Receptor usage dictates HIV-1 restriction by human TRIM5α in dendritic cell subsets

Abstract

The most prevalent route of HIV-1 infection is across mucosal tissues after sexual contact. Langerhans cells (LCs) belong to the subset of dendritic cells (DCs) that line the mucosal epithelia of vagina and foreskin and have the ability to sense and induce immunity to invading pathogens1. Anatomical and functional characteristics make LCs one of the primary targets of HIV-1 infection2. Notably, LCs form a protective barrier against HIV-1 infection and transmission3,4,5. LCs restrict HIV-1 infection through the capture of HIV-1 by the C-type lectin receptor Langerin and subsequent internalization into Birbeck granules5. However, the underlying molecular mechanism of HIV-1 restriction in LCs remains unknown. Here we show that human E3-ubiquitin ligase tri-partite-containing motif 5α (TRIM5α) potently restricts HIV-1 infection of LCs but not of subepithelial DC-SIGN+ DCs. HIV-1 restriction by TRIM5α was thus far considered to be reserved to non-human primate TRIM5α orthologues6,7,8,9, but our data strongly suggest that human TRIM5α is a cell-specific restriction factor dependent on C-type lectin receptor function. Our findings highlight the importance of HIV-1 binding to Langerin for the routeing of HIV-1 into the human TRIM5α-mediated restriction pathway. TRIM5α mediates the assembly of an autophagy-activating scaffold to Langerin, which targets HIV-1 for autophagic degradation and prevents infection of LCs. By contrast, HIV-1 binding to DC-SIGN+ DCs leads to disassociation of TRIM5α from DC-SIGN, which abrogates TRIM5α restriction. Thus, our data strongly suggest that restriction by human TRIM5α is controlled by C-type-lectin-receptor-dependent uptake of HIV-1, dictating protection or infection of human DC subsets. Therapeutic interventions that incorporate C-type lectin receptors and autophagy-targeting strategies could thus provide cell-mediated resistance to HIV-1 in humans.

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Figure 1: Human TRIM5α is a restriction factor for HIV-1 in LCs.
Figure 2: Autophagy restricts HIV-1 infection of LCs.
Figure 3: HIV-1 uptake by Langerin drives human TRIM5α restriction.
Figure 4: Human TRIM5α is a cell-specific restriction factor for HIV-1.

References

  1. 1

    Banchereau, J. et al. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767–811 (2000)

    CAS  Article  Google Scholar 

  2. 2

    Hladik, F. et al. Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1. Immunity 26, 257–270 (2007)

    CAS  Article  Google Scholar 

  3. 3

    Ribeiro, C. M. S., Sarrami-Forooshani, R. & Geijtenbeek, T. B. H. HIV-1 border patrols: Langerhans cells control antiviral responses and viral transmission. Future Virol. 10, 1231–1243 (2015)

    CAS  Article  Google Scholar 

  4. 4

    Sarrami-Forooshani, R. et al. Human immature Langerhans cells restrict CXCR4-using HIV-1 transmission. Retrovirology 11, 52 (2014)

    Article  Google Scholar 

  5. 5

    de Witte, L. et al. Langerin is a natural barrier to HIV-1 transmission by Langerhans cells. Nat. Med. 13, 367–371 (2007)

    CAS  Article  Google Scholar 

  6. 6

    Stremlau, M. et al. The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys. Nature 427, 848–853 (2004)

    CAS  Article  ADS  Google Scholar 

  7. 7

    Sawyer, S. L., Wu, L. I., Emerman, M. & Malik, H. S. Positive selection of primate TRIM5α identifies a critical species-specific retroviral restriction domain. Proc. Natl Acad. Sci. USA 102, 2832–2837 (2005)

    CAS  Article  ADS  Google Scholar 

  8. 8

    Song, B. et al. Retrovirus restriction by TRIM5α variants from Old World and New World primates. J. Virol. 79, 3930–3937 (2005)

    CAS  Article  Google Scholar 

  9. 9

    Sayah, D. M., Sokolskaja, E., Berthoux, L. & Luban, J. Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1. Nature 430, 569–573 (2004)

