Article Figures & Data
Figures
- 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.
- FIG. 2.
UNG is dispensable for infectivity in single-replication assays. Equivalent amounts of virus generated from WT or hUGI-expressing cells were added to the indicated cell types. Infectivity was measured either by expression of a virus-encoded luciferase reporter in the Nef gene (SupT1, H9, and Jurkat cells) or by virus titer in MAGI cells (HeLa cells), measured by number of blue cells per milliliter. Filled boxes represent virus infectivity from virus generated in WT cells, while stippled boxes represent virus infectivity from virus generated in cells that express hUGI. RLU, relative light units.
- FIG. 3.
UNG is dispensable for efficient HIV-1 replication in a spreading infection. HIV-1 was added to either WT T cells or hUGI-expressing T cells and allowed to spread through the culture. HIV production was monitored by the amount of p24gag in the culture supernatant as a function of days after infection.
- FIG. 4.
Virus production and infectivity in UNG−/− cells. (A) 10-fold dilutions of cell lysate from WT or UNG−/− cells were incubated with a labeled U-containing oligonucleotide as described in the legend to Fig. 1 and Materials and Methods. (Inset) Western blot for UNG2 protein in UNG+/+ B cells versus UNG−/− B cells. (B) HIV produced in UNG−/− cells has no detectable UNG activity. Cell-free supernatants were collected from infected cells (WT or UNG−/−) and assayed for UNG enzymatic activity as in Fig. 1C or subjected to Western blot analysis for UNG2. (C) HIV was produced in either WT or UNG−/− cells. Equivalent amounts of p24gag from these cells were used to infect SupT1 T cells. Infectivity was measured by expression of virally encoded luciferase 48 h after infection. (D) VSV-G-pseudotyped HIV-luciferase (LaiΔenvLuc2) was produced in 293T cells, and increasing amounts of virus were used to infect either WT B cells or UNG−/− B cells. Virus infectivity is measured by expression of virally encoded luciferase. oligo, oligonucleotide; RLU, relative light units.
- FIG. 5.
UNG activity does not render HIV-1 more or less sensitive to the antiviral effects of Apobec3G. (A) HIV-1ΔVif that encodes luciferase in place of nef was cotransfected with increasing amounts of Apobec3G in virus-producing cells containing either a WT UNG or the UNG bound to the inhibitor, hUGI. These viruses were then used to infect WT T cells or hUGI-expressing T cells. Virus infectivity is reported as a percentage of infectivity in the absence of Apobec3G. Filled squares, WT/WT as producer/target; open squares, WT/UGI; filled circles, UGI/WT; and open circles, UGI/UGI. (B) Quantitative-real-time PCR analysis of acute HIV-1 infection in SupT1 cells. Equivalent amounts of p24gag of virus from WT or hUGI-expressing cells (with or without Apobec3G) were added to SupT1 cells. Low-molecular-weight DNA was collected at indicated times and analyzed by quantitative real-time PCR. Copies of viral DNA are reported for 105 cells. Filled squares, WT; filled circles, UGI; open squares, WT plus 100 ng Apobec3G; open circles, UGI plus 100 ng Apobec3G.
