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MX2-mediated innate immunity against HIV-1 is regulated by serine phosphorylation

An Author Correction to this article was published on 13 August 2021

This article has been updated

Abstract

The antiviral cytokine interferon activates expression of interferon-stimulated genes to establish an antiviral state. Myxovirus resistance 2 (MX2, also known as MxB) is an interferon-stimulated gene that inhibits the nuclear import of HIV-1 and interacts with the viral capsid and cellular nuclear transport machinery. Here, we identified the myosin light chain phosphatase (MLCP) subunits myosin phosphatase target subunit 1 (MYPT1) and protein phosphatase 1 catalytic subunit-β (PPP1CB) as positively-acting regulators of MX2, interacting with its amino-terminal domain. We demonstrated that serine phosphorylation of the N-terminal domain at positions 14, 17 and 18 suppresses MX2 antiviral function, prevents interactions with the HIV-1 capsid and nuclear transport factors, and is reversed by MLCP. Notably, serine phosphorylation of the N-terminal domain also impedes MX2-mediated inhibition of nuclear import of cellular karyophilic cargo. We also found that interferon treatment reduces levels of phosphorylation at these serine residues and outline a homeostatic regulatory mechanism in which repression of MX2 by phosphorylation, together with MLCP-mediated dephosphorylation, balances the deleterious effects of MX2 on normal cell function with innate immunity against HIV-1.

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Fig. 1: The NTD of MX2 interacts with MYPT1 and PPP1CB.
Fig. 2: MX2 requires functional MLCP for antiviral activity.
Fig. 3: IFN-induced MX2-mediated inhibition of HIV-1 requires MLCP.
Fig. 4: MX2 antiviral activity is antagonized by phosphorylation.
Fig. 5: Phosphorylation of MX2 reduces HIV-1 capsid binding and nuclear envelope accumulation.
Fig. 6: MX2-phosphorylation and nuclear import of cellular cargo.

Data availability

The plasmids generated in this study are available upon request without restriction. All datasets generated in this study are available as source data. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE49 partner repository with the dataset identifier PXD026073 (SILAC data) and PXD026090 and 10.6019/PXD026090 (phosphorylation data). Source data are provided with this paper.

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Acknowledgements

We thank D. Pollpeter, L. Ventimiglia, S. Papaioannou, P. Lehner, J. Cason, C. Mant, M. Kane and P. Bieniasz for the provision of reagents and helpful discussions. The work was supported by the Wellcome Trust (106223/Z/14/Z), the Medical Research Council (MR/M001199/1), the National Institutes of Health (U54 GM103368, AI150472) and the Department of Health via a National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. J.M.J.-G. is a long-term fellow of the European Molecular Biology Organization (ALTF 663-2016).

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G.B. and M.H.M. designed the study and wrote the manuscript with input from all co-authors; G.B. carried out the experiments and analysed the data, with contributions from A.S. in Fig. 1, M.D.J.D. in Fig. 2, J.M.J.-G. in Figs. 3 and 5 and Extended Data Fig. 2, H.K. in Fig. 4, S.L. in Fig. 1 and R.A. in Fig. 4; A.E.S., M.D.J.D. and J.M.J.-G. contributed to the execution of experiments and provided reagents; and M.H.M. supervised all aspects of the project.

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Correspondence to Gilberto Betancor or Michael H. Malim.

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The authors declare no competing interests.

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Peer review information Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Effect of MYPT1 and PPP1CB depletion on MX2-mediated inhibition of HIV-1.

a, Individual siRNAs targeting MYPT1 or PPP1CB, as well as a CTR siRNA were used to transfect U87-MG CD4+ CXCR4+ cells expressing MX2 or Luc. 48 h after challenging with HIV-1/GFP, infectivity was determined by flow cytometry (n = 4 biological replicates mean ± SD; two-tailed unpaired t-test). Immunoblot analyses to the right demonstrate the extents of reductions in protein levels. b, U87-MG CD4+ CXCR4+ cells were transduced with pEasiLV expressing Luc or MX2 and pretreated with doxycycline for 48 h before being doubly transfected with CTR siRNA or siRNAs targeting MYPT1 and/or PPP1CB. Levels of HIV-1/GFP infectivity were determined by flow cytometry at 48 h (n = 4 biological replicates, mean ± SD; two-tailed unpaired t-test). Immunoblot analyses to the right demonstrate the extents of reductions in protein levels.