    CAS  Article  ADS  Google Scholar 

  10. 10

    van den Berg, L. M. et al. Caveolin-1 mediated uptake via Langerin restricts HIV-1 infection in human Langerhans cells. Retrovirology 11, 123 (2014)

    Article  ADS  Google Scholar 

  11. 11

    Stremlau, M. et al. Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5α restriction factor. Proc. Natl Acad. Sci. USA 103, 5514–5519 (2006)

    CAS  Article  ADS  Google Scholar 

  12. 12

    Yap, M. W., Nisole, S. & Stoye, J. P. A single amino acid change in the SPRY domain of human Trim5α leads to HIV-1 restriction. Curr. Biol. 15, 73–78 (2005)

    CAS  Article  Google Scholar 

  13. 13

    Ganser-Pornillos, B. K. et al. Hexagonal assembly of a restricting TRIM5α protein. Proc. Natl Acad. Sci. USA 108, 534–539 (2011)

    CAS  Article  ADS  Google Scholar 

  14. 14

    de Jong, M. A. et al. Mutz-3-derived Langerhans cells are a model to study HIV-1 transmission and potential inhibitors. J. Leukoc. Biol. 87, 637–643 (2010)

    CAS  Article  Google Scholar 

  15. 15

    Mandell, M. A. et al. TRIM proteins regulate autophagy and can target autophagic substrates by direct recognition. Dev. Cell 30, 394–409 (2014)

    CAS  Article  Google Scholar 

  16. 16

    Fujita, N. et al. Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin. J. Cell Biol. 203, 115–128 (2013)

    Article  Google Scholar 

  17. 17

    Moreau, K., Ravikumar, B., Renna, M., Puri, C. & Rubinsztein, D. C. Autophagosome precursor maturation requires homotypic fusion. Cell 146, 303–317 (2011)

    CAS  Article  Google Scholar 

  18. 18

    Gramberg, T. et al. Interactions of LSECtin and DC-SIGN/DC-SIGNR with viral ligands: differential pH dependence, internalization and virion binding. Virology 373, 189–201 (2008)

    CAS  Article  Google Scholar 

  19. 19

    Geijtenbeek, T. B. et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100, 587–597 (2000)

    CAS  Article  Google Scholar 

  20. 20

    Gringhuis, S. I. et al. HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells. Nat. Immunol. 11, 419–426 (2010)

    CAS  Article  Google Scholar 

  21. 21

    Blanchet, F. P. et al. Human immunodeficiency virus-1 inhibition of immunoamphisomes in dendritic cells impairs early innate and adaptive immune responses. Immunity 32, 654–669 (2010)

    CAS  Article  Google Scholar 

  22. 22

    Ward, E. M., Stambach, N. S., Drickamer, K. & Taylor, M. E. Polymorphisms in human Langerin affect stability and sugar binding activity. J. Biol. Chem. 281, 15450–15456 (2006)

    CAS  Article  Google Scholar 

  23. 23

    Gringhuis, S. I., den Dunnen, J., Litjens, M., van der Vlist, M. & Geijtenbeek, T. B. Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori . Nat. Immunol. 10, 1081–1088 (2009)

    CAS  Article  Google Scholar 

  24. 24

    Smith, A. L. et al. Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to the proteasome in dendritic cells. J. Exp. Med. 204, 421–430 (2007)

    CAS  Article  Google Scholar 

  25. 25

    Wu, X., Anderson, J. L., Campbell, E. M., Joseph, A. M. & Hope, T. J. Proteasome inhibitors uncouple rhesus TRIM5α restriction of HIV-1 reverse transcription and infection. Proc. Natl Acad. Sci. USA 103, 7465–7470 (2006)

    CAS  Article  ADS  Google Scholar 

  26. 26

    Perez-Caballero, D., Hatziioannou, T., Yang, A., Cowan, S. & Bieniasz, P. D. Human tripartite motif 5α domains responsible for retrovirus restriction activity and specificity. J. Virol. 79, 8969–8978 (2005)

