Binding of human immunodeficiency virus type 1 nucleocapsid protein to Psi-RNA-SL3

摘要

The interaction of the nucleocapsid protein NCp7, from the pNLA-3 isolate of HIV-1, with psi -RNA-SL3, with the sequence 5'-GGACUAGCGGAGGCUAGUCC, was studied using non-denaturing gel electrophoresis. Two kinds of experiments were performed, using buffered solutions of radiolabeled RNA and unlabeled protein. In the 'dilution' experiments, the total RNA concentration, R-T, was varied for a series of solutions, but kept equal to the total protein concentration, P-T. In the 'titration' experiments, solutions having R-T constant but with varying P-T were analyzed. The solutions were electrophoresed and the autoradiographic spot intensities, proportional to the amounts of the different species present, were measured. The intensities were fit to a number of equilibrium models, differing in species stoichiometries, by finding the best values of the binding constants. It was shown that NCp7 protein and SL3 RNA combine to form at least two complexes. When P-T is below approximately 10 muM, a complex that contains two RNAs and one protein forms. Increasing P-T to approximately 100 muM causes the 2:1 complex to oligomerize, forming a species having eight RNAs and four proteins. For the dilution experiments, run at 5 degreesC at an ionic strength of 31 mM, we found K-1 for the 2:1 complex is - 10(11) M-2 and K, for the 8:4 complex is - similar to 10(16) M-3. Th, titration experiments returned K-1 similar to 10(7) M-2 (poorly determined) and K-2 - 10(19) M-3. The analysis was complicated by the loss of RNA at higher protein concentrations, due to formation of an insoluble species containing both RNA and protein, which does not enter the gel. Correcting for this changes the calculated values of equilibrium constants, but not the molecularities determined by our analysis. The observation that a small complex can oligomerize to form a larger species is consistent with the fact that NCp7 organizes and condenses the genome in the virus particle. (C) 2000 Elsevier Science B.V. All rights reserved. [References: 46]