Establishment and validation of a real-time PCR for HIV-1 integrants in rat cells. (A) Schematic of the generation of Rat2int (rat) and HeLaint (human) cells, carrying HIV-1NL4-3E- GFP, as species-specific HIV-1 integration standards. (B) PCR strategy of the nested rat integration PCR. In the first round of PCR, a segment of integrated HIV-1 cDNA was amplified by one primer annealing in the HIV-1 LTR (primer #1521) and two outward-facing primers targeting the rat ID element (primers #1734 and #1782). To increase specificity, LTR primer #1521 contains a lambda-phage heel sequence at the 5'-end . In a nested, second-round PCR, a lambda-specific primer (primer #1522), a second LTR primer (primer #1523), as well as an HIV-1 LTR-specific probe (probe #1524) were employed to exclusively amplify products generated during the first-round PCR. (C) Technical validation of species-specific integration PCR on Rat2int or HeLaint  cells. Levels of HIV-1 integrants from the complete standard PCR reaction were arbitrarily set to 100%, and levels determined for several specificity controls (omission (w/o) of LTR primer #1521, omission (w/o) of cellular anchor primer pair (BC, #1734 and #1782 (rat) or Alu, #1519 and #1520 (human)), omission (w/o) of first-round PCR reaction) are given relative to that. (D) Validation of rat integration PCR. Parental Rat2 cells were infected with VSV-G pseudotyped HIV-1 GFP vectors carrying either a wildtype integrase (IN wt) or catalytically inactive integrase (IN(D64V)). Where indicated, efavirenz (5 μM) was added 1 h before infection. Cultures of infected Rat2 cells were monitored for the presence of total HIV-1 cDNA on day 1 (left panel) or day 7 (middle panel) post infection. On day 7, cells were also analyzed for the presence of integrated HIV-1 cDNA (right panel).