Activation of the retroviral budding factor ALIX

Abstract

The cellular ALIX protein functions within the ESCRT pathway to facilitate intralumenal endosomal vesicle formation, the abscission stage of cytokinesis, and enveloped virus budding. Here, we report that the C-terminal proline-rich region (PRR) of ALIX folds back against the upstream domains and auto-inhibits V domain binding to viral late domains. Mutations designed to destabilize the closed conformation of the V domain opened the V domain, increased ALIX membrane association, and enhanced virus budding. These observations support a model in which ALIX activation requires dissociation of the autoinhibitory PRR and opening of the V domain arms.

Fig. 1.

Small-angle X-ray scattering analyses of recombinant ALIX. (A) SDS-PAGE analysis (Coomassie blue staining) showing the stepwise expression and purification of full-length human His₆-ALIX protein following baculoviral expression in SF21 insect cells (lane 1), nickel affinity chromatography (lane 2), anion-exchange chromatography (lane 3), and gel filtration chromatography (lane 4). (B) Log I(q) versus q solution small-angle X-ray scattering profile for ALIX (circles) and ALIX_Bro1-VR649E (gray diamonds). Fits to the ALIX scattering data are shown in red (models depicted in panels D and E) or blue (model depicted in F). For clarity, the data have been offset on the I(q) axis. (C) Probable atom-pair distance distributions [P(r) versus r] for ALIX (open circles) and the crystal structure of ALIX_Bro1-V (black squares) and ALIX_Bro1-V,R649E (gray diamonds). The crystal structure P(r) was calculated as for the experimental data except that the intensity profile was generated using CRYSOL26 (34) and the coordinates of ALIX_Bro1-V (7). The areas under the P(r) curves are proportional to I(0) and correctly scaled according to the ratios of the square of the molecular masses of the proteins. (D and E) ALIX models that fit the scattering data (red lines in panel B). The Bro1 domain is shown in light blue, the two arms of the V domain are shown in green and blue, respectively, and PRR dummy atoms are shown in magenta. (F) A model in which the PRR projects into solution does not fit the SAXS data (blue line in panel B).

Fig. 2.

ALIX protein binding to an EIAV p9^Gag late-domain peptide. Isothermal calorimetry titrations of an EIAV p9^Gag peptide (₁₉TQNLYPDLSEIKK₃₁, 750 μM) into 70 μM ALIX_Bro1-V (filled squares) or ALIX (open circles) in a solution of 20 mM sodium phosphate (pH 7.2), 150 mM NaCl, and 1 mM dithiothreitol (DTT) (25°C). The solid line shows the theoretical curve for a 1:1 peptide: ALIX_Bro1-V complex with a dissociation constant of 3.6 μM (N was 1.01 ± 0.004, ΔG_25°C was −7.42 ± 0.02 kcal/mol, ΔH_25°C was −5.55 ± 0.03 kcal/mol, and ΔS_25°C was 6.30 ± 0.17 entropy units [eu], where N, G, H, and S are ligand-binding stoichiometry, free energy, enthalpy, and entropy, respectively [MicroCal Origin software]). The peptide was added in 39 0.5-μl injections (180-s intervals) using a MicroCal iTC₂₀₀.

Fig. 3.

The Arg649Glu mutation activates ALIX for membrane association and virus budding. (A) Flotation analysis showing the degree of membrane association of ALIX and ALIX_R649E. Lanes 1 to 3 show the crude fractionation of 293T cell lysates (lane 1) expressing either ALIX (row 1, WISP03-308), ALIX_R649E (row 2, WISP06-180), or no protein (control, rows 3 and 4). The data demonstrate that neither ALIX nor ALIX_R649E forms insoluble aggregates (compare lanes 2 and 3 and see text for details). Lanes 4 to 6 show the percentages of ALIX (row 1) ALIX_R649E (row 2), aldolase (soluble protein control, row 3), and cadherin (integral membrane protein control, row 4) that partitioned into the membrane-containing (lane 4), soluble (lane 6), or intermediate (row 5) fractions. (B) HIV-1 ΔPTAP viral titers released by 293T cells (six-well plates, 1 μg plasmid DNA) cotransfected with an empty vector control or with the indicated quantities of pCl-neo-FLAG vectors expressing wild-type ALIX (gray triangles, black line) or ALIX_R649E (black crosses, gray line). Titers were measured in triplicate using single-cycle MAGIC infectivity assays. (C) Western blots of supernatants and cells corresponding to the experiment described in panel B, showing levels of virion-associated CA and MA released into the media (panel 1), and cellular levels of viral Gag, (anti-CA [α-CA] and α-MA, panel 2), and ALIX protein levels (α-FLAG, panel 3).

Fig. 4.

Model depicting different stages of ALIX activation. (i) Monomeric ALIX adopts an autoinhibited state in the cytosol in which the two V domain arms (blue and green) adopt a “closed” conformation, and the PRR (red) folds back onto the V domain to occlude the YPXL late-domain binding site (orange) and onto the Bro1 domain (light blue with the CHMP4 binding site shown in purple). ALIX activation requires dissociation of the PRR from the Bro1-V core (ii) (pink arrow), opening of the V domain (iii) (blue arrow), and protein dimerization (iv), denoted in brackets because dimeric species were characterized in references and , not in the present study.