Target cell-mediated editing of HIV-1 cDNA by APOBEC3 proteins in human macrophages

Abstract

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are encapsidated by assembling HIV-1 virions and edit viral cDNA in the next round of infection. Using alpha interferon (IFN-α)-treated monocyte-derived macrophages, we show that infrequent editing of HIV-1 reverse transcripts can also be mediated by APOBEC3 proteins supplied by the targets of infection. Based on the local sequence contexts of these mutations and the established characteristics of APOBEC3 protein expression in myeloid cells, we speculate that APOBEC3A may be responsible for a substantial proportion of this activity.

Fig. 1.

IFN-α induces APOBEC3A expression in monocyte-derived macrophages and promotes resistance to HIV-1 infection. (A) Immunoblot analysis of APOBEC3A and APOBEC3G. Whole-cell lysates of MDMs that had been treated (or not) with IFN-α for 24 h were analyzed using a rabbit polyclonal serum that recognizes APOBEC3A and APOBEC3G or a rabbit anti-HSP90 serum (loading control) (Santa Cruz Biotechnologies), an anti-rabbit secondary antibody conjugated to horseradish peroxidase, and enhanced chemiluminescence (GE Healthcare). (B) qRT-PCR analysis of HIV-1 reverse transcripts. Untreated (diamonds) and IFN-α-treated (squares) cells were challenged with v3SF162/IIIB/567A, and the relative levels of strong-stop DNA at subsequent time points (set to a maximum value of 100) were determined using qRT-PCR (10).

Fig. 2.

Effects of IFN-α on HIV-1 sequence variation during macrophage infection. HIV-1 cDNAs isolated at 3 or 24 h postinfection (pi) (Fig. 1) were subjected to single-molecule amplification. The total number of sequences for each sample is indicated, together with the percentage of sequences containing no changes (mauve) or G-to-A (maroon), C-to-T (yellow), or other (blue) mutations. Based on the low mutational loads seen in the 3-h samples of donors 1, 2, and 3 in the absence of IFN-α, we infer that the cumulative error rate for RNA polymerase II, reverse transcriptase, and Platinum Taq polymerase was ∼5 × 10⁻⁵.