OLIGOMERIZATION OF THE HIV TYPE-2 NEF PROTEIN - MUTATIONAL ANALYSIS OF THE HEPTAD LEUCINE REPEAT MOTIF AND CYSTEINE RESIDUES

The human immunodeficiency virus type 2 (HIV-2) Nef protein expressed in Escherichia coil forms highly stable homooligomeric complexes in vi tro. Similarly, the native protein synthesized in the persistently inf ected H9 T cell line also forms stable homooligomers in vivo. To deter mine whether homooligomer formation is mediated by the leucine zipper- type sequence located in the middle region of the protein, site-direct ed mutagenesis was used to introduce double and triple point mutations at heptad leucine positions L1, L2, and L4 within the HIV-2(NIHZ) Nef protein sequence. Here, we show that substitution of a serine residue for the L1 (residue 108) and L2 (residue 115) heptad leucines, and a glutamine residue for the L4 (residue 129) heptad leucine, did not pre vent Nef homooligomer formation in vitro. However, a more drastic subs titution of alpha-helix-breaking proline residue for the L2 and L4 hep tad leucines significantly abrogated ability of the protein to form st able homooligomers. In addition, because significantly higher levels o f the Nef oligomers were consistently observed under the nonreducing S DS-PAGE condition, site-specific mutagenesis was also used to examine the role of cysteine residues in generating disulfide-linked Nef dimer s in vitro. Here, we also show that single cysteine-to-glycine substit utions at positions 28, 32, or 55 drastically reduced covalent Nef dim er formation and thermal stability of the Nef protein in vitro. Theref ore, these results demonstrate that; the leucine zipper-type motif in the HIV-2 Nef protein mediates stable homooligomer formation in vitro, and also establish a role for covalent disulfide bonds in the formati on of linked Nef dimers and thermal stability of the monomer Nef in vi tro.