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MX2 is an interferon-induced inhibitor of HIV-1 infection

Abstract

HIV-1 replication can be inhibited by type I interferon (IFN), and the expression of a number of gene products with anti-HIV-1 activity is induced by type I IFN1,2. However, none of the known antiretroviral proteins can account for the ability of type I IFN to inhibit early, preintegration phases of the HIV-1 replication cycle in human cells3,4. Here, by comparing gene expression profiles in cell lines that differ in their ability to support the inhibitory action of IFN-α at early steps of the HIV-1 replication cycle, we identify myxovirus resistance 2 (MX2) as an interferon-induced inhibitor of HIV-1 infection. Expression of MX2 reduces permissiveness to a variety of lentiviruses, whereas depletion of MX2 using RNA interference reduces the anti-HIV-1 potency of IFN-α. HIV-1 reverse transcription proceeds normally in MX2-expressing cells, but 2-long terminal repeat circular forms of HIV-1 DNA are less abundant, suggesting that MX2 inhibits HIV-1 nuclear import, or destabilizes nuclear HIV-1 DNA. Consistent with this notion, mutations in the HIV-1 capsid protein that are known, or suspected, to alter the nuclear import pathways used by HIV-1 confer resistance to MX2, whereas preventing cell division increases MX2 potency. Overall, these findings indicate that MX2 is an effector of the anti-HIV-1 activity of type-I IFN, and suggest that MX2 inhibits HIV-1 infection by inhibiting capsid-dependent nuclear import of subviral complexes.

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Figure 1: Differential effects of IFN-α on HIV-1 infection of monocytoid cell lines correlates with MX2 expression.
Figure 2: Inhibition of lentivirus infection by wild-type and mutant MX2, but not other differentially interferon-induced genes.
Figure 3: MX2 inhibits replication-competent HIV-1 and is required for the full antiviral activity of IFN-α.
Figure 4: MX2 activity reduces levels of nuclear HIV-1 DNA, is capsid dependent and is more potent in non-dividing cells.

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Acknowledgements

We thank members of The Rockefeller University Genomics Resource Center for assistance with the microarray experiments and members of the Bieniasz laboratory for discussion and advice. This work was supported by grants from the National Institutes of Health; R37AI64003 (to P.D.B.), R01AI078788 (to T.H.) R01AI100720 (to M.Y.), AI091707 to C.M.R., AI057158 (to I. Lipkin, Northeast Biodefense Center, subcontracted to C.M.R.) and DK095031 to J.W.S., the Greenberg Medical Research Institute and the Starr Foundation (C.M.R.) and by the Howard Hughes Medical Institute.

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Authors and Affiliations

Authors

Contributions

M.K., S.S.Y., J.B., S.B.K., T.Z. and S.J.W. designed and executed the experiments and analysed the data. J.W.S. and C.M.R. provided an interferon-stimulated gene library and advice. M.Y. provided reagents and advice. T.H. provided reagents and advice and supervised the work. P.D.B. conceived the study, supervised the work and wrote the paper, with additional input from all authors.

Corresponding author

Correspondence to Paul D. Bieniasz.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Candidate anti-HIV-1 genes from the microarray analysis.

mRNA levels, determined using Illumina BeadChips and given in arbitrary units, for genes whose differential induction in undifferentiated and PMA-treated THP-1, K562 and U937 cells correlated best with the anti-HIV-1 effect of IFN-α.

Extended Data Figure 2 Additional candidate anti-HIV-1 genes from the microarray analysis.

mRNA levels, determined using Illumina BeadChips and given in arbitrary units, for genes whose differential induction in undifferentiated and MA-treated THP-1, K562 and U937 cells correlated to some degree with the anti-HIV-1 effect of IFN-α.

Extended Data Figure 3 Induction of MX2 by IFN-α in primary CD4+ T cells and macrophages.

Western blot analysis of MX2 and tubulin expression in purified CD4+ T cells, activated with PHA or anti-CD3/CD28, and macrophages treated for 24 h with the indicated doses of IFN-α. Numbers below each lane indicate fluorescence intensity associated with the MX2 band. The second more rapidly migrating MX2 species that was detected inconsistently is of unknown provenance, and may represent a proteolytic breakdown product, or may arise through the use of an alternative start codon at amino acid 25, generating an MX2 protein that lacks the NLS.

Extended Data Figure 4 MX2 is required for the full antiviral activity of IFN-α in HOS cells.

a, Western blot analysis of MX2 expression HOS cells transduced with vectors expressing control or MX2-targeted shRNAs, and treated with IFN-α. Numbers below each lane indicate fluorescence intensity associated with the MX2 band. b, Infectious titre of an HIV-1–GFP reporter virus determined using the shRNA-containing HOS cells from a, with or without IFN-α treatment. Titres are mean + s.d., n = 3 technical replicates, P values calculated using unpaired t-test, representative of three experiments.

Extended Data Figure 5 MX2 activity reduces levels of nuclear HIV-1 DNA in K562 cells.

Quantitative PCR analysis of reverse transcript (left) and 2-LTR circle (right) abundance in empty-vector untreated (none) nevirapine-treated or MX2-expressing K562 cells.

Extended Data Figure 6 Localization of MX2 at nuclear pores.

a, Deconvolution microscopic images (single optical sections) of immunofluorescently stained NUP98 (red), haemagglutinin (HA)-tagged MX2 (green, expressed using CSIB vectors) and DAPI (4′,6-diamidino-2-phenylindole)-stained DNA (blue) in HOS cells. The top set of panels is an optical section approximately through the centre of the vertical dimension of the nucleus, whereas the middle and bottom panels are an optical section approximately coincident with the dorsal surface of the nucleus. The bottom panels are an expanded view of a portion of the centre panels. Scale bars, 10 μm (top), 5 μm (middle) and 1 μm (bottom). b, Pearson’s coefficient for colocalization of MX1 or MX2 and NUP98. Each data point represents an individual cell and the horizontal bar is the mean (n = 6 for MX1, n = 10 for MX2).

Extended Data Figure 7 The N57S capsid mutation reduces HIV-1 sensitivity to IFN-α in HOS cells.

Infectivity of wild-type and N57S CA-mutant HIV-1–GFP reporter viruses in untreated and IFN-α-treated HOS cells. Titres are mean + s.d, n = 3 technical replicates, representative of three experiments. Fold inhibition is the ratio of the titres on untreated and IFN-α-treated cells.

Extended Data Figure 8 Effect of MX2 on HIV-1 and murine leukaemia virus infection in dividing and non-dividing cells.

a, MLV–GFP reporter virus infection of dividing and non-dividing (aphidicolin-treated) vector or MX2-expressing HOS cell clones. b, HIV-1–GFP reporter virus infection of dividing and non-dividing (aphidicolin-treated) vector or MX2-expressing K562 cell clones. c, MLV–GFP reporter virus infection of dividing and non-dividing (aphidicolin-treated) vector or MX2-expressing K562 cell clones.

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Kane, M., Yadav, S., Bitzegeio, J. et al. MX2 is an interferon-induced inhibitor of HIV-1 infection. Nature 502, 563–566 (2013). https://doi.org/10.1038/nature12653

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