Inhibition of HIV-1 enhancer-controlled transcription by artificial enhancer-binding peptides derived from bacteriophage 434 repressor.
An artificial HIV-1 enhancer-binding 42-residue peptide (R42) that had been derived from bacteriophage 434 repressor inhibited the cell-free in vitro transcription of HIV-1 enhancer-containing plasmids [Hehlgans, T., Stolz, M., Klauser, S., Cui, T., Salgam, P., Brenz Verca, S., Widmann, M., Leiser, A., Städler, K. & Gutte, B. (1993) FEBS Lett. 315, 51-55; Caderas, G. (1997) PhD Thesis, University of Zürich]. Here we show that, after N-terminal extension of R42 with a viral nuclear localization signal, the resulting nucR42 peptide was active in intact cells. NucR42 could be detected immunologically in nuclear extracts and produced a 60-70% reduction of the rate of transcription of an HIV-1 enhancer-carrying plasmid in COS-1 cells that had been cotransfected with the HIV enhancer plasmid, an expression plasmid for nucR42, and a control. NucR42 was also synthesized chemically and the synthetic product characterized by HPLC, mass spectrometry, and quantitative amino acid analysis. Band shift, footprint, and in vitro transcription assays in the presence of exogenous NF-kappaBp50 indicated that the binding sites of nucR42 and NF-kappaB on the HIV enhancers overlapped and that a relatively small excess of nucR42 sufficed to displace NF-kappaBp50. Band shift and in vitro transcription experiments showed also that exchange of the 434 repressor-derived nine-residue recognition helix of nucR42 for four glycines abolished the HIV enhancer binding specificity whereas leucine zipper- or retro-leucine zipper-mediated dimerization of R42 analogues increased it suggesting the potential application of such dimeric HIV enhancer-binding peptides as intracellular inhibitors of HIV replication.
Caderas
G
G
Biochemisches Institut der Universität Zürich, Switzerland. gcaderas@ems.salk.edu
Klauser
S
S
Liu
N
N
Bienz
A
A
Gutte
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D003001:Cloning, Molecular; D019281:Dimerization; D015497:HIV-1 / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D019913:Nuclear Localization Signals; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D019913:Nuclear Localization Signals; D010455:Peptides