Animal cells harbour multiple innate effector mechanisms that inhibit virus replication. For the pathogenic retrovirus human immunodeficiency virus type 1 (HIV-1), these include widely expressed restriction factors1, such as APOBEC3 proteins2, TRIM5-α3, BST2 (refs 4, 5) and SAMHD1 (refs 6, 7), as well as additional factors that are stimulated by type 1 interferon (IFN)8,9,10,11,12,13,14. Here we use both ectopic expression and gene-silencing experiments to define the human dynamin-like, IFN-induced myxovirus resistance 2 (MX2, also known as MXB) protein as a potent inhibitor of HIV-1 infection and as a key effector of IFN-α-mediated resistance to HIV-1 infection. MX2 suppresses infection by all HIV-1 strains tested, has equivalent or reduced effects on divergent simian immunodeficiency viruses, and does not inhibit other retroviruses such as murine leukaemia virus. The Capsid region of the viral Gag protein dictates susceptibility to MX2, and the block to infection occurs at a late post-entry step, with both the nuclear accumulation and chromosomal integration of nascent viral complementary DNA suppressed. Finally, human MX1 (also known as MXA), a closely related protein that has long been recognized as a broadly acting inhibitor of RNA and DNA viruses, including the orthomyxovirus influenza A virus15,16, does not affect HIV-1, whereas MX2 is ineffective against influenza virus. MX2 is therefore a cell-autonomous, anti-HIV-1 resistance factor whose purposeful mobilization may represent a new therapeutic approach for the treatment of HIV/AIDS.
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We wish to thank L. Apolonia, A. Cimarelli, B. Hahn, T. Hatziioannou, J. Kappes, P. Mangeot, S. Papaioannou, N. Parrish, N. Sherer and C. Swanson for the provision of reagents and for discussions, and M. Mirza, E. Papouli, Q. Oscar Y. Li and G. Pacini for technical assistance. This work was supported by the UK Medical Research Council, the National Institutes of Health (DA033773), the European Commission’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. PIEF-GA-2009-237501 (to C.G.), a Wellcome Trust Research Training Fellowship (to T.D.) 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.
The authors declare no competing financial interests.
Extended data figures and tables
U87-MG CD4+ CXCR4+ cells were transduced with EasiLV expressing the different candidate cDNAs, CD8, GFP or TRIMCyp cDNA controls and treated with doxycycline for 48 h, left untransduced (Ctrl) or treated with IFN-α for 24 h before infection. The cells were infected with increasing viral inputs of VSV-G-pseudotyped NL4-3/Nef-IRES-Renilla (0.04–5 ng p24Gag) and infection efficiency was monitored at 48 h by measuring Renilla activity. Mean relative infection efficiencies from four independent experiments are shown.
a, CEM-SS cells were transduced with EasiLV expressing CD8 or MX2 and treated with doxycycline for 4–5 days and infected with increasing viral inputs of VSV-G-pseudotyped NL4-3/Nef-IRES-GFP (0.2–25 ng p24Gag). The efficiency of infection was measured at 72 h by scoring the percentage of GFP-expressing cells, among the E2-Crimson-expressing cells, by flow cytometry. Mean percentages of infected cells from independent duplicates are shown. b, 293T cells were transduced with lentiviral vectors containing a bicistronic expression cassette for either CD8 or MX2 with the puromycinR gene, and selected for puromycin resistance. Transduced cells were infected with increasing viral inputs of VSV-G-pseudotyped NL4-3/Nef-IRES-Renilla (0.2–25 ng p24Gag) and Renilla activity was measured at 48 h. Mean relative infection efficiencies from three independent experiments are shown.
RNA was extracted from control (Ctrl) or IFN-α-treated (IFNα) PMA-treated or dividing THP-1 cells, MDMs, primary CD4+ T cells, U87-MG, HT1080, CEM, CEM-SS, HUT78 and Jurkat cells. 500-ng aliquots of RNA were reverse transcribed and the levels of MX2, as well as ISG15, and two endogenous controls, GAPDH and β-actin (ACTB), were analysed by qRT–PCR. The graph shows the mean of relative levels of expression (normalized to both endogenous controls and compared to THP-1 without IFN-α treatment) obtained in three independent experiments.
Extended Data Figure 4 MX2 participates in IFN-α-induced resistance to HIV-1 in monocytic THP-1 cells.
a, THP-1 cells expressing a control shRNA or two different shRNAs targeting MX2 were treated with or without IFN-α (500 U ml−1) for 24 h. Cells were infected with five different doses of NL4-3/Nef-IRES-Renilla (0.04–25 ng p24Gag) for 48 h, and Renilla activity was measured. Mean relative infection efficiencies from two independent experiments are shown. b, Immunoblot analysis of parallel samples from a. Protein levels of MX2 and APOBEC3G (A3G, positive control for IFN-α treatment) were determined, and tubulin served as a loading control.
U87-MG CD4+ CXCR4+ cells were transduced with EasiLV expressing CD8 or MX2 and treated with doxycycline for 48 h, left untransduced (Ctrl) or treated with IFN-α for 24 h before infection. Increasing viral inocula (indicated in ng p24Gag) were used to infect the cells with HIV-1IIIB. The levels of infection were analysed at 48 h by measuring the percentage of cells expressing p24Gag after intracellular staining. The means of three independent experiments are shown. This analysis accompanies the experiments shown in Fig. 3.
Immunoblot analysis of parallel samples from Fig. 4e. Protein levels of Flag-tagged MX1 and MX2 proteins were determined using a Flag-specific antibody and tubulin served as a loading control.
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Goujon, C., Moncorgé, O., Bauby, H. et al. Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection. Nature 502, 559–562 (2013) doi:10.1038/nature12542
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