Rev is a key regulatory protein of HIV-1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA, thereby allowing the production of structural proteins. Based on its essential role in viral replication, Rev is a potential anti-HIV therapeutic target. In our previous work, the structure of Rev was solved for first time by binding it to an antibody fragment (Fab). The Fab stabilized Rev allowing the formation of protein crystals suitable for X-ray structural analysis. Furthermore, the Fab was shown to have anti-HIV activity by binding tightly to Rev and blocking its functional interactions. Regions of the Fab antibody in contact with Rev define the paratope (regions of Rev in contact with the Fab are known as the epitope). The paratope consists of six complementary determining region (CDR); three each from the antibody heavy and light chains. The CDRs are relatively short consisting of 6 to 14 amino acid residues. Analyses of the epitope-paratope interface from the X-ray structure predicted key contacts or binding hot spots among the CDR sequences. Peptides corresponded to the CDRs were cyclized (N- to C-terminal) to give them a structure approximating to their conformation in the intact antibody. Of the CDRs peptides tested, LCDR3 exhibited tight binding to Rev and analogous to the parent antibody was capable of depolymerizing a highly associated form of Rev (filaments) by specifically disrupting Rev protein-protein interactions. The LCDR3 peptide and related peptides selected for their binding to Rev are being modified for cellular uptake and their anti-HIV activities evaluated. Rev functions as a multiprotein protein complex which is formed in the nucleus by binding to RNA followed by Rev oligomerization: the Rev-RNA complex then associates with carrier proteins which mediate export to the cytoplasm. Rev oligomerization is required for biological activity and the structure of oligomeric protein was determined using a combination of structural methods as a first step in understanding assembly of the active complex. This work is now being followed up by structural studies of Rev associated with RNA and accessory proteins. Apart from fundamental information on the mechanism of viral replication, these studies may highlight points of vulnerability which may be suitable targets for therapeutic intervention. HIV protease, a homodimeric protein, is essential in the viral life cycle and a major anti-HIV drug target. We have expressed and purified a number of wild type and drug resistant forms of the protease which have been used in structural studies. Novel drugs which bind to the protease have been studied by co-crystallization and by examining the crystal structures used to rationalize and optimize drug binding. Structural details of interactions between drug moieties and protein have led to the synthesis of new compound with higher anti-HIV potency.

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Support Year
20
Fiscal Year
2015
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Arthritis, Musculoskeletal, Skin Dis
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Dearborn, Altaira D; Eren, Elif; Watts, Norman R et al. (2018) Structure of an RNA Aptamer that Can Inhibit HIV-1 by Blocking Rev-Cognate RNA (RRE) Binding and Rev-Rev Association. Structure 26:1187-1195.e4
Eren, Elif; Watts, Norman R; Dearborn, Altaira D et al. (2018) Structures of Hepatitis B Virus Core- and e-Antigen Immune Complexes Suggest Multi-point Inhibition. Structure 26:1314-1326.e4
Watts, Norman R; Eren, Elif; Zhuang, Xiaolei et al. (2018) A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for nuclear export. J Struct Biol 203:102-108
DiMattia, Michael A; Watts, Norman R; Cheng, Naiqian et al. (2016) The Structure of HIV-1 Rev Filaments Suggests a Bilateral Model for Rev-RRE Assembly. Structure 24:1068-80
Wingfield, Paul T (2015) Overview of the purification of recombinant proteins. Curr Protoc Protein Sci 80:6.1.1-35
Stahl, Stephen J; Watts, Norman R; Wingfield, Paul T (2014) Generation and use of antibody fragments for structural studies of proteins refractory to crystallization. Methods Mol Biol 1131:549-61
Zhuang, Xiaolei; Stahl, Stephen J; Watts, Norman R et al. (2014) A cell-penetrating antibody fragment against HIV-1 Rev has high antiviral activity: characterization of the paratope. J Biol Chem 289:20222-33
Yedidi, Ravikiran S; Garimella, Harisha; Aoki, Manabu et al. (2014) A conserved hydrogen-bonding network of P2 bis-tetrahydrofuran-containing HIV-1 protease inhibitors (PIs) with a protease active-site amino acid backbone aids in their activity against PI-resistant HIV. Antimicrob Agents Chemother 58:3679-88
Yedidi, Ravikiran S; Maeda, Kenji; Fyvie, W Sean et al. (2013) P2' benzene carboxylic acid moiety is associated with decrease in cellular uptake: evaluation of novel nonpeptidic HIV-1 protease inhibitors containing P2 bis-tetrahydrofuran moiety. Antimicrob Agents Chemother 57:4920-7
Ben-Sasson, Shlomo Z; Hogg, Alison; Hu-Li, Jane et al. (2013) IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells. J Exp Med 210:491-502

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