Abstract
The HIV-1 capsid (CA) protein lattice encases viral genomic RNA and regulates steps essential to target-cell invasion1. Cyclophilin A (CypA) has interacted with the CA of lentiviruses related to HIV-1 for millions of years2,3,4,5,6,7. Disruption of the CA−CypA interaction decreases HIV-1 infectivity in human cells8,9,10,11,12 but stimulates infectivity in non-human primate cells13,14,15. Genetic and biochemical data suggest that CypA protects HIV-1 from a CA-specific restriction factor in human cells16,17,18,19,20. Discovery of the CA-specific restriction factor tripartite-containing motif 5α (TRIM5α)21 and multiple, independently derived, TRIM5−CypA fusion genes4,5,15,22,23,24,25,26 pointed to human TRIM5α being the CypA-sensitive restriction factor. However, HIV-1 restriction by human TRIM5α in tumour cell lines is minimal21 and inhibition of such activity by CypA has not been detected27. Here, by exploiting reverse genetic tools optimized for primary human blood cells, we demonstrate that disruption of the CA−CypA interaction renders HIV-1 susceptible to potent restriction by human TRIM5α, with the block occurring before reverse transcription. Endogenous TRIM5α associated with virion cores as they entered the cytoplasm, but only when the CA−CypA interaction was disrupted. These experiments resolve the long-standing mystery of the role of CypA in HIV-1 replication by demonstrating that this ubiquitous cellular protein shields HIV-1 from previously inapparent restriction by human TRIM5α.
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Data availability
The plasmids described in Supplementary Table 1 are available at https://www.addgene.org/Jeremy_Luban/. All data generated or analysed during this study are presented in the Letter or Supplementary Information, or are available from the corresponding author on request.
References
Yamashita, M. & Engelman, A. N. Capsid-dependent host factors in HIV-1 infection. Trends Microbiol. 25, 741–755 (2017).
Luban, J., Bossolt, K. L., Franke, E. K., Kalpana, G. V. & Goff, S. P. Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell 73, 1067–1078 (1993).
Goldstone, D. C. et al. Structural and functional analysis of prehistoric lentiviruses uncovers an ancient molecular interface. Cell Host Microbe 8, 248–259 (2010).
Malfavon-Borja, R., Wu, L. I., Emerman, M. & Malik, H. S. Birth, decay, and reconstruction of an ancient TRIMCyp gene fusion in primate genomes. Proc. Natl Acad. Sci. USA 110, E583–E592 (2013).
Mu, D. et al. Independent birth of a novel TRIMCyp in Tupaia belangeri with a divergent function from its paralog TRIM5. Mol. Biol. Evol. 31, 2985–2997 (2014).
Gilbert, C., Maxfield, D. G., Goodman, S. M. & Feschotte, C. Parallel germline infiltration of a lentivirus in two Malagasy lemurs. PLoS Genet. 5, e1000425 (2009).
Katzourakis, A., Tristem, M., Pybus, O. G. & Gifford, R. J. Discovery and analysis of the first endogenous lentivirus. Proc. Natl Acad. Sci. USA 104, 6261–6265 (2007).
Braaten, D., Franke, E. K. & Luban, J. Cyclophilin A is required for an early step in the life cycle of human immunodeficiency virus type 1 before the initiation of reverse transcription. J. Virol. 70, 3551–3560 (1996).
Franke, E. K., Yuan, H. E. & Luban, J. Specific incorporation of cyclophilin A into HIV-1 virions. Nature 372, 359–362 (1994).
Thali, M. et al. Functional association of cyclophilin A with HIV-1 virions. Nature 372, 363–365 (1994).
Braaten, D. & Luban, J. Cyclophilin A regulates HIV‐1 infectivity, as demonstrated by gene targeting in human T cells. EMBO J. 20, 1300–1309 (2001).
Sokolskaja, E., Sayah, D. M. & Luban, J. Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity. J. Virol. 78, 12800–12808 (2004).
Towers, G. J. et al. Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors. Nat. Med. 9, 1138–1143 (2003).
