Specific and distinct determinants mediate membrane binding and lipid raft incorporation of HIV-1(SF2) Nef.
Membrane association is believed to be a prerequisite for the biological activity of the HIV-1 pathogenicity factor Nef. Attachment to cellular membranes as well as incorporation into detergent-insoluble microdomains (lipid rafts) require the N-terminal myristoylation of Nef. However, this modification is not sufficient for sustained membrane association and a specific raft-targeting signal for Nef has not yet been identified. Using live cell confocal microscopy and membrane fractionation analyses, we found that the N-terminal anchor domain (aa 1-61) is necessary and sufficient for efficient membrane binding of Nef from HIV-1(SF2). Within this domain, highly conserved lysine and arginine residues significantly contributed to Nef's membrane association and localization. Plasma membrane localization of Nef was also governed by an additional membrane-targeting motif between residues 40 and 61. Importantly, two lysines at positions 4 and 7 were not essential for the overall membrane association but critically contributed to Nef's incorporation into lipid raft domains. Cell surface receptor downmodulation was largely unaffected by mutations of all N-terminal basic residues, while the association of Nef with Pak2 kinase activity and its ability to augment virion infectivity correlated with its lysine-mediated raft incorporation. In contrast, all basic residues were required for efficient HIV-1 replication in primary human T lymphocytes but did not contribute to the incorporation of Nef into HIV-1 virions. Together, these results unravel that Nef's membrane association is governed by a complex pattern of signature motifs that differentially contribute to individual Nef activities. The identification of a critical raft targeting determinant and the functional characterization of a membrane-bound, non-raft-associated Nef variant indicate raft incorporation as a regulatory mechanism that determines the biological activity of distinct subpopulations of Nef in HIV-infected cells.
Giese
Simone I
SI
Department of Virology, University of Heidelberg, 69120 Heidelberg, Germany.
Woerz
Ilka
I
Homann
Stefanie
S
Tibroni
Nadine
N
Geyer
Matthias
M
Fackler
Oliver T
OT
D020816:Amino Acid Motifs; D019677:Artificial Gene Fusion; D015153:Blotting, Western; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D015693:Gene Products, nef / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D019065:Virus Assembly; D014779:Virus Replication; D054311:nef Gene Products, Human Immunodeficiency Virus; D054462:p21-Activated Kinases
D015693:Gene Products, nef; D021382:Protein Sorting Signals; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D049452:Green Fluorescent Proteins; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases