2014 Nov 12
Structural Insight Into HIV-1 Restriction by MxB
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Structural Insight Into HIV-1 Restriction by MxB
Cell Host Microbe
The myxovirus resistance (Mx) proteins are interferon-induced dynamin GTPases that can inhibit a variety of viruses. Recently, MxB, but not MxA, was shown to restrict HIV-1 by an unknown mechanism that likely occurs in close proximity to the host cell nucleus and involves the viral capsid. Here, we present the crystal structure of MxB and reveal determinants involved in HIV-1 restriction. MxB adopts an extended antiparallel dimer and dimerization, but not higher-ordered oligomerization, is critical for restriction. Although MxB is structurally similar to MxA, the orientation of individual domains differs between MxA and MxB, and their antiviral functions rely on separate determinants, indicating distinct mechanisms for virus inhibition. Additionally, MxB directly binds the HIV-1 capsid, and this interaction depends on dimerization and the N terminus of MxB as well as the assembled capsid lattice. These insights establish a framework for understanding the mechanism by which MxB restricts HIV-1.
Copyright © 2014 Elsevier Inc. All rights reserved.
Structure of MxB 84YRGK and its antiviral activity
(A) Structure of the MxB dimer shown in two orientations, with protomers 1 and 2 colored in purple and yellow, respectively. (B) Schematic (left) and structure (right) of an MxB protomer with residues of domain boundaries denoted and colored. The arrows in the schematic denote the first and last visible residues in the structure. (C) Superposition of protomer 1 and protomer 2 in two views. (D) Cells expressing HA-tagged WT or MxB
YRGK were analyzed for MxB expression. Total cellular proteins were extracted, resolved by SDS-PAGE, and visualized by western blotting with anti-HA antibody. WT MxB expression was set to 1. Results are the mean of 3 independent experiments, with error bars denoting standard error. (E) Immunofluorescent microscopy of untransduced or MxB-expressing cells. Blue, nuclear DNA. (F) Susceptibility of WT or MxB YRGK-expressing versus control (non-transduced) HOS cells to HIV-1 (dark gray), EIAV (light gray), or FIV (striped gray) infection. Error bars denote 95% confidence intervals derived from 7 independent experiments.
Figure 2. MxB hinge mutations do not abolish antiviral activity
(A) Superposition of MxA monomer (PDBID: 3SZR; red) and MxB protomer 1 (purple), based on either GTPase domain (left) or stalk domain (right). (B) Zoomed in view of BSE domain with hinges and key residues highlighted. (C-E) Cells expressing WT, E681A, or R689A mutant MxB were analyzed for (C) total expression, (D) subcellular localization, and (E) antiviral activity as described in Figure 1. Relative expression values are an average of at least 3 independent blotting experiments, with error bars denoting standard error. Infection values are the mean of 5 independent experiments, with error bars denoting 95% confidence intervals.
Figure 3. The MxB dimer is required for antiviral activity
(A) Dimer of MxB (top) with zoomed in view of dimer interface in surface representation (bottom). (B) Zoomed view of dimer interface with key residues shown as sticks. (C) Size exclusion chromatography analysis of MxB
84YRGK (green), which elutes at a volume corresponding to a dimer, and MxB 84YRGK/ M574D (cyan), a majority of which elutes at a volume corresponding to a monomer. Inset, SDS-PAGE of peak fractions corresponding to monomeric (lane 2 of each set) and dimeric (lane 1) MxB. (D) Antiviral activities of WT and interface 2 mutants of MxB. Activity assays are as described in Figure 1F. Results are an average of at least 4 independent experiments, with error bars denoting 95% confidence intervals. (E) Total mutant MxB expression levels relative to WT MxB (set to 1). Results are an average of 3 independent experiments, with error bars denoting standard error. (F) WT and mutant MxB localization as determined by confocal microscopy following antibody staining.
Figure 4. Higher-order oligomerization of MxB is not required for antiviral activity
(A) Two adjacent MxB dimers representing formation of higher-order oligomers in two views (left) and a zoomed in view of higher-order interface 1 (right). Protomer 1 and 2 of one dimer are colored as in Figure 3 while protomer 1’ and 2’ are colored in dark grey and light grey, respectively. Important interface residues are shown in sticks. (B) CryoEM images of full-length MBP-MxB WT (6 μM) and the interface mutant MBP-MxB
YRGK/IKM (3 μM). Short filaments (marked by arrows) and circular structures (marked by triangles) are seen for WT MBP-MxB. (C) Size exclusion chromatography analysis of MBP-MxB 1-715 (green), which elutes close to the void volume, and MBP-MxB 1-715 IK/DD (red) and MBP-MxB 1-715 IKM/DDD (blue), which elute at volumes corresponding to dimers. Inset, SDS-PAGE of peak fractions. (D) Antiviral activities of WT and MxB interface 1 mutants. Results are an average of at least 4 independent experiments, with error bars denoting 95% confidence intervals. (E) Total mutant MxB expression levels relative to WT MxB (set to 1). Results are an average of at least 3 independent experiments, with error bars denoting standard error. (F) WT and mutant MxB localization as determined by confocal microscopy.
