-3 and the position of the <i>cis</i>-acting instability elements (INS) are illustrated. The arrows indicate the mutated sites in the <i>pol</i> (sites “a” and “b”) and <i>env</i>-<i>nef</i> (sites “c”, “d” and “e”) regions. The number and bar pattern corresponds to the graph below. (B) The effects of multiple mutations in the <i>pol</i> region (sites “a” and “b”; elements 1 and 7) were assessed in Jurkat cells. Six independent experiments were performed. The bar patterns correspond to the mutational patterns shown in (F). The “2 m” indicates that the vector has two mutation sites in the repressive sequence. (C) The effects of multiple mutations in the <i>pol</i> region were assessed in M4C8 cells. (D) The effects of multiple mutations introduced into the <i>env</i>-<i>nef</i> region (sites “c”, “d” and “e”; elements 15 and 16) were assessed in Jurkat cells. Four independent experiments were performed. The bar patterns correspond to the mutational patterns shown in (G). The “1 m”, “2 m” and “3 m” indicate that the vector has one, two and three mutation sites individually. The red and blue bars indicate the more derepressed mutational patterns. (E) The effects of multiple mutations introduced into the <i>env</i>-<i>nef</i> region were assessed in M4C8 cells. The psiCHECK was used as a control, and the results are expressed as the mean ± S.D. as a percentage of the control. ***P<0.001, **P<0.005 and *P<0.05. (F) Combinations of mutated patterns in the <i>pol</i> region and the bar patterns in graph B and C. (G) Combinations of mutated patterns in the <i>env</i>-<i>nef</i> region and the bar patterns in graph D and E. The gray characters represent unchanged residues. The asterisk denotes the mutational pattern used for the generation of mutant viruses.