PROPERTIES AND GROWTH-MECHANISM OF THE ORDERED AGGREGATION OF A MODELRNA BY THE HIV-1 NUCLEOCAPSID PROTEIN - AN ELECTRON-MICROSCOPY INVESTIGATION

NCp7, the nucleocapsid protein of the human immunodeficiency virus typ e I, indices an ordered aggregation of RNAs, a mechanism that is thoug ht to be involved in the NCp7-induced promotion of nucleic acid anneal ing. To further investigate this aggregation, the morphology and the p roperties of the NCp7-induced aggregates of the model RNA homoribopoly mer, polyA, were investigated by electron microscopy in various condit ions. In almost all the tested conditions, the aggregates were spheric al and consisted of a central dense cope surrounded by a less dense ha lo made of NCp7-covered polyA molecules. The formation of these aggreg ates with a narrow distribution of sizes constitutes a distinctive fea ture of NCp7 over other single-stranded nucleic acid binding proteins. In most conditions, at the shortest rimes that can be reached experim entally, all the polyA molecules were already incorporated in small ag gregates, suggesting that the nucleation step and the first aggregatio n events took place rapidly. The aggregates then orderly grew with tim e by fusion of the smaller aggregates to give larger ones. The aggrega te halo was important in the fusion process by initiating the bridging between the colliding aggregates. In the presence of an excess of pro tein, the aggregates grew rapidly but were loosely packed and dissocia ted easily, suggesting adverse protein-protein interactions in the agg regates obtained in these conditions. In the presence of an excess of nucleotides, the presence of both amorphous nonspherical and slowly gr owing spherical aggregates suggested some changes in the mechanism of aggregate growth due to an incomplete covering of polyA molecules by N Cp7. Finally, we showed that in the absence of added salt, the aggrega te fusions were unfavored but not the initial events giving the first aggregates, the reverse being tote in the presence of high salt concen trations ( greater than or equal to 300 mM). (C) 1998 John Wiley & Son s, Inc.