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A triple-arginine motif in the amino-terminal domain and oligomerization are required for HIV-1 inhibition by human MX2.

Goujon C, Greenbury RA, Papaioannou S, Doyle T, Malim MH - J. Virol. (2015)

Bottom Line: First, we describe an essential triple-arginine motif in the amino-terminal domain.Second, we demonstrate that this 91-residue domain mediates antiviral activity when appended to heterologous proteins, and we provide genetic evidence that protein oligomerization is required for MX2 function.These insights will facilitate future work aiming to elucidate MX2's mechanism of action.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom cgoujon@cpbs.cnrs.fr michael.malim@kcl.ac.uk.

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The amino-terminal domain of MX2 inhibits HIV-1 infection when transferred to heterologous scaffolds. (A) Fusion of the amino-terminal domain of MX2 to the amino terminus of Fv1b. U87-MG/CD4/CXCR4 cells were transduced with EasiLV expressing CD8 (Neg Ctrl), MX2, or amino-terminally Flag-tagged Fv1b, NMX2-Fv1b, or NMX2/RRR11–13A-Fv1b; treated with doxycycline for 3 days; and challenged with HIV-1/GFP. The percentage of infected cells was analyzed 2 days later by flow cytometry. Mean percentages of transduced cells from four independent experiments are shown. (Lower panel) Immunoblot analysis of parallel samples from the upper panel was performed as for Fig. 1A. (B) The NMX2-Fv1b fusions inhibit infection by N-MLV. 293T cells were transduced with EasiLV expressing CD8 (Neg Ctrl), Fv1b, NMX2-Fv1b, or NMX2/RRR11–13A-Fv1b and treated with doxycycline (2.5 μg/ml) for 3 days. The cells were then challenged with similar volumes (4 μl) of GFP-encoding N-MLV and B-MLV viral stocks (produced by 293T cotransfection of pMD.G, p13077, and pCIG3N or pCIG3B expression plasmids, respectively) (21, 27), and infection efficiency was analyzed by flow cytometry 2 days later. Mean percentages of transduced cells from three independent experiments are shown. (C) Dimeric and trimeric leucine zipper fusion proteins containing the amino-terminal domain of MX2. U87-MG/CD4/CXCR4 cells were transduced with EasiLV expressing CD8 (Neg Ctrl), MX2, and carboxy-terminally Flag-tagged fusion proteins containing the amino-terminal domain of MX2 grafted to different versions of the GCN4 leucine zipper, forming either dimers, trimers, or monomers (NMX2-LZdim, NMX2-LZtrim, and NMX2-LZmon, respectively); treated with doxycycline for 3 days; and challenged with HIV-1/GFP. The percentage of infected cells was analyzed 2 days later by flow cytometry. Mean percentages of transduced cells from three independent experiments are shown. (Lower panel) Immunoblot analysis of parallel samples from the upper panel was performed as for Fig. 1A.
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Figure 4: The amino-terminal domain of MX2 inhibits HIV-1 infection when transferred to heterologous scaffolds. (A) Fusion of the amino-terminal domain of MX2 to the amino terminus of Fv1b. U87-MG/CD4/CXCR4 cells were transduced with EasiLV expressing CD8 (Neg Ctrl), MX2, or amino-terminally Flag-tagged Fv1b, NMX2-Fv1b, or NMX2/RRR11–13A-Fv1b; treated with doxycycline for 3 days; and challenged with HIV-1/GFP. The percentage of infected cells was analyzed 2 days later by flow cytometry. Mean percentages of transduced cells from four independent experiments are shown. (Lower panel) Immunoblot analysis of parallel samples from the upper panel was performed as for Fig. 1A. (B) The NMX2-Fv1b fusions inhibit infection by N-MLV. 293T cells were transduced with EasiLV expressing CD8 (Neg Ctrl), Fv1b, NMX2-Fv1b, or NMX2/RRR11–13A-Fv1b and treated with doxycycline (2.5 μg/ml) for 3 days. The cells were then challenged with similar volumes (4 μl) of GFP-encoding N-MLV and B-MLV viral stocks (produced by 293T cotransfection of pMD.G, p13077, and pCIG3N or pCIG3B expression plasmids, respectively) (21, 27), and infection efficiency was analyzed by flow cytometry 2 days later. Mean percentages of transduced cells from three independent experiments are shown. (C) Dimeric and trimeric leucine zipper fusion proteins containing the amino-terminal domain of MX2. U87-MG/CD4/CXCR4 cells were transduced with EasiLV expressing CD8 (Neg Ctrl), MX2, and carboxy-terminally Flag-tagged fusion proteins containing the amino-terminal domain of MX2 grafted to different versions of the GCN4 leucine zipper, forming either dimers, trimers, or monomers (NMX2-LZdim, NMX2-LZtrim, and NMX2-LZmon, respectively); treated with doxycycline for 3 days; and challenged with HIV-1/GFP. The percentage of infected cells was analyzed 2 days later by flow cytometry. Mean percentages of transduced cells from three independent experiments are shown. (Lower panel) Immunoblot analysis of parallel samples from the upper panel was performed as for Fig. 1A.

