Intermolecular binding sites of human immunodeficiency virus type 1 Rev protein determined by protein footprinting.
Human immunodeficiency virus encodes the regulatory protein Rev, which is required for expression of viral structural proteins. It binds to an RNA element (RRE) in the viral transcript and up-regulates the cytoplasmic appearance of unspliced and singly spliced viral mRNA. We have studied the structure of Rev alone and complexed with the RRE and two monoclonal antibodies, using a protein footprinting approach. The method involves radioactive labeling at the C-terminal end of Rev fusion protein followed by limited proteolysis under native conditions, using 10 different proteinases. Rev protein was mainly cleaved within the basic domain and in the C-terminal part. The periodicity of the proteolytic cleavages within the basic domain strongly suggests that it forms an alpha-helical structure with one side facing the solvent. In the presence of RRE, these cleavages became significantly reduced. In addition, strong protection was observed at position 66 outside the basic domain. As a control for the specificity of the footprinting reaction, we confirmed the position of the epitopes for two monoclonal antibodies. This protein footprinting methodology is generally applicable to other proteins for which terminal modifications are acceptable, and provides a useful tool for mapping structure, substrate binding, and conformational changes.
Jensen
T H
TH
Department of Molecular Biology, University of Aarhus, Denmark.
Leffers
H
H
Kjems
J
J
D001483:Base Sequence; D001665:Binding Sites; D018604:Epitope Mapping; D015694:Gene Products, rev / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015497:HIV-1; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D012313:RNA