Solution oligomerization of the rev protein of HIV-1: implications for function.
rev is an RNA-binding protein of human immunodeficiency virus-1 and is required for the expression of incompletely spliced viral transcripts. Oligomerization of rev is thought to be associated with RNA binding and rev function. Here, we have characterized the oligomerization of rev using equilibrium analytical centrifugation. rev is predominantly monomeric at low concentrations, but reversibly polymerizes to produce large aggregates at higher concentrations. The data fit well to an unlimited isodesmic self-association model in which the association constants for the addition of a monomer to each aggregate are equal [K = 1.08 x 10(6) M-1 at 4 degrees C]. The association constant is essentially independent of monovalent salt concentration from 0.15 to 2 M at pH 6-9. Thermodynamic parameters derived from the temperature dependence of the association constant over the limited range of 0-30 degrees C reveal that the primary contribution to the free energy of oligomerization is a large negative enthalpy. Binding of rev to the rev-responsive element of RNA was characterized under the same conditions as the centrifugation experiments using a nitrocellulose filter assay. rev binds to the RRE at a protein concentration where rev is predominantly monomeric, suggesting that solution multimerization of rev is not required for rev function.
Cole
J L
JL
Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486.
Gehman
J D
JD
Shafer
J A
JA
Kuo
L C
LC
D003001:Cloning, Molecular; D004926:Escherichia coli; D015694:Gene Products, rev / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions; D013816:Thermodynamics; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D046911:Macromolecular Substances; D012367:RNA, Viral; D011994:Recombinant Proteins; D012996:Solutions; D054321:rev Gene Products, Human Immunodeficiency Virus