HIV-1 Nef membrane association depends on charge, curvature, composition and sequence

Abstract

Nef-mediated internalization of T-cell receptor molecules from the surface of an infected cell is required for the pathogenicity of HIV and disease progression to AIDS. This function depends on the N-terminal myristoylation of Nef, a lipid modification that targets the protein to membranes. We have analyzed how specific membrane properties and sequence motifs within Nef determine this interaction. Using time-resolved techniques we find that the association with membranes is a biphasic process with a fast rate for an electrostatic-driven protein-liposome interaction and a slow rate for the formation of an amphipathic helix. The rate of myristate insertion into liposomes depends on membrane curvature, while changes in the lipid composition with respect to phosphoinositides, cholesterol or sphingomyelin did not significantly alter the interaction. Moreover, Nef binding to membranes requires negatively charged liposomes, and mutations of basic and hydrophobic residues strongly diminished the association and changed the binding kinetics differently.

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Figure 1: Nef binding to lipid membranes is a biphasic process.
Figure 2: An acidic membrane surface is required for Nef binding.
Figure 3: Membrane association of Nef correlates with liposome curvature.
Figure 4: N-terminal residues in Nef sustain the membrane association.
Figure 5: Kinetics of Nef mutants revealed different contributions of basic and hydrophobic residues for the two phases of membrane binding.
Figure 6: Kinetic model for Nef membrane binding.

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Acknowledgements

We thank S. Gentz (Max Planck Institute for Molecular Physiology) for peptide synthesis, G. Holtermann for expert technical support and O. Fackler for stimulating discussions. F. Thorwirth, P. Verveer and P. Bastiaens are kindly acknowledged for help and advice with fluorescence imaging.

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H.G. performed most of the experiments with support of V.L. and C.F.W.B.; S.M. contributed to tubulation experiments. R.S.G. supervised the kinetic data analyses. M.G. designed the study and wrote the manuscript together with R.S.G., with the support of S.M. and C.F.W.B. All authors discussed the results and commented on the manuscript.

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Correspondence to Matthias Geyer.

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Gerlach, H., Laumann, V., Martens, S. et al. HIV-1 Nef membrane association depends on charge, curvature, composition and sequence. Nat Chem Biol 6, 46–53 (2010). https://doi.org/10.1038/nchembio.268

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