Cyclophilin A protects HIV-1 from restriction by human TRIM5α

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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Fig. 1: Disruption of the CA−CypA interaction in primary human blood cells renders HIV-1 susceptible to restriction by TRIM5.
Fig. 2: Human TRIM5α is sufficient to explain the inhibition of reverse transcription that results from disruption of CA−CypA interaction.
Fig. 3: Endogenous TRIM5α in primary human macrophages associates with HIV-1 CA after acute challenge but only when the CA−CypA interaction is disrupted.
Fig. 4: Endogenous TRIM5α suppresses the spread of HIV-1 infection in primary human macrophages and CD4+ T cells when the CA−CypA interaction is disrupted.

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.

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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.

Author information

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.

Correspondence to Jeremy Luban.

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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 Information

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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