Mentions: Given that transfer of the amino-terminal 91 amino acids of MX2 onto MX1 bestows full anti-HIV-1 function (13), we wished to determine whether this domain would still have this capability when appended to an entirely unrelated protein. We initially chose to use mouse Fv1b as the substrate since this protein is an inhibitor of retrovirus infection (N-tropic NLV but not HIV-1) (20, 21), suppression is manifested as a lack of viral cDNA integration (22, 23), and it is naturally oligomeric (24). Remarkably, fusing residues 1 to 91 of MX2 to the amino terminus of Fv1b (NMX2-Fv1b) conferred potent HIV-1-inhibitory activity, whereas the parental Fv1b protein had no effect (Fig. 4A). Importantly, a genetic constraint of MX2 function was faithfully preserved, as introduction of the RRR11–13A mutation into this chimeric protein abrogated activity. As a confirmation of Fv1b functionality, both fusion proteins suppressed N-MLV (but not B-tropic MLV) infection as efficiently as the wild-type protein (Fig. 4B). Therefore, the amino-terminal domain of human MX2 is the only element of MX1/MX2 that is necessary for HIV-1 inhibition in the context of a heterologous fusion partner.


A triple-arginine motif in the amino-terminal domain and oligomerization are required for HIV-1 inhibition by human MX2.

Goujon C, Greenbury RA, Papaioannou S, Doyle T, Malim MH - J. Virol. (2015)

