NUCLEIC-ACID BINDING-PROPERTIES OF RECOMBINANT ZN-2 HIV-1 NUCLEOCAPSID PROTEIN ARE MODULATED BY COOH-TERMINAL PROCESSING

The nucleocapsid protein (NC) of all animal retroviruses is the major structural protein of the core ribonucleoprotein complex, bound to gen omic RNA in mature virions. In a previous report, we showed that recom binant NC protein from HIV-1, a 71-amino-acid protein (NC71), is appar ently able to form two types of protein-nucleic acid complexes under l ow [NaCl], pH 8.3 and 25 degrees C. These appeared to differ in occlud ed apparent site size, n(app), forming n = 8 and n = 14 complexes on p oly(A) (Dib-Hajj, F., Khan, R., and Giedroc, D. P. (1993) Protein Sci. 2, 331-243) under conditions of high and low protein-nucleotide ratio s, respectively. Here we show that both NC71-poly(A) complexes strongl y scatter light under these solution conditions. Examination of the wa velength dependence of the light scattering at lambda less than or equ al to 320 nm indicates that each complex is characterized by a differe nt scattering coefficient. Optical density measurements suggest that u pon formation of the saturated n = 8 complex, additional polynucleotid e is not incorporated into the complex over a period of hours, i.e. th e n = 14 complex is not formed via redistribution of the n = 8 complex under low salt conditions, 25 degrees C. In contrast, the n = 14 comp lex readily incorporates additional protein until that sufficient to f orm the n = 8 complex is present. The n = 14 complex efficiently preci pitates poly(A) and shows spectral characteristics expected for an ext ensively charge-neutralized nucleic acid complex. At [NC71] in excess of that required to form the n = 8 complex, this n = 14 complex is bes t described as a kinetic intermediate on the pathway to the n = 8 comp lex, which forms over a period of hours under low salt conditions, 25 degrees C. This slow kinetics of binding provides a possible explanati on for the finding that the previously observed moderate cooperativity of Zn-2 NC71 binding to poly(A) (w approximate to 200) at pH 8.3 and 0.29 M NaCl (Khan, R., and Giedroc, D. P. (1992) J. Biol. Chen. 267, 6 689-6695) is shown here to represent a nonequilibrium phenomenon, appa rently converting to a low or no cooperativity complex over a period o f hours. Proteolytic removal of the COOH-terminal 14 amino acids from NC71, forming a 57-amino-acid protein (denoted NC57), removes this app arent binding site size heterogeneity of NC71 on poly(A). At 20 mM NaC l, NC57 binds with n = 6-7 nucleotides, in a manner which is independe nt of the protein-poly(A) nucleotide ratio. The implications of these findings on processing of the gag precursor which leads to mature NC i n HIV-1 virions is discussed.