PMC

Mutations in the Dimerization Interface Do Not Disturb the Three-Dimensional Fold of Nef Required for SH3-binding Activity. (a) Crystal structure of a high-affinity Nef:SH3 complex where the SH3 domain is shown in green and the Nef core monomer in blue (PDB:1EFN). Key Nef residues involved in this interaction are indicated and include Nef PxxP helix and residues Phe 90, Trp 113 and Tyr 120 that form a hydrophobic pocket in the folded Nef core. This Nef pocket interacts with SH3 RT loop residue Ile-96 and is essential for high-affinity docking of the SH3 domain. (b) SH3-binding activity of wild-type and dimerization-defective Nef mutants revealed an equivalent recovery of the GST-SH3 fusion proteins from each reaction. Nef proteins tested in this experiment included wild-type (WT), D123A (DA), D123N (DN), D123V (DV), and 4D (I109D/L112D/Y115D/F121D).

Nef reduces binding of p53 to DNA, p53-mediated transcriptional activation, and p53 protein levels. (a) Nef reduces p53 binding to DNA. Nuclear extracts prepared from MOLT-4 cells were preincubated with increasing amounts of purified recombinant Nef or GST proteins before reaction with [α-³²P]dATP-labeled human p53 consensus site DNA binding or random control oligonucleotides. DNA binding events were analyzed by EMSA. Samples include nuclear extracts incubated with either the labeled consensus p53 binding site oligonucleotide (lane 2), the labeled consensus p53 binding site oligonucleotide and an excess of the same unlabeled “cold” oligonucleotide (lane 3), the labeled consensus p53 binding site oligonucleotide and excess unlabeled random-sequence oligonucleotides (lane 4), increasing amounts of purified Nef protein (lanes 6 to 8), or GST (lane 5) prior to incubation with the labeled consensus p53 binding site oligonucleotide. Lane 1, labeled oligonucleotide only. Nef added to nuclear extracts prior to formation of p53-DNA complexes inhibited specifically the formation of these complexes, while addition of GST had no effect. (b) Nef reduces p53-mediated transcriptional activation. A luciferase reporter under the control of a p53-dependent WAF 1 promoter (WWP-Luc) was introduced into Saos-2 cells with or without p53 as indicated. Twenty-four hours after transfection the cells were electroporated with increasing amounts of purified recombinant Nef 1-206 (0.1 to 3.0 μM), Nef 20-206 (3.0 μM), Nef 1-57 (3.0 μM), or GST (3.0 μM). Mock-electroporated cells were used as a control. The luciferase activities measured represent average values of three experiments. (c) Nef reduces p53 protein levels. The nuclear extracts prepared from the MOLT-4 cells incubated either alone (no treatment; lane 1) or with GST (3.0 μM; lane 2) or increasing amounts of purified Nef protein (1.0 to 3.0 μM; lanes 3 to 6) were separated by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose filters were immunoblotted with anti-p53 (top), anti-Oct-1 (middle), or anti-Nef (bottom). (d) Lysates were prepared from Saos-2 cells which had been transfected for p53 and WWP-Luc expression and subsequently electroporated with Nef 1-206 (0.1 to 3.0 μM; lanes 3 to 6), Nef 20-206 (3.0 μM; lane 2), Nef 1-57 (3.0 μM; lane 7), or GST (3.0 μM; lane 1). The lysates were separated by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose filters were immunoblotted with anti-p53 (top) or antiactin (middle). Lysates derived from cells electroporated with Nef 1-206 (3.0 μM), GST (3.0 μM), Nef 20-206 (3.0 μM), or Nef 1-57 (3.0 μM) were also immunoblotted with anti-Nef (bottom). (e) WAF 1 levels are reduced during HIV-1 infection of MOLT-4 cells with HIV-1 NL4-3. MOLT-4 cells were infected with HIV-1 NL4-3 (lane 2) or HIV-1 NL4-3-nef-stop (lane 3) or were mock infected (lane 1). After a determination by immunofluorescence staining of HIV antigens that similar numbers of cells (∼50% of the population) were infected by each virus, whole-cell extracts were prepared. The extracts were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-WAF 1 (top) or antiactin (bottom).

(a) Nef reaction. (b) Nef reaction via reductive methods. (c) Nef reaction catalyzed by old yellow enzymes.

Nef interacts with PAK1 in vivo and in vitro. (A) Purification of the NAK complex by MonoQ ion exchange chromatography. Nef complexes were isolated from Jurkat cells expressing the hybrid CN using the MonoQ column. Anti-CD8 immunoprecipitations from different fractions were examined for NAK activity (IVKA) and for the presence of Nef and PAK1 (Western blot). (B) Nef from HIV interacts with PAK1 in COS cells. The hybrid CN (left panel), hybrid Nef.GFP (right panel, + in table), CD8 truncated proteins or GFP alone (− in table) were coexpressed with PAK1 in COS cells. Cellular lysates were incubated with the anti-N20 (right panel, IP:αPAK1) or the anti-CD8 antibody (left panel, IP:αCD8), and immunoprecipitations were analyzed by Western blotting by using anti-Nef (lanes 3 and 4) or anti-N20 (lanes 1 and 2) antibodies, respectively (upper panel, IP). Western blotting of input Nef and PAK1 proteins prior to the immunoprecipitation are presented in the bottom panel (input). (C) Nef binds to PAK1 in vitro. Nef and the mutant Nef^RR-LL protein were purified from insect cells by using baculovirus, were incubated with the hybrid GST-PAK1 protein or GST alone, and were passed over glutathione beads. The bound Nef was analyzed by Western blotting by using the anti-Nef antibody (Western blot). Nef proteins (10% of that used in the binding reaction) were loaded on the gel as input control (10% input).