Human APOBEC3G-mediated editing can promote HIV-1 sequence diversification and accelerate adaptation to selective pressure

Abstract

Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, hereinafter referred to as A3G) is an innate virus restriction factor that inhibits human immunodeficiency virus type 1 (HIV-1) replication and induces excessive deamination of cytidine residues in nascent reverse transcripts. To test the hypothesis that this enzyme can also help generate viral sequence diversification and the evolution of beneficial viral variants, we have examined the impact of A3G on the acquisition of (-)2',3'-dideoxy-3'-thiacytidine (3TC) resistance in vitro. That characteristic resistance mutations are rapidly fixed in the presence of A3G and 3TC suggests that A3G-mediated editing can be an important source of genetic variation on which natural selection acts to shape the structure of HIV-1 populations.

FIG. 1.

Immunoblot analysis of A3G protein in modified CEM-SS cells and relative replication efficiencies of HIV-1 grown in control and A3G-modified CEM-SS cells. (A) The cells were stably transduced with a retroviral vector encoding A3G or a corresponding control parental vector (8). Whole-cell lysates were analyzed by immunoblotting using primary antibodies to A3G (α-A3G) or heat shock protein 90 (HSP90)-α/β (Santa Cruz Biochemicals), followed by horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (14). HSP90 was used as a loading control. The level of A3G protein in these cells closely matched that detected in primary human CD4⁺ T cells that had been activated with anti-CD3 and anti-CD28 antibodies (14). (B and C) Replication efficiencies of HIV-1 (copies of viral RNA per microliter of culture supernatant) in control or A3G-modified CEM-SS cell cultures with 3TC and without 3TC [3TC (−)]. Cells were infected with HIV-1_NL4-3 at a MOI of 0.01. HIV-1 gag in the cell culture supernatants from four independent experiments was measured by real-time quantitative reverse transcription-PCR. The horizontal axis indicates the days after infection with wild-type HIV-1_NL4-3. The vertical axis indicates the amount of viral RNA per microliter of CEM-SS cell culture supernatant. The means and standard deviations of the viral RNA values from four independent experiments are shown.

FIG. 2.

Frequencies of mutations that confer 3TC resistance in A3G-modified CEM-SS cells grown in the presence and absence of the drug. (A) The frequencies of mutations at position 184 in the RT region of pol in the virus population at days 3, 7, 9, and 13 following infection of A3G-modified CEM-SS cells with wild-type HIV-1_NL4-3 in the presence of 3TC (IC₉₀) are shown. The virus population was subjected to in-depth analysis by highly parallel DNA sequencing. To reduce the number of pyrosequencing artifacts, we screened the processed reads for the pattern of light intensity by using quality filters. DNA sequences were removed from the analysis because of more than one mismatch (insertion, deletion, or substitution) in the MID region, a quality score of <25, or mismatches (except for G-to-A changes) in the two flanking amino acids on either side. The pyrosequencing data were analyzed with the assumption that the observations were drawn independently and randomly from the population of viruses. Exact binomial 95% confidence intervals were calculated using the statistical package R on the underlying frequency under this assumption. A background mutation rate determined in the absence of A3G and 3TC was 0.001. The dominant base replacement errors were transitions (G/A interchange and C/T interchange) at a level of 10⁻³; transversions were rare (unpublished data). In three of four independent experiments, a mutation at position 184 in the RT region of pol emerged by day 9 and reached fixation soon thereafter. In one of four experiments, the M184I mutation that conferred 3TC resistance arose only at day 16. (B) The frequencies of mutations in the RT region of pol in the virus population at days 3, 7, 9, and 13 following infection of control [A3G (−)] and A3G-modified [A3G (+)] CEM-SS cells with wild-type HIV-1_NL4-3 in the absence of 3TC are shown. In A3G-modified CEM-SS cells, the frequency of G-to-A changes increased over time.