Berthoux, L., Sebastian, S., Sokolskaja, E. & Luban, J. Cyclophilin A is required for TRIM5α-mediated resistance to HIV-1 in Old World monkey cells. Proc. Natl Acad. Sci. USA 102, 14849–14853 (2005).
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).
Luban, J. Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus type 1 infection. J. Virol. 81, 1054–1061 (2007).
Sayah, D. M. & Luban, J. Selection for loss of Ref1 activity in human cells releases human immunodeficiency virus type 1 from cyclophilin A dependence during infection. J. Virol. 78, 12066–12070 (2004).
Sebastian, S. & Luban, J. TRIM5α selectively binds a restriction-sensitive retroviral capsid. Retrovirology 2, 40 (2005).
Sebastian, S., Sokolskaja, E. & Luban, J. Arsenic counteracts human immunodeficiency virus type 1 restriction by various TRIM5 orthologues in a cell type-dependent manner. J. Virol. 80, 2051–2054 (2006).
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).
Stremlau, M. et al. The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys. Nature 427, 848–853 (2004).
Wilson, S. J. et al. Independent evolution of an antiviral TRIMCyp in rhesus macaques. Proc. Natl Acad. Sci. USA 105, 3557–3562 (2008).
Virgen, C. A., Kratovac, Z., Bieniasz, P. D. & Hatziioannou, T. Independent genesis of chimeric TRIM5-cyclophilin proteins in two primate species. Proc. Natl Acad. Sci. USA 105, 3563–3568 (2008).
Newman, R. M. et al. Evolution of a TRIM5-CypA splice isoform in old world monkeys. PLoS Pathog. 4, e1000003 (2008).
Brennan, G., Kozyrev, Y. & Hu, S.-L. TRIMCyp expression in Old World primates Macaca nemestrina and Macaca fascicularis. Proc. Natl Acad. Sci. USA 105, 3569–3574 (2008).
Boso, G. et al. Evolution of the rodent Trim5 cluster is marked by divergent paralogous expansions and independent acquisitions of TrimCyp fusions. Sci. Rep. 9, 11263 (2019).
Sokolskaja, E., Berthoux, L. & Luban, J. Cyclophilin A and TRIM5α independently regulate human immunodeficiency virus type 1 infectivity in human cells. J. Virol. 80, 2855–2862 (2006).
Pertel, T. et al. TRIM5 is an innate immune sensor for the retrovirus capsid lattice. Nature 472, 361–365 (2011).
Fellmann, C. et al. An optimized microRNA backbone for effective single-copy RNAi. Cell Rep. 5, 1704–1713 (2013).
McCauley, S. M. et al. Intron-containing RNA from the HIV-1 provirus activates type I interferon and inflammatory cytokines. Nat. Commun. 9, 5305 (2018).
Yurkovetskiy, L. et al. Primate immunodeficiency virus proteins Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex. Nat. Microbiol. 3, 1354–1361 (2018).
Adachi, A. et al. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59, 284–291 (1986).
Salazar-Gonzalez, J. F. et al. Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection. J. Exp. Med. 206, 1273–1289 (2009).
Mackman, R. L. et al. Discovery of a potent and orally bioavailable cyclophilin inhibitor derived from the sanglifehrin macrocycle. J. Med. Chem. 61, 9473–9499 (2018).
Husi, H. & Zurini, M. G. Comparative binding studies of cyclophilins to cyclosporin A and derivatives by fluorescence measurements. Anal. Biochem. 222, 251–255 (1994).
Varshavsky, A. Ubiquitin fusion technique and related methods. Methods Enzymol. 399, 777–799 (2005).
De Iaco, A. & Luban, J. Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus. Retrovirology 8, 98 (2011).
Wu, X., Anderson, J. L., Campbell, E. M., Joseph, A. M. & Hope, T. J. Proteasome inhibitors uncouple rhesus TRIM5 restriction of HIV-1 reverse transcription and infection. Proc. Natl Acad. Sci. USA 103, 7465–7470 (2006).
Colgan, J. et al. Cyclophilin A regulates TCR signal strength in CD4+ T cells via a proline-directed conformational switch in Itk. Immunity 21, 189–201 (2004).
