Moderate influence of human APOBEC3F on HIV-1 replication in primary lymphocytes

Abstract

Multiple APOBEC3 proteins are expressed in HIV-1 target cells, but their individual contributions to viral suppression when expressed at endogenous levels remain largely unknown. We used an HIV NL4-3 mutant that selectively counteracts APOBEC3G (A3G) but not APOBEC3F (A3F) to dissect the relative contribution of A3F to the inhibition of HIV-1 replication in primary human lymphocytes (peripheral blood mononuclear cells [PBMCs]). This HIV Vif mutant replicated similarly to wild-type virus in PBMCs, suggesting that the effect of A3F on HIV restriction in these cells is limited. The different A3F variants found in PMBC donors displayed either comparable activity or less activity than wild-type A3F. Lastly, the endogenous A3F mRNA and protein expression levels in PBMCs were considerably lower than those of A3G. Our results suggest that A3F neutralization is dispensable for HIV-1 replication in primary human T-lymphocytes.

FIG. 1.

(A) Schematic representation of HIV-1 Vif domain organization, including the domains that provide selective A3G neutralization activity. (B) Infectivity of WT, W11R mutant, and SLQ144-146AAA Vif full-length NL4-3 viruses (HIV WT, HIV 11R, and HIV ΔSLQ, respectively) produced in the presence or absence of A3F, A3G, and A3B, measured as β-galactosidase expression levels in TZM-bl cells. Data are shown relative to the infectivity of viruses produced without any APOBEC3. Error bars show standard deviations. (C) Levels of degradation of A3F, A3G, and A3B achieved by the different Vif variants in the context of the full-length virus. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 2.

(A) Schematic representation of the anti-APOBEC3 activities of WT HIV, HIV 11R, and HIV ΔSLQ. The symbol ⊥ indicates inhibition. (B and C) Replication kinetics of the full-length HIV-1 NL4-3 W11R variant (HIV 11R) compared to those of WT virus and negative control HIV ΔSLQ in MT2 and MT4 T-cell lines (B) and PBMCs (C). Infections were carried out using 20 ng p24 equivalents for each virus in PHA-stimulated PBMCs from 5 anonymous donors. CA-p24 production was measured every 2 to 4 days for 12 to 15 days. The results shown are representative of two independent replication experiments.

FIG. 3.

(A) Measurement of the restriction activities of the different A3F variants (A3F WT, 108-231, and 307) on the infectivity of HIV WT, HIV 11R, and HIV ΔSLQ full-length viruses. Viruses were produced in the absence or presence of increasing amounts (50 ng, 100 ng, and 200 ng) of the A3F plasmids, and infectivity measured as β-galactosidase expression in TZM-bl cells. Data are shown relative to the infectivity of viruses produced without any A3F. The averages ± standard deviations of the results of two independent experiments are shown. (B) Protein expression levels of the different A3F haplotypes upon transfection of 100 ng of each plasmid into HEK293T cells. A3 proteins were detected with anti-FLAG antibody.

FIG. 4.

Expression of APOBEC3G and APOBEC3F. (A to C) Endogenous transcript expression levels of A3B, A3G, and A3F in PHA-IL-2-stimulated PBMCs, as well as in MT2 and MT4 T-cell lines. These cells were used for the HIV-1 replication experiments described in the legend to Fig. 2B and C. mRNA quantification was performed using TaqMan gene expression assays (Applied Biosystems) specific for human A3B (Hs00358981_m1), A3F (Hs00736570_m1), and A3G (Hs00222415_m1). Data are expressed as the number of molecules per 10 ng of reverse-transcribed RNA, with error bars representing the standard deviations from the means. The results shown represent two independent experiments. (D) Endogenous expression levels of A3G and A3F proteins in the same PBMCs and T-cell lines used for the experiments whose results are shown in panel A.