AMINO-ACID-REQUIREMENTS OF THE NUCLEOCAPSID PROTEIN OF HIV-1 FOR INCREASING CATALYTIC ACTIVITY OF A KI-RAS RIBOZYME IN-VITRO

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic ac id binding protein with several functions such as specific recognition , dimerization and packaging of viral RNA, tRNA annealing to viral RNA and protection against nucleases. Since some of these functions invol ve annealing and double-stranded RNA-melting activity we applied the n ucleocapsid protein to a hammerhead ribozyme specific for the activate d Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU si te. A synthetic ribozyme containing 2'-O-allyl-modified nucleotides an d alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observe d at 37 degrees C. Presence of a synthetic nucleocapsid protein signif icantly increases the catalytic activity of the ribozyme. Kinetic anal yses by means of single and multiple turnover reactions performed at v arious substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constant s in multiple turnover reactions, however, are stimulated up to 17-fol d by the presence of the nucleocapsid protein. The activating region o f the nucleocapsid protein was characterized by a number of mutants. T he mutants demonstrate that activation requires both basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate th at the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly red uces the enhancing effect of the nucleocapsid protein on the ribozyme.