BINDING OF THE HIV-1 NUCLEOCAPSID PROTEIN TO THE PRIMER TRNA(3)(LYS),IN-VITRO, IS ESSENTIALLY NOT SPECIFIC

The nucleocapsid protein NCp7 of human immunodeficiency virus, type 1, is a key component in the viral life cycle, Since, the first common s tep of all reported NCp7 activities corresponds to a nucleic acid-bind ing step, the NCp7 binding parameters to the natural primer tRNA(3)(Ly s) were investigated. Using NCp7 intrinsic fluorescence, we found that (i) in 0.1 hr NaCl, NCp7 bound noncooperatively to tRNA(3)(Lys) with a K-obs = 3.2 x 10(6) M(-1) association constant and a n = 6 binding s ite size, (ii) four ionic interactions were formed in the NCp7.tRNA(3) (Lys) complex, and (iii) nonelectrostatic factors provided about 60% o f the binding energy. These binding parameters were not significantly altered when the natural tRNA(3)(Lys) was replaced by either an in vit ro synthetic tRNA(3)(Lys) transcript, the heterologous yeast tRNA(Phe) or the structurally unrelated 5 S RNA from Escherichia coil. Moreover , the environment of the intrinsic fluorescent reporters (Trp(37) and Trp(61)) was similar in the various complexes. Finally, experiments pe rformed at low protein concentration provide no evidence of high affin ity binding sites. Taken together, our data strongly suggested an esse ntially nonspecific binding of NCp7 to tRNA(3)(Lys) and thus did not s eem to support a direct role of NCp7, per se, in the selection of tRNA (3)(Lys) from the pool of cellular tRNAs.