    CAS  Article  Google Scholar 

  27. 27

    Stremlau, M., Perron, M., Welikala, S. & Sodroski, J. Species-specific variation in the B30.2(SPRY) domain of TRIM5α determines the potency of human immunodeficiency virus restriction. J. Virol. 79, 3139–3145 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Song, B. et al. The B30.2(SPRY) domain of the retroviral restriction factor TRIM5α exhibits lineage-specific length and sequence variation in primates. J. Virol. 79, 6111–6121 (2005)

    CAS  Article  Google Scholar 

  29. 29

    Shi, J. & Aiken, C. Saturation of TRIM5α-mediated restriction of HIV-1 infection depends on the stability of the incoming viral capsid. Virology 350, 493–500 (2006)

    CAS  Article  Google Scholar 

  30. 30

    Kootstra, N. A., Munk, C., Tonnu, N., Landau, N. R. & Verma, I. M. Abrogation of postentry restriction of HIV-1-based lentiviral vector transduction in simian cells. Proc. Natl Acad. Sci. USA 100, 1298–1303 (2003)

    CAS  Article  ADS  Google Scholar 

  31. 31

    Arrighi, J. F. et al. DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells. J. Exp. Med. 200, 1279–1288 (2004)

    CAS  Article  Google Scholar 

  32. 32

    Björndal, A. et al. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J. Virol. 71, 7478–7487 (1997)

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Setiawan, L. C. & Kootstra, N. A. Adaptation of HIV-1 to rhTrim5α-mediated restriction in vitro . Virology 486, 239–247 (2015)

    CAS  Article  Google Scholar 

  34. 34

    Eng, K. E., Panas, M. D., Karlsson Hedestam, G. B. & McInerney, G. M. A novel quantitative flow cytometry-based assay for autophagy. Autophagy 6, 634–641 (2010)

    CAS  Article  Google Scholar 

  35. 35

    Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544 (2012)

    CAS  Article  Google Scholar 

  36. 36

    Chan, L. L. et al. A novel image-based cytometry method for autophagy detection in living cells. Autophagy 8, 1371–1382 (2012)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to the members of the Host Defense group and Laboratory for Viral Immune Pathogenesis (Department of Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands) for their input and D. Picavet (van Leeuwenhoek Centrum for Advanced Microscopy, Academic Medical Center, Amsterdam, The Netherlands) for technical assistance during confocal experiments. We wish to thank the Boerhaave Medical Centre (Amsterdam, The Netherlands) and A. Knottenbelt (Flevoclinic, Almere, The Netherlands) for the provision of human skin tissues. This work was supported by the Dutch Scientific Organization NWO (VENI 863.13.025 and VICI 918.10.619), Aids Fonds (2010038) and European Research Council (Advanced grant 670424).

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Authors

Contributions

C.M.S.R. designed, performed and interpreted most experiments and prepared the manuscript; R.S.F. assisted with the lentiviral transductions and the confocal experiments; L.C.S. assisted with culturing the CD4+CCR5+ U87 cell lines and with the lentiviral transductions; E.M.Z.W. cultured MUTZ-LCs and helped with immunoblotting; J.L.v.H. assisted with primary cell isolation and silencing experiments; W.T. and N.N.v.d.W. performed the EM microscopy; N.A.K. and S.I.G. helped prepare the manuscript and T.B.H.G. supervised all aspects of the project.

Corresponding authors

Correspondence to Carla M. S. Ribeiro or Teunis B. H. Geijtenbeek.

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The authors declare no competing financial interests.

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Nature thanks J. Luban, C. Munz and G. Towers for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Langerin in MUTZ-LCs restricts HIV-1 integration, infection and transmission to CD4+ T cells.

a, b, HIV-1NL4.3 integration (a) and infection (b) of MUTZ-LCs after Langerin silencing, determined by Alu-PCR (a) and intracellular p24 staining (b). c, HIV-1NL4.3-BaL transmission by MUTZ-LCs after Langerin silencing, determined in LC and T-cell coculture by intracellular p24 staining. d, e, Silencing was confirmed by real-time PCR (d) or by flow cytometer (e; representative of n = 3). mRNA expression was normalized to β-actin (d) and set at 1 in control-siRNA treated cells. *P < 0.05 (two-tailed t-test). Data are mean ± s.d. of three (a, c, d) and four (b) independent experiments.

