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HIV–host interactome revealed directly from infected cells

Nature Microbiology volume 1, Article number: 16068 (2016) Cite this article

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Abstract

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.

Due to its small genome, many biological processes that occur during HIV-1 infection and propagation are probably achieved by a heavy reliance on its host. In turn, some of these cellular factors impart antiviral defences. Elucidating these virus–host protein interactions—the viral interactome—may therefore help fully reveal how HIV-1 gains control of the host and how the cell attempts to counteract infection.

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Figure 1: Selection of replication-competent, tagged derivatives of HIV-1.
Figure 2: Permissible sites of insertion within Env and Vif and visualization of each tagged protein during infection.
Figure 3: Reciprocal mass spectrometric isotopic differentiation of interactions as random or targeted (MS I-DIRT) (ref. 19) plot for Env (including inset of region from 0.97 to 1.0) and histogram of the number of proteins versus average I-DIRT ratios for Env.
Figure 4: Vif interactors identified by reciprocal MS I-DIRT analysis and documentation of differential binding kinetics of its interactors.
Figure 5: Experimental documentation for select interactors.
Figure 6: Summary of biological subclasses of cellular interactors engaged with Vif and Env during active viral growth.

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Acknowledgements

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.

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Author notes

  1. Tommy Tong

    Present address: †Present address: Department of Biological Sciences, Faculty of Science and Technology, Sunway University, Jalan University, No. 5, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia,

  2. Yang Luo, Erica Y. Jacobs and Todd M. Greco: These authors contributed equally to this work.

Authors and Affiliations

  1. Aaron Diamond AIDS Research Center, 455 1st Avenue, New York, 10016, New York, USA

    Yang Luo, Kevin D. Mohammed & Mark A. Muesing

  2. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Avenue, New York, 10065, New York, USA

    Erica Y. Jacobs & Brian T. Chait

  3. Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, 08540, New Jersey, USA

    Todd M. Greco & Ileana M. Cristea

  4. San Diego Biomedical Research Institute, 10865 Road to the Cure, San Diego, 92121, California, USA

    Tommy Tong & James M. Binley

  5. Department of Biochemistry, New York University Langone Medical Center, 227 East 30th Street, New York, 10016, New York, USA

    Sarah Keegan & David Fenyö

  6. Laboratory of Cellular and Structural Biology, The Rockefeller University, 1230 York Avenue, New York, 10065, New York, USA

    Michael P. Rout

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Contributions

Y.L. and M.A.M. designed and developed the genetic strategy used to select epitope-tagged, replication-competent viruses. I.M.C., E.Y.J., M.P.R. and B.T.C. designed and developed the proteomic and MS approaches described. Y.L. generated all libraries and performed the genetic, virological, immunofluorescent, electron microscopic studies and immunoisolations. T.M.G. performed the mass spectrometric analyses. D.F., T.M.G., E.Y.J. and Y.L. compiled and evaluated the MS data. S.K., D.F., E.Y.J. and Y.L. analysed the deep sequencing results of the mutagenic landscape. Y.L. performed molecular characterization of tagged viral clones. T.T. and J.M.B. determined the oligomeric status of the HIV-1 tagged Env pullouts. Y.L. and K.D.M. performed the neutralization and super-pseudotyping experiments, and assayed Notch1 processing and phosphorylation, while E.Y.J. conducted Env reciprocal immunoprecipitations and Vif co-transfection experiments. Y.L., E.Y.J., T.M.G., T.T., J.M.B., M.P.R., B.T.C. and M.A.M. wrote the manuscript, with assistance from all authors.

Corresponding author

Correspondence to Mark A. Muesing.

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Supplementary Figures 1-17 and Supplementary Tables 1 and 2. (PDF 1780 kb)

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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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