Article

HIV–host interactome revealed directly from infected cells

  • Nature Microbiology 1, Article number: 16068 (2016)
  • doi:10.1038/nmicrobiol.2016.68
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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.

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References

  1. 1.

    et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science 319, 921–926 (2008).

  2. 2.

    et al. Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathogens 5, e1000437 (2009).

  3. 3.

    et al. Global analysis of host–pathogen interactions that regulate early-stage HIV-1 replication. Cell 135, 49–60 (2008).

  4. 4.

    et al. Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe 4, 495–504 (2008).

  5. 5.

    & Mass spectrometry-based proteomic approaches for discovery of HIV–host interactions. Future Virol. 9, 979–992 (2014).

  6. 6.

    et al. Global landscape of HIV–human protein complexes. Nature 481, 365–370 (2012).

  7. 7.

    et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science 342, 1477–1483 (2013).

  8. 8.

    et al. Structural basis for hijacking CBF-β and CUL5 E3 ligase complex by HIV-1 Vif. Nature 505, 229–233 (2014).

  9. 9.

    et al. Structure and immune recognition of trimeric pre-fusion HIV-1 Env. Nature 514, 455–461 (2014).

  10. 10.

    et al. Immunogenicity of stabilized HIV-1 envelope trimers with reduced exposure of non-neutralizing epitopes. Cell 163, 1702–1715 (2015).

  11. 11.

    et al. CD4-induced activation in a soluble HIV-1 Env trimer. Structure 22, 974–984 (2014).

  12. 12.

    & Infectivity and neutralization of simian immunodeficiency virus with FLAG epitope insertion in gp120 variable loops. J. Virol. 81, 10838–10848 (2007).

  13. 13.

    , , & The human immunodeficiency virus type 1 envelope spike of primary viruses can suppress antibody access to variable regions. J. Virol. 83, 1649–1659 (2009).

  14. 14.

    , , , & Antibody binding is a dominant determinant of the efficiency of human immunodeficiency virus type 1 neutralization. J. Virol. 80, 11404–11408 (2006).

  15. 15.

    et al. The SOCS-box of HIV-1 Vif interacts with ElonginBC by induced-folding to recruit its Cul5-containing ubiquitin ligase complex. PLoS Pathogens 6, e1000925 (2010).

  16. 16.

    , , , & Gene loss and adaptation to hominids underlie the ancient origin of HIV-1. Cell Host Microbe 14, 85–92 (2013).

  17. 17.

    , , , & Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif. Nucleic Acids Res. 39, 2404–2415 (2011).

  18. 18.

    , & Human retroviral host restriction factors APOBEC3G and APOBEC3F localize to mRNA processing bodies. PLoS Pathogens 2, e41 (2006).

  19. 19.

    et al. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J. Proteome Res. 4, 1752–1756 (2005).

  20. 20.

    et al. 4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proc. Natl Acad. Sci. USA 107, 4967–4972 (2010).

  21. 21.

    et al. Molecular cloning of ADIR, a novel interferon responsive gene encoding a protein related to the torsins. Genomics 79, 315–325 (2002).

  22. 22.

    , , , & The torsin activator LULL1 is required for efficient growth of HSV-1. J. Virol. 89, 8444–8452 (2015).

  23. 23.

    & MHC class I antigen presentation: learning from viral evasion strategies. Nature Rev. Immunol. 9, 503–513 (2009).

  24. 24.

    & HIV's evasion of the cellular immune response. Immunol. Rev. 168, 65–74 (1999).

  25. 25.

    et al. The HLA-ER/HLA-ER genotype affects the natural course of hepatitis C virus (HCV) infection and is associated with HLA-E-restricted recognition of an HCV-derived peptide by interferon-γ-secreting human CD8+ T cells. J. Infect. Dis. 200, 1397–1401 (2009).

  26. 26.

    et al. Cell–cell transmission enables HIV-1 to evade inhibition by potent CD4bs directed antibodies. PLoS Pathogens 8, e1002634 (2012).

  27. 27.

    , , , & Inefficient human immunodeficiency virus replication in mobile lymphocytes. J. Virol. 81, 1000–1012 (2007).

  28. 28.

    et al. Partial rescue of V1V2 mutant infectivity by HIV-1 cell–cell transmission supports the domain inverted question marks exceptional capacity for sequence variation. Retrovirology 11, 75 (2014).

  29. 29.

    , & Mechanisms of enhanced HIV spread through T-cell virological synapses. Immunol. Rev. 251, 113–124 (2013).

  30. 30.

    et al. High-multiplicity HIV-1 infection and neutralizing antibody evasion mediated by the macrophage–T cell virological synapse. J. Virol. 88, 2025–2034 (2014).

  31. 31.

    et al. Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission. J. Exp. Med. 210, 2813–2821 (2013).

  32. 32.

