HIV-1 Nef perturbs artificial membranes: investigation of the contribution of the myristoyl anchor.
Nef, an accessory protein from human immunodeficiency virus type 1, is critical for optimal viral replication and pathogenesis. Here, we analyzed the influence of full-length myristoylated and nonmyristoylated Nef on artificial lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By means of cosedimentation assays, we found that neither nonmyristoylated nor myristoylated Nef stably binds to POPC unilamellar vesicles. Time-resolved ellipsometry rather indicates that the proteins perturb the assembly of POPC planar bilayers. This observation was corroborated by fluorescence and scanning force microscopy, suggesting that membrane disordering occurs upon interaction of full-length myristoylated and nonmyristoylated Nef with planar POPC membranes immobilized on SiO(2) surfaces resulting in loss of material from the surface. The membrane perturbations were further investigated by vesicle release experiments, demonstrating that the disordering results in defects through which the fluorophor carboxyfluorescein can pass. From these results, we conclude that Nef is capable of disordering and perturbing lipid membranes and that the myristoyl group is not the decisive determinant for the action of the protein on lipid membranes.
Szilluweit
Ruth
R
Institute of Organic and Biomolecular Chemistry, Georg-August University, 37077 Göttingen, Germany.
Boll
Annegret
A
Lukowski
Sonja
S
Gerlach
Holger
H
Fackler
Oliver T
OT
Geyer
Matthias
M
Steinem
Claudia
C
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015497:HIV-1 / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism*; D010784:Photomicrography; D011485:Protein Binding / Q000502:physiology; D012822:Silicon Dioxide; D053835:Unilamellar Liposomes / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
D010713:Phosphatidylcholines; D053835:Unilamellar Liposomes; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D012822:Silicon Dioxide; C028694:1-palmitoyl-2-oleoylphosphatidylcholine