FIG. 4.
The presence of IN within the viral particle is required to allow the incorporation of UDG into viral particles. (a) 293 cells were transfected with wild-type, Vpr-defective, IN-defective, or IN-Vpr double-defective molecular clones, and viruses produced in cell-free supernatant were treated with subtilisin and collected by ultracentrifugation. Virions were then resolved on a linear 20 to 60% sucrose density gradient, and gradient fractions coinciding with the peak of reverse transcriptase activity were pooled and normalized for reverse transcriptase activity. Virus pellets were then solubilized in sample buffer, and viral lysates were analyzed by Western blotting for the presence of Gag and UDG products. M, molecular mass markers (in kilodaltons). (b) Evaluation of the enzymatic activity of UDG within virions by using a nicking assay. A 32P-labeled single-stranded 34-mer oligonucleotide containing one uracil in the middle of its sequence was incubated with either recombinant purified UDG (0.005 to 0.1 units) or purified virions (3 × 106 cpm of reverse transcriptase activity). Upon incubation, DNAs were recovered and separated by electrophoresis on denaturing polyacrylamide gels.