Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen–host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention.
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This work was supported by grants from the National Institutes of Health: R01AI047054, R21AI065321, R01AI081615 and R21AI097233 (to M.A.M.), P41GM103314 (to B.T.C. and D.F.), U54GM103511 (to M.P.R., B.T.C., M.A.M. and D.F.), P41GM109824 (to M.P.R. and B.T.C.), R01AI093278 and R33AI084714 (to J.M.B.) and DP1DA026192 and R21AI102187 (to I.M.C.). The authors thank V. Sahi (Aaron Diamond AIDS Research Center) for all flow cytometry employed in this study, C. Zhao and C. Schlieker (Yale University) for the TOR1AIP2 (LULL1) CRISPR/Cas9 knockout HeLa cell line, P. Nahirney (Rockefeller EM Resource Center) for imaging, M. Nussenzweig (Rockefeller University) for a trimerized derivative of YU-2 gp140 Env, K. Jacobs, J. Boland and D. Roberson of the NCI Core Genotyping Facility for ion torrent sequencing, and contributors to the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, for specified reagents described in the Supplementary Methods.
The authors declare no competing financial interests.
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Luo, Y., Jacobs, E., Greco, T. et al. HIV–host interactome revealed directly from infected cells. Nat Microbiol 1, 16068 (2016). https://doi.org/10.1038/nmicrobiol.2016.68
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