Demonstration of a molecular interaction between thioredoxin and the pro-fs gene product of HIV-1, a mimic of NF kappa B
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A programmed –1 frameshift in the HIV-1 protease gene has been previously demonstrated. The sequence encoded in the overlapping –1 reading frame, named pro-fs, has significant similarity to the DNA binding loop of NF-êB, which is also the peptide known to bind thioredoxin (Trx) as part of the process of NF-êB activation. The hypothesis that the putative HIV-1 pro-fs gene product functions by mimicry of NF-êB via interacting with Trx was tested by coimmunoprecipitation and GST-pull down assay in vitro. Both experiments consistently showed that pro-fs binds human Trx in vitro; and the binding affinity is apparently stronger with Trx-wt than with Trx-CS, in which the two Cys residues in the active center of Trx were mutated to Ser. The fact that pro-fs interacts with Trx-CS rules out the possibility that the interaction between pro-fs and Trx is just due to cross-linkage via formation of an intermolecular disulfide bond between the Cys residues of pro-fs (the Sec residues in pro-fs sequence are mutated to Cys for bacterial expression) and the Cys residues in the active site of Trx. Due to the nature of the –1 frameshift mechanism, pro-fs and the HIV-1 protease share the first 20 amino acid residues at the N-terminus. Since both the retroviral protease and NF-êB function as dimers, we investigated whether pro-fs also dimerizes in living mammalian cells by fluorescent resonance energy transfer (FRET) analysis using confocal microscopy. Fluorescence microscopy was utilized to investigate the hypothesis that pro-fs is a nuclear protein, based upon its high pI and mimicry of NF-êB. The results demonstrated that pro-fs localizes in cell nuclei and forms oligomers. FRET analysis was also used to study the interaction between pro-fs and Trx in living cells. The results showed that phorbol 12-myristate 13-acetate (PMA) treatment of the 293T cells induces the nuclear translocation of Trx, and pro-fs binds to both Trx-wt and Trx-CS in PMA-stimulated cells. Together with the results of co-immunoprecipitation and GST-pull down assay, it suggests that Trx-pro-fs binding is a structurally specific interaction that involves multiple amino acid residues in the interactive region.