Abstract
Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription (“uracilation”). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that the kinetics for MC infection is compatible with their lifetime in vivo and their near absence of hUNG2 activity is consistent with the retention of viral dUMP at high levels at least until differentiation into macrophages, where UBER becomes possible. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.
Highlights
We have previously reported that HIV DNA obtained from alveolar macrophages and circulating blood monocytes from drug naïve and ART patients both contained high levels of dUMP, while T cells from the same patients did not [4]
When dUTPase is added before the reaction, the extension can only arise from dTTP and the level of dUTP-associated extension is determined by the difference in the fractional extension between the +/dUTPase reactions
DNA repair could repair plus strand mismatches, the low repair capacity of nondividing cells makes this unlikely compared to the simple minus strand mechanism. Consistent with this idea, a recent deep sequencing study concluded that minus strand deamination events catalyzed by A3G were only inefficiently repaired by uracil base excision repair (UBER) or mismatch repair in T cells, which have much greater repair activity than monocyte-derived macrophages (MDM) [44]. These in vitro results inform on the previous detection of dUMP in both alveolar macrophages and peripheral blood monocytes of HIV patients on ART [4]
Summary
CellsMonocytes (MC) were purified from peripheral blood mononuclear cells (PBMC) of HIV negative consenting donors under Johns Hopkins University IRB approval (IRB00038590) using a Ficoll-Hypaque density gradient followed by negative selection using a Pan monocyte isolation kit (Miltenyi Biotech). To prevent cell adherence and differentiation into monocytederived macrophages (MDM), MC were cultured up to 7 days in suspension using ultra-low adherence 96-well plates (Corning) using RPMI 1640 (Gibco) supplemented with 10% donor autologous serum, 100 μg/ mL penicillin, 100 μg/mL streptomycin (HyClone), 0.3 mg/ml glutamine and 10 mM HEPES (RPMI-AS). T cells were first cultured for three days using stimulating conditions (RPMI supplemented with 10% (vol/vol) donor autologous serum, 100 μg/ mL penicillin, 100 μg/mL streptomycin (HyClone), 0.3 mg/ml glutamine 1 mg/ml of Phytohemagglutinin (Gibco) and, ciprofloxacin (5 μg/ml)). The culture media for the dividing cells (HI-10) consisted of RPMI-1640 medium + 10% heat inactivated FBS (Sigma), 100 μg/mL penicillin and 100 μg/mL streptomycin. The cells were released from the flask using 5 mL of 0.05% trypsin-EDTA (Gibco), followed by washing three times with PBS (Gibco) without CaCl2 or MgCl2
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