Abstract
Viruses target host proteins for degradation to enhance their replication and transmission, and identifying these targets has provided key insights into the host-pathogen interaction. Here, we use complementary unbiased mass spectrometry-based approaches to dissect the widespread proteome 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. This protein regulation required recruitment of the DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex and the substrate binding surfaces of Vpr, but was mostly independent of Vpr mediated cell cycle arrest. Combined approaches of 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. Thus, whilst other HIV-1 accessory proteins downregulate a small number of specific host factors, Vpr degrades multiple protein targets, causing systems-level changes to the cellular proteome. Depletion of a subset of the novel cellular targets identified in this study, including KIF18A/B, ZNF512B and ESCO2, was conserved across Vpr variants from diverse primate lentiviral lineages, confirming their biological importance in vivo. Further, antagonism of multiple novel Vpr targets identified here can explain previously described cellular Vpr phenotypes such as modulation of NF-κB signalling, premature chromatid segregation, and cell cycle arrest.
Significance Statement HIV-1 encodes four ‘accessory proteins’, required for replication in vivo but not in vitro. The functions of three of these proteins are well characterised – they degrade a small number cellular proteins, that reflect the critical pathways HIV must antagonise to replicate within the host. The fourth accessory protein, Vpr, is much less understood. Here we show that unlike the other accessory proteins, Vpr causes hundreds of changes to the cellular proteome, by directly degrading at least 38 proteins. In fact, almost all protein-level changes that occur in an HIV infected cell require Vpr. These complex changes explain why understanding the function of Vpr has been problematic, and provide a dramatically different new context for existing and future studies of Vpr.