The amino-terminal domain of MX2 inhibits HIV-1 infection when transferred to heterologous scaffolds. (A) Fusion of the amino-terminal domain of MX2 to the amino terminus of Fv1b. U87-MG/CD4/CXCR4 cells were transduced with EasiLV expressing CD8 (Neg Ctrl), MX2, or amino-terminally Flag-tagged Fv1b, NMX2-Fv1b, or NMX2/RRR11–13A-Fv1b; treated with doxycycline for 3 days; and challenged with HIV-1/GFP. The percentage of infected cells was analyzed 2 days later by flow cytometry. Mean percentages of transduced cells from four independent experiments are shown. (Lower panel) Immunoblot analysis of parallel samples from the upper panel was performed as for Fig. 1A. (B) The NMX2-Fv1b fusions inhibit infection by N-MLV. 293T cells were transduced with EasiLV expressing CD8 (Neg Ctrl), Fv1b, NMX2-Fv1b, or NMX2/RRR11–13A-Fv1b and treated with doxycycline (2.5 μg/ml) for 3 days. The cells were then challenged with similar volumes (4 μl) of GFP-encoding N-MLV and B-MLV viral stocks (produced by 293T cotransfection of pMD.G, p13077, and pCIG3N or pCIG3B expression plasmids, respectively) (21, 27), and infection efficiency was analyzed by flow cytometry 2 days later. Mean percentages of transduced cells from three independent experiments are shown. (C) Dimeric and trimeric leucine zipper fusion proteins containing the amino-terminal domain of MX2. U87-MG/CD4/CXCR4 cells were transduced with EasiLV expressing CD8 (Neg Ctrl), MX2, and carboxy-terminally Flag-tagged fusion proteins containing the amino-terminal domain of MX2 grafted to different versions of the GCN4 leucine zipper, forming either dimers, trimers, or monomers (NMX2-LZdim, NMX2-LZtrim, and NMX2-LZmon, respectively); treated with doxycycline for 3 days; and challenged with HIV-1/GFP. The percentage of infected cells was analyzed 2 days later by flow cytometry. Mean percentages of transduced cells from three independent experiments are shown. (Lower panel) Immunoblot analysis of parallel samples from the upper panel was performed as for Fig. 1A.
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Figure 4: The amino-terminal domain of MX2 inhibits HIV-1 infection when transferred to heterologous scaffolds. (A) Fusion of the amino-terminal domain of MX2 to the amino terminus of Fv1b. U87-MG/CD4/CXCR4 cells were transduced with EasiLV expressing CD8 (Neg Ctrl), MX2, or amino-terminally Flag-tagged Fv1b, NMX2-Fv1b, or NMX2/RRR11–13A-Fv1b; treated with doxycycline for 3 days; and challenged with HIV-1/GFP. The percentage of infected cells was analyzed 2 days later by flow cytometry. Mean percentages of transduced cells from four independent experiments are shown. (Lower panel) Immunoblot analysis of parallel samples from the upper panel was performed as for Fig. 1A. (B) The NMX2-Fv1b fusions inhibit infection by N-MLV. 293T cells were transduced with EasiLV expressing CD8 (Neg Ctrl), Fv1b, NMX2-Fv1b, or NMX2/RRR11–13A-Fv1b and treated with doxycycline (2.5 μg/ml) for 3 days. The cells were then challenged with similar volumes (4 μl) of GFP-encoding N-MLV and B-MLV viral stocks (produced by 293T cotransfection of pMD.G, p13077, and pCIG3N or pCIG3B expression plasmids, respectively) (21, 27), and infection efficiency was analyzed by flow cytometry 2 days later. Mean percentages of transduced cells from three independent experiments are shown. (C) Dimeric and trimeric leucine zipper fusion proteins containing the amino-terminal domain of MX2. U87-MG/CD4/CXCR4 cells were transduced with EasiLV expressing CD8 (Neg Ctrl), MX2, and carboxy-terminally Flag-tagged fusion proteins containing the amino-terminal domain of MX2 grafted to different versions of the GCN4 leucine zipper, forming either dimers, trimers, or monomers (NMX2-LZdim, NMX2-LZtrim, and NMX2-LZmon, respectively); treated with doxycycline for 3 days; and challenged with HIV-1/GFP. The percentage of infected cells was analyzed 2 days later by flow cytometry. Mean percentages of transduced cells from three independent experiments are shown. (Lower panel) Immunoblot analysis of parallel samples from the upper panel was performed as for Fig. 1A.
Mentions: Given that transfer of the amino-terminal 91 amino acids of MX2 onto MX1 bestows full anti-HIV-1 function (13), we wished to determine whether this domain would still have this capability when appended to an entirely unrelated protein. We initially chose to use mouse Fv1b as the substrate since this protein is an inhibitor of retrovirus infection (N-tropic NLV but not HIV-1) (20, 21), suppression is manifested as a lack of viral cDNA integration (22, 23), and it is naturally oligomeric (24). Remarkably, fusing residues 1 to 91 of MX2 to the amino terminus of Fv1b (NMX2-Fv1b) conferred potent HIV-1-inhibitory activity, whereas the parental Fv1b protein had no effect (Fig. 4A). Importantly, a genetic constraint of MX2 function was faithfully preserved, as introduction of the RRR11–13A mutation into this chimeric protein abrogated activity. As a confirmation of Fv1b functionality, both fusion proteins suppressed N-MLV (but not B-tropic MLV) infection as efficiently as the wild-type protein (Fig. 4B). Therefore, the amino-terminal domain of human MX2 is the only element of MX1/MX2 that is necessary for HIV-1 inhibition in the context of a heterologous fusion partner.

Bottom Line: First, we describe an essential triple-arginine motif in the amino-terminal domain.Second, we demonstrate that this 91-residue domain mediates antiviral activity when appended to heterologous proteins, and we provide genetic evidence that protein oligomerization is required for MX2 function.These insights will facilitate future work aiming to elucidate MX2's mechanism of action.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom cgoujon@cpbs.cnrs.fr michael.malim@kcl.ac.uk.

Show MeSH
Related in: MedlinePlus