Fig 1.
G-to-A mutational load and mutation location in HIV-1 proviruses with multiple G-to-A mutations. (A) G-to-A mutational load in HIV-1 proviruses from CEM-GFP, U373-MAGI, and 293T cells. Each provirus with a mutant HSA sequence containing with multiple G-to-A mutations is indicated by a black circle (CEM-GFP), black square (MAGI), or black triangle (293T). The average G-to-A mutational load and standard deviation are indicated. (B) Location of G-to-A mutations in recovered proviruses harboring multiple G-to-A mutations. The red, green, and blue circles above G residues indicate the locations of G-to-A mutations (one circle per mutation identified) in proviruses recovered from CEM-GFP, U373-MAGI, and 293T, respectively. The start and stop codons of the HSA gene are identified by black rectangular boxes. (C) Quantitative RT-PCR was performed to determine the relative levels of APOBEC3 mRNA expression among the cell lines under investigation (i.e., CEM-GFP, SupT1, U373-MAGI, and 293T). The asterisk indicates that the APOBEC3C level from 293T cells was set to 1, and all other values are relative to this measurement. The mRNA expression levels were normalized to TATA-binding protein (TBP) mRNA levels. For these analyses, data with a difference in efficiencies between the APOBEC3 standard and TBP standard were ≤10% and R2 ≥0.98. Threshold cycle (CT) values were determined using the regression method, and data were analyzed by E−ΔCT. Experiments were conducted in triplicate with the standard deviation indicated.