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Abstract:
The nef genes, derived from two different human immunodeficiency-virus-type-1 (HIV-1) strains, were expressed in procaryotic cells (Escherichia coli) and in eucaryotic cells (insect cells infected with nef-containing baculovirus). The oligomerization of recombinant Nef protein was studied by NMR spectroscopy and immunoblotting under various experimental conditions. 1H-NMR spectroscopy shows that native folded protein has the tendency to polymerize under low-salt conditions. These oligomers become covalently linked by disulfide bonds after decreasing the reduction potential, a process which is fully reversible. Cross-linking studies with bis(sulfo-succinimidyl)suberate and alkylation with iodoacetic acid under non-reducing and reducing conditions document for the first time that Nef can also form homomeric structures including monomers, dimers, trimers and tetramers in cell lysates and intact cells. We found disulfide-linked as well as non-covalently associated oligomers. Since the Nef molecules are not exclusively found in the cytoplasm of HIV infected cells and since the reduced glutathione concentration in lymphocytes of virus infected persons is known to be unusually low, it might be possible that these Nef oligomers have a biological function in vivo as well.
