Amino Acid Requirements of the Nucleocapsid Protein of HIV-1 for Increasing Catalytic Activity of a Ki-ras Ribozyme in Vitro

Abstract

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic acid binding protein with several function such as specific recognition, dimerization and packaging of vital RNA, tRNA annealing to vital RNA and protection against nucleases. Since some of these functions involve annealing and double-stranded RNA-melting activity we applied the nucleocapsid protein to a hammerhead ribozyme specific for the activated Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU site. A synthetic ribozyme containing 2′-O-allyl-modified nucleotides and alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observed at 37°C. Presence of a synthetic nucleocapsid protein significantly increases the catalytic activity of the ribozyme. Kinetic analyses by means of single and multiple turnover reactions performed at various substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constants in multiple turnover reactions, however, are stimulated up to 17-fold by the presence of the nucleocapsid protein. The activating region of the nucleocapsid protein was characterized by a number of mutants. The mutants demonstrate that activation requires both Basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate that the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly reduces the enhancing effect of the nucleocapsid protein on the ribozyme.