HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B

Author summaryHIV-1 spreads efficiently through virological synapses (VSs), cell-cell interfaces induced by interaction of HIV-1 envelope protein on infected cells and the CD4 receptor on uninfected target cells. Using mass spectrometry, we identified changes in the levels and phosphorylation of many cellular proteins in HIV-1-infected cells after co-culture with CD4+ cells. We identified alterations in numerous cellular pathways, including striking effects on genes regulating the cell cycle. Notably, the key cell cycle regulatory kinase Aurora kinase B (AURKB) showed a dramatic decrease in kinase activity. We found that AURKB specifically inhibits HIV-1 Env-mediated cell fusion and cell-to-cell transmission. This suppression is specific to HIV-1 Env and alters Env's membrane fusion activity. We found that, in T cell lines and primary T cells, HIV-1 envelope-CD4 engagement prevents this suppression by causing premature relocalization of AURKB to nuclear centrosomes, but does not alter the cell cycle state of the infected cell. Thus, we identified cellular signaling pathways altered by co-culturing HIV-1+ and CD4+ T cells, novel effects of HIV-1 on cell cycle regulatory kinases and a previously unknown role for AURKB at the plasma membrane. The results have significant implications for understanding HIV-1 and cell processes and for controlling HIV-1. HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.