Updated publication reference for PubMed record(s): 27690223. HIV accessory proteins manipulate host factors to evade cellular restriction and enhance viral replication. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to gain a comprehensive, time-based overview of proteins and processes subverted during HIV infection. To make specific functional and mechanistic predictions and systematically identify candidate accessory protein targets, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. As well as depleting APOBEC family members, we found Vif to be necessary and sufficient for CUL5-dependent degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A. Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PP2A subunits spans primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function.