Figure 5. Interactions with capsid assemblies by WT and mutant MxB proteins
(A) Cell extracts containing HA-tagged MxB were tested for binding to cross-linked CA tubular assemblies. Pelleted proteins resolved by SDS-PAGE were visualized by western blotting (MxB) or stained with Coomassie Blue (CA). Results are an average of 5 independent experiments with standard errors plotted. Lane indicators above the representative western blot: i, 20% of input cell lysate in absence of CA; -, pellets from binding reactions in the absence of CA; +, reactions in the presence of CA. Lane 1 of lower panel, 20% of input CA. (B) Scatterplot of WT and mutant MxB-HA binding (x-axis) versus normalized level of HIV-1 infectivity (y-axis). Points denote the geometric mean of each data set. The comparison exhibited a negative correlation with a significant Spearman rank correlation (
P = 0.028). (C) Binding of purified MxB (with or without CypA or CPSF6 313-327), maltose binding protein (MBP) (negative control), or a region of TRIMCyp (CC-Cyp) (positive control) to cross-linked CA assemblies. Total (T), soluble (S), and pellet (P) fractions resolved by SDS-PAGE were visualized with Coomassie staining and quantified with ImageJ. Three CA variants were analyzed for binding, A14C/E45C (CA), A14C/E45C/G89V (G89V) and A14C/E45C/N74D (N74D). Quantification of the binding, with standard errors, from 3 independent experiments is plotted below the gels. P values from two sided, unequal variance t-tests are shown for the pelleting comparison for each MxB construct with or without CA. (D) Visualization of the interaction of MxB constructs with HIV-1 CA tubes. CryoEM images of reaction mixtures containing cross-linked CA assemblies (10 μM) and MBP-MxB 1-715YRGK/IKM (5 μM) or MBP-MxB 84-715YRGK/IKM (5 μM) shows additional protein density decorating the tubes (middle and right panels) that is not observed in the control tubes without MxB (left). Substantially more decoration of the CA tubes occurs with the full-length MxB construct (middle) than with the N-terminal truncation construct (right). (E) Size exclusion chromatograms of purified MBP-MxB YRGK/IKM (blue), cross-linked CA hexamers (red), and their mixture (green). CA hexamers and MBP-MxB YRGK/IKM do not bind, as the elution profile of the mixture is the exact superposition of the individual ones. The SDS-PAGE analysis of the peaks (labeled 1, 2 and 3) is shown.
Figure 6. Conceptual binding model of the MxB dimer to HIV capsid
(A) The dimension of the MxB dimer matches the spacing (marked by the lines) between either trimers (3-fold axes marked by triangles) or dimers (2-fold axes marked by eye-shaped symbols) of CA hexamers. The MxB dimer (top) is colored with both the GTPase and the BSE domains in red and yellow, respectively, and with the stalk domain of each protomer in green or cyan. The N-terminus of MxB is indicated by an oval. The capsid model (bottom) was created by docking the crystal structure of HIV-1 CA hexamer (PDBID: 3H4E) to the EM map of HIV-CA helical tube (EMDB accession code: EMD-5136). (B) Two orthogonal views of a possible binding mode of the MxB dimer to the capsid at the interfaces of a trimer of CA hexamers. The GTPase domains of MxB are oriented such that the N-termini (ovals) of MxB can extend and interact with residues known to be important (207/210, asterisks) at the hexamer interfaces and the sites (marked by the # signs) where binding of the CPIPB inhibitor led to competitive inhibition of MxB binding to capsid. The flexibility of the MxB N-terminus and the hinge regions may allow MxB to adjust to the changing curvature of the HIV-1 capsid. The pink colored hexamers are removed in the side view for clarity. (B) Potential MxB binding to the interfaces of a dimer of CA hexamers. The tan colored hexamers are removed in the side view for clarity.
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Gene Expression Regulation
Myxovirus Resistance Proteins / chemistry
Myxovirus Resistance Proteins