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. 2008 Dec 5;4(12):e1000231. doi: 10.1371/journal.ppat.1000231

Figure 3. Endogenous A3G has the same effect on early cDNA accumulation as equivalent amounts of A3G expressed in 293T cells.

Figure 3

(A) Immunoblot analysis of A3G expression in uninfected CEM cells, CEM cells infected with either WT HIV-1 or HIV-1/Δvif, or 293T cells transfected with a fixed amount of HIV-1/Δvif proviral plasmid (3 µg) and increasing amounts of A3G plasmid (0, 0.03, 0.1, 0.3 or 1 µg). Below the blot, the graph shows the quantity of A3G expressed relative to HSP90. (B) Viral incorporation of A3G was measured by immunoblot analysis of purified virions harvested from the cells in (A). Below the blot, the graph shows the quantity of A3G incorporated relative to HIV-1 CA-p24. (C) WT HIV-1 or HIV-1/Δvif were passaged through CEM cells and used in natural endogenous reverse transcription reactions. DNA was harvested at the indicated times after the addition of dNTPs, and the relative amounts of early reverse transcription products (strong stop) were measured using quantitative PCR analysis. (D) The virus preparations from CEM cells described in (C) were used in parallel to challenge TZM β-gal indicator cells, and productive infection was measured as the induction of β-galactosidase activity, monitored using a chemiluminescent substrate. Infectivity is reported as counts per sec.