Fig 1.

Analysis of catalytically inactive mutants of UNG2 for modulation of HIV-1 mutation rate. (A and C) The ability of wt or mutated Vpr or VprW54R-UNG2 fusions to complement a vpr-defective HIV-1 (Δvpr) was analyzed in a single-cycle replication assay for mutant frequencies. The plasmids for expression of HA-tagged forms of Vpr, VprW54R, or VprW54R-UNG2 fusions were cotransfected with helper packaging plasmids into cells containing a single integrated HIV-1 vector provirus containing the lacZ gene as a mutation target. The viruses produced were then used to infect permissive HeLa cells, which allowed for a determination of the virus mutant frequency per round of replication. A total of 421 colonies were screened, and the average mutant frequency of the vpr-null mutant HIV-1 in the absence of Vpr transcomplementation was 0.15 (63/421) mutant/cycle. Values are the means of three independent experiments. Error bars represent one standard deviation (SD) from the mean. (B and D) Virion incorporation and enzymatic activity of the VprW54R-UNG2 fusion proteins. 293T cells were cotransfected with a vpr-defective HIV-1-based vector in combination with plasmids for expression of HA-tagged VprW54R-UNG2 mutated fusions. Virions were collected from cell supernatants, and proteins from cell and virion lysates were analyzed by Western blotting with anti-HA and anti-CAp24. In panel B (right side), UNG activity from the VprW54R-UNG2 fusions incorporated into virions was assayed with a 25-bp single-stranded DNA oligonucleotide substrate containing a uracil base at position 13, and apurinic sites were then cleaved by adding NaOH and EDTA and boiling for 30 min. The samples were run on a polyacrylamide denaturing gel. The gel was stained with SYBR Gold, and nucleic acids were visualized with an UV transilluminator. The control lane contains untreated DNA substrate.