APOBEC3 antagonism fully explains HIV-1 Vif essentiality in THP-1 cells under interferon and differentiation conditions

HIV-1 virion infectivity factor (Vif) counteracts APOBEC3 (A3) proteins by targeting them for proteasomal degradation, thereby preventing lethal G-to-A mutations and loss of infectivity. However, Vif also degrades additional cellular proteins, raising the possibility that its essential role in infectious virion production may extend beyond A3 antagonism, particularly under inflammatory or differentiation conditions. Whether such conditions reveal additional essential Vif targets remains unresolved. Here, using interferon (IFN)-stimulated or phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytic cells, we directly addressed this question. Although type I IFN and PMA markedly suppressed viral production through Vif-independent mechanisms, ΔVif viruses produced from stimulated parental cells exhibited severely reduced infectivity. In contrast, disruption of A3A-A3G fully restored ΔVif infectivity to wild-type levels under all conditions tested. G-to-A mutations were attributable exclusively to A3 proteins under both IFN and PMA stimulation, whereas IFN-induced, A3-independent blocks to reverse transcription were not antagonized by Vif. Moreover, Vif-mediated degradation of the non-A3 targets PPP2R5 family proteins did not measurably contribute to virion infectivity under these conditions. Across diverse HIV-1 strains, the requirement for Vif in this experimental system was strictly dependent on A3 family proteins. These findings indicate that, under the conditions examined here, the essential role of Vif is fully explained by antagonism of A3-mediated restriction. Our results further suggest that counteracting A3-mediated restriction and mutagenesis represents a major evolutionary pressure shaping Vif.IMPORTANCEA3 proteins are potent intrinsic antiviral factors that restrict HIV-1 by inducing lethal mutations and impairing reverse transcription. The viral protein Vif counteracts A3-mediated restriction, yet it also targets additional host proteins, raising questions about whether its essential role extends beyond A3 antagonism under inflammatory conditions. Myeloid cells mount interferon (IFN) responses and undergo transcriptional reprogramming upon differentiation, environments in which additional Vif functions could emerge. Using an A3A-A3G-null monocytic cell model, we show that even under IFN stimulation or phorbol 12-myristate 13-acetate (PMA)-induced differentiation, the requirement for Vif in maintaining HIV-1 infectivity is due to A3 proteins. In contrast, Vif does not counteract IFN-induced, A3-independent restriction pathways, highlighting functional specificity. These findings establish the A3-Vif axis as the central determinant of Vif dependency and suggest that counteracting A3-mediated restriction and mutagenesis represents a major evolutionary pressure shaping Vif. Targeting Vif may therefore expose HIV-1 to intrinsic inactivation, even in inflammatory environments.