Source data

Extended Data Fig. 2 Role of functional MLCP in viral infection.

a, U87-MG CD4+ CXCR4+ cells were doubly transduced with pEasiLV expressing Flag-tagged Luc or MX2 and also with pEasiLV-BFP expressing Flag-tagged Luc, MYPT1 or PPP1CB. Infectivity data with HIV-1/GFP are shown (above) and immunoblotting was used to determine the expression of each construct (below) (n = 6 biological replicates, mean ± SD; two-tailed unpaired t-test). b, 293T cells doubly transfected with pCAGGs plasmids expressing HA-tagged MX2 and either Flag-tagged MX1 or Flag-tagged MYPT1 were lysed, MX2 was immunoprecipitated using an anti-HA antibody and recovered proteins analyzed by immunoblot using anti-PPP1CB, anti-HA or anti-Flag antibodies. All experiments were done at least 4 times. c, Titration of calyculin A and okadaic acid in U87-MG CD4+ CXCR4+ expressing Luc or MX2 and challenged with MLV/GFP. The number of infected cells was determined by enumeration of GFP expressing cells by flow cytometry 48 h after challenging (n = 5 biological replicates for calyculin A; n = 3 biological replicates for okadaic acid, mean ± SD, (ns) non-significant; two-tailed unpaired t-test).

Source data

Extended Data Fig. 3 MX2, MYPT1 and PPP1CB do not participate in IFN-mediated suppression of MLV infection.

U87-MG CD4+ CXCR4+ cells depleted of MX2 using CRISPR-Cas9 genome editing (bulk population and clone MX2 CRISPR_2) and CTR CRISPR cells were transduced with pEasiLV expressing Luc or MX2, incubated in the presence or absence of 500 U/ml IFNα, and challenged with MLV/GFP. Infectivity was determined 48 h later by flow cytometry (n = 4 biological replicates, mean ± SD; two-tailed unpaired t-test).

Source data

Extended Data Fig. 4 Phosphorylation of MX2.

a, Example of LC-MSMS data from LysC generated MX2 peptides spanning A4 to K20, acquired on an Orbitrap Lumos and processed in PEAKS 8. The spectrum on the left shows the unphosphorylated form of the peptide and the spectrum on the right shows the corresponding peptide with putative phosphorylation at S14. b, 293T cells expressing C-terminally Flag-tagged MX2 were treated with 3 nM calyculin-A. After 6 h, cells were lysed, MX2 immunoprecipitated and the resulting protein digested with LysC. LC-MSMS data were processed in PEAKS 8. Sum peak areas for peptides spanning from M1 to K20 and covering serines 14, 17 and 18 were calculated using the areas under the curves, where the x-axis represents time and the y-axis represents intensity.

Source data

Extended Data Fig. 5 Phosphorylation of serines 14, 17 and 18 antagonizes the antiviral activity of MX2.

a, Residues S14, S17 and S18 in the N-terminal domain of the chimeras MX1 (NTDMX2) or Fv1b (NTDMX2) were substituted for alanine or aspartic acid and the effects on HIV-1/GFP infectivity measured at 48 h by flow cytometry (n = 4 biological replicates for Luc, MX2, MX1 (NTDMX2) and MX1 (NTDMX2) S14, 17-18A; n = 5 biological replicates for MX1 (NTDMX2) S14, 17-18D, Fv1b (NTDMX2), Fv1b (NTDMX2) S14, 17-18A and Fv1b (NTDMX2) S14, 17-18D, mean ± SD). Protein expression was confirmed by immunoblot. b, Residues 14, 17 and 18 of Flag-tagged MX2 were substituted for aspartic acid individually, in pairs or in triplicate. U87-MG CD4+ CXCR4+ cells expressing Luc, wild type or mutant proteins were challenged with HIV-1/GFP and infectivity was determined at 48 h by flow cytometry (n = 5 biological replicates, mean ± SD). Below, immunoblot showing protein expression.

Source data

Extended Data Fig. 6 Gating strategy for flow cytometry analysis of GFP+ infected cells.

a, Cells were distinguished from debris using the forward scatter (FCS, related to the cell size) and side scatter (SSC, related to the cell granularity). b, Selected population was gated for GFP+ (FITC channel) using uninfected cells as negative control. c, HIV-1/GFP infected cells were then gated for GFP. A representative plot showing GFP+ cells is shown.

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Betancor, G., Jimenez-Guardeño, J.M., Lynham, S. et al. MX2-mediated innate immunity against HIV-1 is regulated by serine phosphorylation. Nat Microbiol 6, 1031–1042 (2021). https://doi.org/10.1038/s41564-021-00937-5

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