Extended Data Figure 2 Silencing of TRIM5α, Atg5, Atg16L1 and LSP-1 by RNA interference.

ak, Indicated proteins were silenced using specific SMARTpools and non-targeting siRNA as a control. Silencing was confirmed by real-time PCR (ag) or by immunoblotting (β-actin served as loading control; hk) in MUTZ-LCs (a, d, e, h, i, j), primary LCs (b), DCs (c), CD4+CCR5+ U87 parental cells (f) or CD4+CCR5+ U87 cells transduced with either Langerin (f, k) or rhesus TRIM5α (g). mRNA expression was normalized to β-actin (a, d, e) or GAPDH (b, c, f, g) and set at 1 in cells treated with control siRNA. Relative abundance of indicated proteins was quantified by normalizing to β-actin and set at 1 in control siRNA treated cells. Representative of n = 2 (d, e, hk). For gel source data, see Supplementary Fig. 1. Data are mean ± s.d. of three (a, c, f, g) and six (b) independent experiments.

Extended Data Figure 3 Human TRIM5α-mediated restriction in LCs or, the lack thereof in DCs, is independent of virus tropism.

ad, HIV-1NL4.3 (X4, CXCR4-tropic virus) or HIV-1NL4.3-BaL (R5, CCR5-tropic virus) integration (a, c) and infection (b, d) of primary LCs (a, b) or DCs (b, d) after TRIM5α silencing determined by Alu-PCR (a, c) and intracellular p24 staining (b, d). *P < 0.05, **P < 0.01 (two-tailed t-test). Data are mean ± s.d. of three (ad) independent experiments.

Extended Data Figure 4 ULK1 complex-dependent autophagy restricts HIV-1 integration in LCs and human TRIM5α restriction is dependent on Atg5 function.

a, TRIM5α, p62 and Atg16L1 in whole-cell lysates of uninfected MUTZ-LCs before (input) or after immunoprecipitation with Atg16L1, p62, TRIM5α, rabbit IgG control (as control for Atg16L1 and TRIM5α IP) or mouse IgG2a isotype control (as control for p62 immunoprecipitation), determined by immunoblotting (n.d., not determined). b, Autophagy induction in primary LCs pre-treated with bafilomycin followed by incubation with HIV-1NL4.3, determined by immunoblotting for LC3. For gel source data, see Supplementary Fig. 1. c, HIV-1NL4.3 infection of MUTZ-LCs after Atg5 or Atg16L1 silencing, determined by intracellular p24 staining. d, HIV-1NL4.3 integration into MUTZ-LCs after Atg13 or FIP200 silencing, determined by Alu-PCR. e, f, HIV-1NL4.3 integration (e) or infection (f) of MUTZ-LCs after Atg5, TRIM5α silencing or simultaneously with Atg5 and TRIM5α silencing, determined by Alu-PCR (e) and intracellular p24 staining (f). Data are representative of three (a) or two (b, df) experiments and mean ± s.d. of four independent experiments (c).

Extended Data Figure 5 Increased Atg5 recruitment into TRIM5α–Atg16L1 complex scaffold in CD4+CCR5+ U87 transfectants.

a, Atg5, TRIM5α and Atg16L1 in whole-cell lysates of CD4+CCR5+ U87 parental cells (U87) or transduced with either human TRIM5α (U87 hu5α) or rhesus TRIM5α (U87 rh5α) infected with HIV-1NL4.3-BaL before (input) or after immunoprecipitation with Atg16L1 or rabbit IgG control, determined by immunoblotting (n.d., not determined). b, Autophagy induction in U87 transfectants with bafilomycin followed by incubation with HIV-1SF162, determined by immunoblotting for LC3 (autophagy induction in control CD4+CCR5+ U87 parental cells presented in Fig. 3o). Relative abundance of LC3 II determined by normalizing to β-actin. Representative of n = 2 (a, b). For gel source data, see Supplementary Fig. 1. c, HIV-1SF162 infection of CD4+CD5+ U87 cells transduced with rhesus TRIM5α after Atg16L1 silencing, determined by intracellular p24 staining. *P < 0.05 (t-test). Data are mean ± s.d. of three (c) independent experiments.

