STRUCTURAL, BIOCHEMICAL, AND BIOLOGICAL CHARACTERIZATION OF HIV NEF AND VPU

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A recombinant HIV-2 (NIH-Z) Nef protein synthesized in E. coli, and purified by the detergent/chaotrope extraction technique and preparative SDS-PAGE is: immunologically reactive on Western blots with anti-Nef antibodies; an excellent substrate in vitro for phosphorylation both by purified protein kinase C (PKC) and the maturation promoting factor (MPF) kinase purified from Xenopus oocytes; exhibits an intrinsic low-level autokinase activity, and forms stable homodimers and homotetrameric complexes in vitro, which are significantly increased in the absence of sulfhydryl-reducing agents. Preliminary results of in vivo phosphory- lation and oligomerization experiments suggest that the native 25 kD HIV-2 (NIH-Z) Nef protein in infected T-lymphocytes in culture becomes highly phosphorylated following stimulation with calcium ionophores, and the dimeric (50 kD) and tetrameric (100 kD) forms of this protein can be radioimmunoprecipitated with specific anti-HIV-2 Nef monoclonal antibodies. In addition, evidence of a putative Nef cellular protein complex was also obtained. Structural studies have also revealed the presence of a leucine zipper-like repeat structure at the conserved central "core" region of the Nef proteins, with a characteristic 4,3 repeat similar to the heptad leucine repeat motif of the bZIP factors. Moreover, at the C-terminus of the Nef proteins is a highly acidic sequence (net charge of -5 to -8) stretched over 40 amino acids, and it contains two predicted alpha-helices separated by a predicted beta-turn structure, with homology to known acidic activation domains of transcriptional activation factors. Biochemical characterization of individual biological Nef clones expressing only p27nef (clone 3B-3) or p25nef (clone 3B-5), isolated by limiting dilution of HTLV-IIIB-infected H9 cells, are continuing. In addition, a baculovirus vector-expressed HIV-1 Vpu protein, which contains a potential calcium binding or calcium channel-like sequence similar to one of the S-IV sequences of the dihydropyridine (DHP)-receptor, is being purified for use in calcium channel drug-binding studies.