    , & Cell–cell spread of human immunodeficiency virus type 1 overcomes tetherin/BST-2-mediated restriction in T cells. J. Virol. 84, 12185–12199 (2010).

  33. 33.

    et al. Cell-to-cell transmission can overcome multiple donor and target cell barriers imposed on cell-free HIV. PLoS ONE 8, e53138 (2013).

  34. 34.

    et al. Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy. Nature 477, 95–98 (2011).

  35. 35.

    , & Structural and functional mechanisms of CRAC channel regulation. J. Mol. Biol. 427, 77–93 (2014).

  36. 36.

    et al. DANGER, a novel regulatory protein of inositol 1,4,5-trisphosphate-receptor activity. J. Biol. Chem. 281, 37111–37116 (2006).

  37. 37.

    et al. Insulin-like growth factor 2 receptor is an IFNγ-inducible microglial protein that facilitates intracellular HIV replication: implications for HIV-induced neurocognitive disorders. Am. J Pathol. 177, 2446–2458 (2010).

  38. 38.

    et al. Characterization of host-cell line specific glycosylation profiles of early transmitted/founder HIV-1 gp120 envelope proteins. J. Proteome Res. 12, 1223–1234 (2013).

  39. 39.

    , & The Notch signalling system: recent insights into the complexity of a conserved pathway. Nature Rev. Genet. 13, 654–666 (2012).

  40. 40.

    et al. The immunological synapse: the gateway to the HIV reservoir. Immunol. Rev. 254, 305–325 (2013).

  41. 41.

    & CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency. EMBO J 26, 4985–4995 (2007).

  42. 42.

    , & Phosphorylation-dependent regulation of Notch1 signaling: the fulcrum of Notch1 signaling. BMB Rep. 48, 431–437 (2015).

  43. 43.

    et al. The tumor suppressor Ikaros shapes the repertoire of Notch target genes in T cells. Sci. Signal. 7, ra28 (2014).

  44. 44.

    , , , & Dynamics of the cellular metabolome during human cytomegalovirus infection. PLoS Pathogens 2, e132 (2006).

  45. 45.

    et al. CBFβ stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression. Mol. Cell 49, 632–644 (2013).

  46. 46.

    et al. Vif hijacks CBF-β to degrade APOBEC3G and promote HIV-1 infection. Nature 481, 371–375 (2012).

  47. 47.

    , , , & T-cell differentiation factor CBF-β regulates HIV-1 Vif-mediated evasion of host restriction. Nature 481, 376–379 (2012).

  48. 48.

    et al. Assembly of HIV-1 Vif-Cul5 E3 ubiquitin ligase through a novel zinc-binding domain-stabilized hydrophobic interface in Vif. Virology 349, 290–299 (2006).

  49. 49.

    , , , & Selective assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and upstream cysteines. Genes Dev. 18, 2867–2872 (2004).

  50. 50.

    et al. Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science 302, 1056–1060 (2003).

  51. 51.

    , & Multiple epitope tagging of expressed proteins for enhanced detection. Biotechniques 28, 789–793 (2000).

  52. 52.

    et al. Natural polymorphisms of human immunodeficiency virus type 1 integrase and inherent susceptibilities to a panel of integrase inhibitors. Antimicrob. Agents Chemother. 53, 4275–4282 (2009).

  53. 53.

    , & Sequential deletion of the integrase (Gag-Pol) carboxyl terminus reveals distinct phenotypic classes of defective HIV-1. J. Virol. 85, 4654–4666 (2011).

  54. 54.

    et al. Posttranslational acetylation of the human immunodeficiency virus type 1 integrase carboxyl-terminal domain is dispensable for viral replication. J. Virol. 81, 3012–3017 (2007).

  55. 55.

    & Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells. J. Virol. 69, 376–386 (1995).

  56. 56.

    et al. A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins. mBio 5, e01469 (2014).

  57. 57.

    , , & Dynamics of HIV-1 recombination in its natural target cells. Proc. Natl Acad. Sci. USA 101, 4204–4209 (2004).

  58. 58.

    Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365–369 (1967).

  59. 59.

    et al. Nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1. J. Virol. 80, 2515–2528 (2006).

  60. 60.

    et al. Characterizing anti-HIV monoclonal antibodies and immune sera by defining the mechanism of neutralization. Hum. Antibodies 14, 101–113 (2005).

  61. 61.

    et al. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J. Virol. 74, 627–643 (2000).

  62. 62.

    et al. The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J. Virol. 76, 7293–7305 (2002).

  63. 63.

    et al. The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha1→2 mannose residues on the outer face of gp120. J. Virol. 76, 7306–7321 (2002).

  64. 64.

    et al. Stability of a receptor-binding active human immunodeficiency virus type 1 recombinant gp140 trimer conferred by intermonomer disulfide bonding of the V3 loop: differential effects of protein disulfide isomerase on CD4 and coreceptor binding. J. Virol. 81, 4604–4614 (2007).