Bosco, D. A., Eisenmesser, E. Z., Pochapsky, S., Sundquist, W. I. & Kern, D. Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A. Proc. Natl Acad. Sci. USA 99, 5247–5252 (2002).
Bock, M., Bishop, K. N., Towers, G. & Stoye, J. P. Use of a transient assay for studying the genetic determinants of Fv1 restriction. J. Virol. 74, 7422–7430 (2000).
Laguette, N. et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654–657 (2011).
Hrecka, K. et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658–661 (2011).
Petrillo, C. et al. Cyclosporine H overcomes innate immune restrictions to improve lentiviral transduction and gene editing in human hematopoietic stem cells. Cell Stem Cell 23, 820–832 (2018).
Reinhard, C., Bottinelli, D., Kim, B. & Luban, J. Vpx rescue of HIV-1 from the antiviral state in mature dendritic cells is independent of the intracellular deoxynucleotide concentration. Retrovirology 11, 12 (2014).
Butler, S. L., Hansen, M. S. & Bushman, F. D. A quantitative assay for HIV DNA integration in vivo. Nat. Med. 7, 631–634 (2001).
Acknowledgements
We thank T. Cihlar, B. Hahn, S. Hausmann, E. Hunter, R. Mackman, M. Pizzato and S. Yant for reagents. We are also grateful to anonymous blood donors who contributed leukocytes to this study. This work was supported by NIH grant nos. 5R01AI111809, 5DP1DA034990, 1R01AI117839 and 1R37AI147868 to J.L.
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K.K. and J.L. designed the experiments. K.K. conducted and analysed most experiments. S.M.M., C.C. and W.E.D. cloned the plasmids used in this study. A.D., S.M.M., and L.Y. performed the HIV-1 spreading infections. S.K. acquired and analysed PLA samples. C.S.-D.-C. and E.M.C. provided advice and technical expertise for the imaging experiments. K.K. and J.L. wrote the manuscript with input from all authors.
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Extended data
Extended Data Fig. 1 Gating strategy for flow cytometry experiments assessing single cycle infectivity.
Macrophage and dendritic cell populations, previously enriched as per the methods, were gated by SSC-A vs. FSC-A, as indicated, and then the GFP+ population was plotted vs. FSC-A. Enriched CD4+ T cells were gated by SSC-A vs FSC-A, as indicated, then a singlet population was gated from FSC-H vs. FSC-A, and finally GFP+ cells were plotted vs. FSC-A.
Extended Data Fig. 2 Assessment of shRNA-mediated knockdown in primary human blood cells.
a-c, Lentiviral vectors containing puromycin N- acetyltransferase (PuroR) and shRNA targeting TRIM5 or Luc were used to transduce macrophages (a), dendritic cells (b), or CD4+ T cells (c). At 3 days post-transduction, cells were selected with puromycin for 3 days. Total RNA was isolated from the macrophages and dendritic cells, followed by cDNA synthesis, and qPCR with TaqMan detection of TRIM5 and the housekeeping gene OAZ1, for normalization (mean ± SEM, n = 3 independent samples). Significance was determined by two-tailed, unpaired t-test (a and b). The selected CD4+ T cells were challenged with N- or B-MLV vector harboring GFP reporter for 3 days. Flow cytometry was used to assess the percentage of GFP+ cells. The infectivity of each vector in TRIM5 knockdown cells was normalized to the Luc control condition. Shown is mean ± SD (n = 3 donors for each). Significance was determined by two-tailed, paired t-test (c). d. Macrophages were simultaneously transduced with two lentiviral vectors, the first expressing shRNA targeting TRIM5 or Luc with PuroR, and the second expressing shRNA targeting CypA or Luc with blasticidin S-deaminase. After selection with both antibiotics, CypA and β-actin proteins were detected by western blot. Data shown is representative of three independent experiments using cells from three blood donors.
