MAPPING THE RNA-BINDING SITES FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1GAG AND NC PROTEINS WITHIN THE COMPLETE HIV-1 AND HIV-2 UNTRANSLATED LEADER REGIONS

Encapsidation of HIV-1 genomic RNA is mediated by specific interaction s between the RNA packaging signal and the Gag protein. During maturat ion of the virion, the Gag protein is processed into smaller fragments , including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathion e-S-transferase (GST) Gag and NC fusion proteins from HIV-1, to the en tire HIV-1 and -2 leader RNA encompassing the packaging signal. We hav e mapped the binding sites at conditions where only about two complexe s are formed and find that GST-Gag and GST-NC fusion proteins bind spe cifically to discrete sites within the leader. Analysis of the HIV-1 l eader indicated that GST-Gag strongly associates with the PSI stem-loo p and to a lesser extent with regions near the primer binding site. GS T-NC binds the same regions but with reversed preferences. The HIV-1 p roteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context.