The amino terminal domain of HIV-1 Rev is required for discrimination of the RRE from nonspecific RNA.
The ability of HIV-1 Rev to successfully discriminate between specific Rev-responsive elements (RRE) and nonspecific binding sites in the presence of excess nonspecific RNA was examined using filter binding, gel shift, and gel filtration techniques, using purified M4 Rev mutant protein and endoproteinase Lys-C cleaved wild-type Rev. The M4 Rev displayed a slightly reduced binding affinity to the RRE, as well as a tenfold decrease in its ability to discriminate the RRE from non-specific RNA compared to the wild-type Rev. Gel shift and gel filtration chromotography data also showed decreased ability of the mutant to multimerize in the absence or presence of the RRE. The Lys-C cleaved Rev, which lacks the amino-terminal 20 amino acids of the protein, displayed less ability to discriminate the RRE from nonspecific RNA compared to either the wild-type or the M4 mutant Rev and appeared unable to form protein-protein interactions, yet still bound sense and antisense RNA species with high affinity (Kd was in the nanomolar concentration range). A 40 amino acid peptide containing the arginine-rich RRE binding domain of Rev was also observed to interact with both the RRE and antisense RNA fragments with a binding constant of about 1 x 10(-9) M. However, the peptide displayed almost no ability to discriminate between the RRE and a comparably sized antisense RRE. The loss in ability to discriminate correct from incorrect binding sites correlates with overall decreases in the alpha-helical character of the protein and perturbations within the amino terminus. The amino terminus of Rev is likely to maintain the conformational integrity of the arginine rich RRE binding domain which is required for specific RNA binding site discrimination or stabilization of specific Rev-RRE interactions.
Daly
T J
TJ
Repligen Corporation, Cambridge, MA 02139, USA.
Doten
R C
RC
Rusche
J R
JR
Auer
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D015694:Gene Products, rev / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008961:Models, Structural; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D016372:RNA, Antisense / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D046911:Macromolecular Substances; D010446:Peptide Fragments; D016372:RNA, Antisense; D012367:RNA, Viral; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus