Validation of the quantitative PCR for the detection of HIV-1 integrants in the mouse genome. (A) Technical validation of species-specific HIV-1 integration PCRs on genomic DNA from mouse NIH3T3int cells or human HeLaint cells (20). Levels of HIV-1 integrants from the complete standard PCR reaction were set to 100% and levels determined for several specificity controls are given relative to that (omission (-) of LTR primer #1521, omission (-) of cellular anchor primer pair (B1, #2194 and #2231 (mouse) or Alu, #1519 and #1520 (human), omission (-) of first round PCR reaction). (B, C) Validation of the mouse integration PCR in a dynamic infection context. Parental NIH3T3 cells were infected with VSV-G HIV-CMV-GFP, carrying either a wildtype integrase (IN wt) or a catalytically inactive integrase mutant (IN(D64V)). Where indicated, the NNRTI efavirenz was added 1 h prior to infection. (B) Infected NIH3T3 cells were monitored for levels of total HIV-1 cDNA on day 1 p.i. (C) On day 7 p.i., cells were analyzed for the presence of integrated HIV-1 cDNA applying the mouse integration PCR.