Updated publication reference for PubMed record(s): 31042482. Viruses target host proteins for degradation to enhance their replication and transmission, and identifying these targets has provided key insights into the host-pathogen interaction1-3. Here, we use complementary unbiased mass spectrometry-based approaches to dissect the widespread proteomeic remodelling seen in HIV-1 infected T-cells. Remarkably, the HIV accessory protein Vpr is both necessary and sufficient to cause the vast majority of these changes. Protein regulation requires recruitment of the DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex, and pulsed-Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) and immunoprecipitation-mass spectrometry (IP-MS) identified at least 38 cellular proteins directly targeted for degradation by Vpr. Whilst other HIV-1 accessory proteins downregulate a small number of specific host factors, Vpr depletes multiple protein targets, causing systems-level changes to the cellular proteome. A subset of the novel cellular targets identified in this study are depleted by Vpr variants from across HIV-1/SIVcpz and other primate lentiviral lineages, confirming their biological importance in vivo.