The specific aims of this study are the elucidation of the determinants of the structural polymorphism manifested by the 5'-untranslated region (5'-UTR) of Human Immunodeficiency Virus type 1 (HIV-1), the dentification of the binding sites of the nucleocapsid (NC) domain of Gag on the different forms assumed by the 5'-UTR fold, and the evaluation of deoxyribozymes as functional probes and possible interferents of 5'- UTR dynamics. 5'-UTR is involved in key steps of viral replication, including reverse transcription, splicing, translation, genome recognition, dimerization, and packaging. Facilitated by the chaperone activity of NC, 5'-UTR can fold into polymorphic forms defined by the different pairing arrangement of complementary sequences ocated in distal positions of the HIV-1 leader. The equilibrium between alternative conformations has been interpreted as a possible riboswitch mechanism for regulating the 5'-UTR functions. Understanding the riboswitch determinants and the role played by NC in switch actuation would enable the development of new antiviral strategies aimed at disrupting the 5'-UTR processes. We propose to investigate the 5'-UTR polymorphism in vitro using an approach based on bifunctional crosslinking, chemical footprinting, and high-resolution mass spectrometry (MS3D). Not limited by considerations of size and crystallization behavior, this approach will provide valuable information on the spatial organization and long range interactions between secondary structures formed by the different conformers of leader RNA. The remodeling of local hairpins and loops with formation of new base pairs and tertiary interactions will be investigated in the context of full-length 5'-UTR mutants that fold in the specific conformations. The effects of NC on the stability of the new structures will be investigated through binding and crosslinking experiments. These experiments will also enable the possible identification of new NC binding sites formed by distal regions of RNA, which are brought into close proximity by the global fold of 5'- UTR. The effects of deoxyribozyme interference on the observed RNA-RNA and protein-RNA interactions will be determined to elucidate the mechanism of riboswitch actuation and support the design of new 5'-UTR inhibitors. The long term goal is to understand the molecular basis for the 5'-UTR processes and the mechanism regulating its different roles during viral replication. New strategies that interfere with 5'-UTR function and regulation would provide a much needed complement to the current treatments based on protease and reverse-transcriptase inhibitors, which are particularly affected by the emergence of drug-resistant strains.
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