Fusogenic activity of amino-terminal region of HIV type 1 Nef protein.
We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.
Biomolecular Research Institute, Parkville, Victoria, Australia.
C C
CC
Curtain
F
F
Separovic
D
D
Rivett
A
A
Kirkpatrick
A J
AJ
Waring
L M
LM
Gordon
A A
AA
Azad
D015060:1,2-Dipalmitoylphosphatidylcholine; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry; D015693:Gene Products, nef / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D008027:Light; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D054311:nef Gene Products, Human Immunodeficiency Virus
D000942:Antigens, Bacterial; D015693:Gene Products, nef; D008081:Liposomes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D015060:1,2-Dipalmitoylphosphatidylcholine; C043431:M antigen, Brucella melitensis