Extended Data Fig. 3 CA-CypA interaction promotes HIV-1 transduction by inhibiting TRIM5 activity in primary human blood cells.
a, Raw infectivity data for single cycle viruses, before normalization of infectivity to control condition. Shown are representative of three independent experiments using cells from three blood donors for each condition. b, Macrophages expressing shRNA targeting TRIM5 or Luc were challenged with single-cycle, VSV G-pseudotyped, HIV-1NL4-3GFP in the presence of 8 µM CsA, 8 µM CsH, or DMSO solvent (mean ± SEM, n = 2 donors). c and d, TRIM5 or Luc knockdown CD4+ T cells were challenged with single-cycle HIV-1NL4-3GFP (c) or HIV-1Z331M-TFGFP (d) in the presence of 2.5 µM GS-CypAi3 or DMSO solvent alone (mean ± SEM, n = 3 donors for each). e, HIV-1NL4-3GFP was used to challenge TRIM5 or Luc knockdown CD4+ T cells with 2.5 µM GS-CypAi48 or DMSO solvent alone (mean ± SEM, n = 2 donors). Flow cytometry was used to measure the percentage of GFP+ cells, followed by normalization to WT in Luc knockdown cells. Significance was determined by two-tailed, paired t- test for data generated with at least three donors (n = 3).
Extended Data Fig. 4 Saturation of TRIM5α-mediated restriction in primary human macrophages.
Luc or TRIM5 knockdown macrophages were simultaneously challenged with a constant amount of single-cycle, VSV G-pseudotyped HIV-1NL4-3GFP containing CA-P90A and the indicated quantities of HIV- 1NL4-3 VLPs harboring either WT CA or CA-P90A. The percentage of GFP+ cells was assessed by flow cytometry at day 3 post-challenge. Data shown here are representative of four independent experiments performed on cells from four blood donors.
Extended Data Fig. 5 CA-CypA interaction prevents association of endogenous TRIM5α with HIV-1 CA in primary human macrophages.
a-d, TRIM5 or Luc knockdown macrophages from a different blood donor than that used in Fig. 3 were challenged with VSV G-pseudotyped, HIV-1NL4-3GFP in the presence of 5 µM CsA or DMSO solvent for 2 hrs (a and b), or challenged with HIV-1NL4-3GFP bearing WT CA or CA-P90A (c and d). PLA was then performed using anti-CA (p24) and anti-TRIM5α antibodies. Representative images (a and c) show PLA puncta (red), nuclei stained with Hoechst (blue), and actin filaments stained with phalloidin (green). The plots (b and d) are the number of PLA puncta per cell in the PLA with mean ± SEM. b, Luc KD + CsA No Virus, n = 45 cells analyzed; Luc KD + DMSO + HIV-1, n = 45; Luc KD + CsA + HIV-1, n = 80; TRIM5 KD + CsA + HIV-1, n = 45. d, Luc KD + WT HIV-1, n = 20; Luc KD + CA-P90A HIV-1, n = 30; TRIM5 KD + CA-P90A HIV-1, n = 20; TRIM5 KD + WT HIV-1, n = 20. Significance was determined by two-tailed, unpaired t-test. Scale bars in a and c are 5 μm.
Extended Data Fig. 6 The effect of proteasome inhibitor treatment on the proximity ligation assay for HIV-1 CA and endogenous TRIM5α.
Luc control knockdown macrophages treated with 5 µM CsA were challenged with VSV G-pseudotyped HIV-1NL4- 3GFP in the presence of 2 µM MG132 or DMSO solvent. Cells were fixed and proximity ligation assay (PLA) was performed with anti-CA (p24) and anti-TRIM5α antibodies. Representative images show PLA puncta (red), nuclei stained with Hoechst (blue), and actin filaments stained with phalloidin (green). Scale bars are 5 μm. The graph on the right shows the number of puncta per cell in the PLA, after analysis of 30 cells per condition (mean ± SEM). Significance was determined by two-tailed, unpaired t- test.
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Supplementary Tables 1–3.
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Source Data Extended Data Fig. 2
Original blot of Extended Data Fig. 2d.
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Kim, K., Dauphin, A., Komurlu, S. et al. Cyclophilin A protects HIV-1 from restriction by human TRIM5α. Nat Microbiol 4, 2044–2051 (2019). https://doi.org/10.1038/s41564-019-0592-5
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DOI: https://doi.org/10.1038/s41564-019-0592-5
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