FIG. 1.

hUGI inhibits UNG activity in cells and in HIV-1 produced from those cells. (A) Cell lines expressing the UNG inhibitor hUGI have a greater than 100-fold decrease in UNG activity. Increasing amounts of cell lysate for each cell line were incubated with 0.05 pmol of labeled oligonucleotide containing a uracil, as described in Materials and Methods. The amount of uracil DNA glycosylase activity present in the lysates is denoted in picomoles of a 19-mer oligonucleotide cleaved at the uracil base per minute (A, lower). Cleavage results in generation of a 9-mer oligonucleotide. NEG, oligonucleotide in the absence of lysate and therefore our detection limit (depicted on the graphic representation of the data below the panel as a dotted line); POS, oligonucleotide in the presence of recombinant UDG (New England BioLabs) (used as a positive control). Lines with filled squares represent activity of lysates from WT cells, while lines with open circles represent activity of lysates from cells that express hUGI. (B) Western blot analysis of concentrated virus generated in WT or hUGI-expressing cells with antibodies specific for UNG2 or p24 capsid. (C) Concentrated HIV virions from panel B were assayed for UNG activity in vitro as in panel A. oligo, oligonucleotide.