  65. 65.

    et al. Rapid development of glycan-specific, broad, and potent anti-HIV-1 gp120 neutralizing antibodies in an R5 SIV/HIV chimeric virus infected macaque. Proc. Natl Acad. Sci. USA 108, 20125–20129 (2011).

  66. 66.

    et al. Mechanism of neutralization by the broadly neutralizing HIV-1 monoclonal antibody VRC01. J. Virol. 85, 8954–8967 (2011).

  67. 67.

    et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856–861 (2010).

  68. 68.

    et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477, 466–470 (2011).

  69. 69.

    et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009).

  70. 70.

    et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature 491, 406–412 (2012).

  71. 71.

    , & gp41: HIV's shy protein. Nature Med. 10, 133–134 (2004).

  72. 72.

    et al. Epitope mapping and topology of baculovirus-expressed HIV-1 gp160 determined with a panel of murine monoclonal antibodies. AIDS Res. Hum. Retroviruses 10, 371–381 (1994).

  73. 73.

    , , & Generation of human monoclonal antibodies to human immunodeficiency virus. Proc. Natl Acad. Sci. USA 86, 1624–1628 (1989).

  74. 74.

    , , , & Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies. J. Virol. 65, 4832–4838 (1991).

  75. 75.

    et al. Nonneutralizing HIV-1 gp41 envelope cluster II human monoclonal antibodies show polyreactivity for binding to phospholipids and protein autoantigens. J. Virol. 85, 1340–1347 (2011).

  76. 76.

    et al. The Vif and Gag proteins of human immunodeficiency virus type 1 colocalize in infected human T cells. J. Virol. 71, 5259–5267 (1997).

  77. 77.

    , , & Human immunodeficiency virus type 1 Vif does not influence expression or virion incorporation of gag-, pol-, and env-encoded proteins. J. Virol. 70, 8263–8269 (1996).

  78. 78.

    , , , & Complementation of vif-defective human immunodeficiency virus type 1 by primate, but not nonprimate, lentivirus vif genes. J. Virol. 69, 4166–4172 (1995).

  79. 79.

    , & Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. Biotechniques 16, 730–735 (1994).

  80. 80.

    , , & Fluorescent proteins as proteomic probes. Mol. Cell. Proteom. 4, 1933–1941 (2005).

  81. 81.

    et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature 458, 636–640 (2009).

  82. 82.

    et al. A method for identification of HIV gp140 binding memory B cells in human blood. J. Immunol. Methods 343, 65–67 (2009).

  83. 83.

    , , & Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins. J. Virol. 74, 5716–5725 (2000).

  84. 84.

    et al. Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors. PLoS ONE 7, e50859 (2012).

  85. 85.

    et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–1857 (2012).

  86. 86.

    et al. Integrase inhibitors and cellular immunity suppress retroviral replication in rhesus macaques. Science 305, 528–532 (2004).

  87. 87.

    , , , & Aurora B-dependent regulation of class IIa histone deacetylases by mitotic nuclear localization signal phosphorylation. Mol. Cell. Proteom. 11, 1220–1229 (2012).

  88. 88.

    et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, D447–D456 (2016).

  89. 89.

    , , , & Improvements to the percolator algorithm for peptide identification from shotgun proteomics data sets. J. Proteome Res. 8, 3737–3745 (2009).

  90. 90.

    et al. Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis. Cell 159, 1096–1109 (2014).

  91. 91.

    & Mislocalization to the nuclear envelope: an effect of the dystonia-causing torsinA mutation. Proc. Natl Acad. Sci. USA 101, 847–852 (2004).

  92. 92.

    & AAA+ proteins: have engine, will work. Nature Rev. Mol. Cell Biol. 6, 519–529 (2005).

  93. 93.

    , , & TorsinA in the nuclear envelope. Proc. Natl Acad. Sci. USA 101, 7612–7617 (2004).

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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.

Author information

Author notes

    • Tommy Tong

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

    • Yang Luo
    • , Erica Y. Jacobs
    •  & Todd M. Greco

    These authors contributed equally to this work.

Affiliations

  1. Aaron Diamond AIDS Research Center, 455 1st Avenue, New York, New York 10016, 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, New York 10065, USA

    • Erica Y. Jacobs
    •  & Brian T. Chait
  3. Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08540, USA

    • Todd M. Greco
    •  & Ileana M. Cristea
  4. San Diego Biomedical Research Institute, 10865 Road to the Cure, San Diego, California 92121, USA

    • Tommy Tong
    •  & James M. Binley
  5. Department of Biochemistry, New York University Langone Medical Center, 227 East 30th Street, New York, New York 10016, USA

    • Sarah Keegan
    •  & David Fenyö
  6. Laboratory of Cellular and Structural Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, 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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mark A. Muesing.

Supplementary information

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  1. 1.

    Supplementary information

    Supplementary Figures 1-17 and Supplementary Tables 1 and 2.