FIG. 3.

Proviral DNA analysis and requirement for IN activity in the transduction of human macrophages. (A) Day 4 DCs and macrophages were transduced with the same amount of F-MLV vectors at an MOI 10 and lysed 24 h postinfection for the analysis of late reverse transcription products that were full-length (FL) and 2LTR circles. Analysis was performed on threefold dilution of the sample with primers that allow the specific amplification of each form followed by agarose gel migration and detection. (B) A single point mutation was introduced in the catalytic aspartic acid of the F-MLV IN (D1513A) by site-directed mutagenesis. Mutant and wild-type (WT) viruses were produced in parallel, and their infectivity was tested on growing HeLa cells after normalization by exogenous (Exo) RT activity (right panel). Upon normalization of their infectious titer, wild-type and mutant viral preparations were used to infect macrophages (left panel). (C) VSVg- and RD114-pseudotyped MLV vectors were similarly produced and tested on macrophages at equal MOIs. The infectivity of RD114-pseudotyped MLV vectors is shown with respect to that of VSVg-MLV, set arbitrarily at 1.