Figure 3.

Decreased viral DNA production by IN-defective viruses is concomitant with an abnormal accumulation of LTR-LTR junctions. Quantification of viral DNA synthesis was carried out by real-time PCR amplification of total DNA extracts from U373-MG infected cells (equal virion levels as measured by reverse transcriptase activity), collected 3, 6, 10, and 24 hours post-infection. An m.o.i. of 1 for the WT infection as determined by the FAB assay was used. Data are presented for 10⁶ cells as measured by quantification of the nuclear β-globin gene and standard deviations representing variations between two quantifications of the same sample are given. To ensure that only freshly synthesized DNA, and not contaminating DNA contained in the viral particles input, was analyzed, all infections were performed in parallel control experiments under AZT treatment that inhibits viral neosynthesis. Representative kinetics from 4 independent experiments is presented. (A) Total viral DNA was detected using primers allowing amplification of the region of the PFV cDNA at the 5' end of the gag gene [40]. (B) Viral DNA with 2-LTR junctions was measured using primers that cross the junction between the two LTRs as previously described [40]. (C) The abundance of 2-LTR molecules is expressed as the percentage of 2-LTR copies relative to the total viral DNA (gag) at each infection time-point.