Probing the RNA Binding Surface of the HIV-1 Nucleocapsid Protein by Site-Directed Mutagenesis

Abstract

The HIV-1 nucleocapsid protein is highly conserved and plays roles in many stages of HIV infection. NC binds genomic RNA and distinguishes it from other RNA's in order to form new virus particles. NC recognizes a packaging signal known as SL3 in the 5’-UTR of genomic RNA. Previous NMR work on the NCp7-SL3 complex has provided detailed information, including the protein side chains that contact RNA. Site-directed mutagenesis was used to construct 18 mutants of NCp7 to eliminate RNA-side chain contacts or to switch salt-bridge partners. The protein variants were subjected to MALDI mass spectrometry and analytical ultracentrifugation for integrity and homogeneity checks. The dissociation constants of the mutants for SL3 RNA were measured using a Trp-fluorescence assay and/or isothermal titration calorimetry. A few of the variants we tested, such as F16A, showed dramatic reductions in affinity, and others showed moderate reductions, including I24A and Q45A. The affinity of most variants, including N5A, V13A and N17A, were not significantly affected by the mutation, even though the replaced side-chains are predicted to interact directly with RNA. The bridge-switch mutants showed small to moderate reductions in affinity, confirming that the residues involved contribute mainly to stabilizing the protein internal structure through salt bridges, and that the disposition of the side chains is less important. As most mutations have relatively minor effects on stability of the complex, we conclude that (1) In wild-type SL3-NC, many of the H-bond and steric contacts either contribute little to stability or can be compensated by new contacts in the mutants, and (2) Conservation of many of these residues arise from NC's interactions with other proteins or in other aspects of its many roles in HIV infection. Supported in part by NIH grant R01 GM32691.