FIG 6.

Hili inhibits the replication of IAP. (A) Schematic representation of pFL, which contains the full coding IAP genome, where an interrupted EGFP reporter gene was inserted in the opposite orientation at the 5′ end (indicated with reversed type). The 5′ LTR contains the cytomegalovirus (CMV) enhancer/chicken β-actin promoter for higher levels of expression in cells. The EGFP gene in the antisense orientation was interrupted by an intron in the sense orientation (arrow). It was transcribed from an eIF1α promoter. Other notations are as follows: PBS, primer binding site; PPT, polypurine tract. Upon transcription of IAP, the intron in EGFP was removed and the new integrated provirus expressed EGFP. EGFP-positive cells were then counted. The white arrow points to a green cell(s). The white bar represents 10 μm. (B) Hili inhibits IAP retrotransposition in 293T cells. The number of EGFP-positive cells in the presence of Hili relative to such cells transfected with an empty vector is presented (bar 2). (C) Hili inhibits the expression of IAP proteins in 293 T cells. Expression of IAP Gag proteins is presented below the Hili Western blot. A >5-fold decrease in expression of IAP proteins was observed in the presence of Hili in these cells. Expression of actin served as the internal control. (D) Levels of IAP transcripts are equivalent in 293T cells that do or do not express Hili. Relative levels of IAP gag RNA in the presence of Hili are presented in bar 2. The primers used for RT-qPCR are presented in Fig. 1A. (E) Reverse transcription of IAP is decreased in the presence of Hili. A 200-nucleotide (nt) product of RT was detected in control 293T cells transfected with an empty vector but not in Hili-expressing 293T cells (lanes 2 and 3).