Extended Data Figure 6 Human TRIM5α induces autophagy upon HIV-1 exposure in Langerin+ U87 transfectant and interacts with Langerin through LSP-1, but not Atg16L1.

a, Atg5, TRIM5α and Atg16L1 in whole-cell lysates of CD4+CCR5+ U87 parental cells (U87) or transduced with Langerin (U87 Langerin) before (input) or after immunoprecipitation with Atg16L1 or rabbit IgG control, determined by immunoblotting (n.d., not determined). b, Autophagy levels in Langerin+ U87 transfectant after TRIM5α silencing, pre-treated with bafilomycin followed by incubation with HIV-1NL4.3-BaL, determined by intracellular LC3 II levels by flow cytometer. c, LSP-1 in whole-cell lysates of MUTZ-LCs infected with HIV-1NL4.3 before (input) or after immunoprecipitation with TRIM5α or rabbit IgG control. d, TRIM5α in whole-cell lysates of Langerin+ U87 transfectant after Atg16L1 silencing before (input) or after immunoprecipitation with Langerin, determined by immunoblotting. For gel source data, see Supplementary Fig. 1. e, Autophagy induction in Langerin+ U87 transfectant pre-treated with bafilomycin followed by incubation with VSV-G-pseudotyped HIV-1, determined by intracellular LC3 II levels. Data are representative of two experiments (ae).

Extended Data Figure 7 Proteosome inhibition does not relieve Langerin-mediated restriction of HIV-1 reverse-transcription products nor infection.

ad, R/gag proviral DNA levels (a, c) and HIV-1 infection (b, d) in CD4+CCR5+ U87 parental cells (U87) or cells transduced with either rhesus TRIM5α (U87 rh5α) or Langerin (U87 Lang) after pre-treatment with proteosome inhibitor MG-132 and infected with VSV-G-pseudotyped HIV-1 (a, b; VSV-G) or HIV-1NL4.3-BaL (c, d; HIV-1), determined by qPCR (a, c) and intracellular p24 staining (b, d). Data are representative of two experiments (ad).

Extended Data Figure 8 Langerin-controlled human TRIM5α restriction mechanism in Langerhans cells.

a, HIV-1 binding to Langerin in Langerhans cells drives human TRIM5α-mediated restriction of viral integration, HIV-1 infection and HIV-1 transmission to CD4+ T cells. b, Langerin associates at steady-state with LSP-1–TRIM5α–Atg16L1 complex. Capture of HIV-1 by Langerin targets internalization of the incoming virus into Birbeck granules. Upon viral fusion, human TRIM5α mediates recruitment of Atg5 to TRIM5α–Atg16L1–HIV-1p24 capsid complex, which promotes lipidation of LC3 (LC3 II) and thereby elicit autophagosome formation. Vesicles containing Langerin–HIV-1 capsid complexes are subsequently targeted into autophagosomes for lysosomal degradation, which prevents infection of Langerhans cells.

Extended Data Table 1 Primer sequences used for mRNA expression and HIV-1 integration assay

Supplementary information

Supplementary Information

DESCRIPThis file contains Supplementary Figure 1, gel source data with size marker indications for Figure 2a; Figure 3e,g,o; Figure 4e,i; Extended Data Fig. 2h-k, Extended Data Fig. 4a,b, Extended Data Fig. 5a,b and Extended Data Fig. 6a,c,d. It also contains Supplementary Table 1, primary data of HIV-1 integration Alu-PCR assay and calculation of relative HIV-1 integration; see also Figure 1a.TION (PDF 2796 kb)

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Ribeiro, C., Sarrami-Forooshani, R., Setiawan, L. et al. Receptor usage dictates HIV-1 restriction by human TRIM5α in dendritic cell subsets. Nature 540, 448–452 (2016). https://doi.org/10.1038/nature20567

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