Figure 5.

Amino acid Y1180 is not required for the RT-medicated cDNA synthesis by ORF2p. (A) Western blot analysis of the Gal4-tagged ORF2, ORF2 Y1180A, and ORF2 Y1189A transiently transfected in HeLa cells. ORF2p variants detected using anti-Gal4 antibodies. Protein products of expected size denoted by * for each ORF2 construct. Molecular weight markers denoted on the left. GAPDH used as loading control. (B) Alu retrotransposition assay results. Colony counts normalized to wild-type Gal4-tagged ORF2p-driven Alu retrotransposition levels. Neo^r colonies correspond to de novo retrotransposition events. Error bars denote SD determined using results from three independent experiments. Representative flasks are shown above corresponding graph bars. (C) LEAP analysis of wild-type and Y1180A Gal-4 tagged ORF2p. Displayed at the top is a schematic of the LEAP assay. Western blot analysis using anti-Gal4 antibodies of LEAP preparations shows that protein levels between the two ORF2p variants (wt and Y:A, *) are equivalent. Blank (Bl) is loaded with loading buffer only and control (C) is LEAP prepared on cells transfected with empty vector. LEAP analysis shows the presence of the PCR band of expected size in the ORF2p LEAP sample containing functional Y1180 (wt) and mutated Y1180 (Y:A) (*). MW, molecular weight marker; wt, wild type.