Integration of IN-defective viruses . (A) A quantitative assay based on a real-time RACE-PCR reaction was designed, amplifying Alu-LTR junctions between the cell genome and integrated proviruses twenty-four hours post-infection. PCR amplifications of existing Alu-PFV-1 LTR junctions were subjected to a second quantitative round of real time PCR with PFV-1 LTR-specific primers. Fluorogenic hybridization probes were used to quantify the amplification products. Infected cells with known copy numbers of integrated proviruses were used as quantification standards. The assay is highly sensitive since it allows detecting 25 proviruses copies in 50,000 human cells. Control reactions are detailed in the Material and methods section. (B) Detection of integrated viral DNA following infection of IN-mutated viruses. Quantitation of viral DNA accumulated in PFV-1 infected cells was carried out by real-time PCR of total DNA extracts from U373-MG infected cells (m.o.i. of 1) collected at the completion of the first viral replication cycle, 24 hours post-infection. Total viral DNA (gag quantifications) and integrated proviruses were quantified in duplicate using real-time PCRs. Data obtained in one representative infection from four independent experiments are expressed as integrated DNA copies per million cells (logarithmic scale) as determined by a human β-globin quantification in cell extracts ("Integrated provirus" panel). Total DNA copies per million cells (logarithmic scale) present in the same extracts are presented in the lower panel. Standard deviations representing variations between two quantifications of the same sample are given. (C) Integration efficiency in PFV-1 infected cells. Integration efficiency was determined by normalizing the number of integrated proviruses (mean of duplicates) with the total number of viral DNA molecules (mean of duplicates) present in the same extract. Raw LightCycler data from four independent experiments are presented in the upper table. Mean of integration efficiencies from these four experiments are figured in the lower histogram.