Investigation of a catalytic role for cyclophilin A in HIV-1 replication

Human cyclophilin A is a member of the peptidyl prolyl isomerase superfamily, which represents a class of enzymes that catalyze the cis/trans isomenzation of prolyl peptide bonds. Cyclophilin A is required for efficient HIV-1 replication, and is incorporated into HIV-1 virions through a binding interaction at the Gly-Pro90 bond within the N-terminal domain of the HIV-1 capsid protein. The goal of this dissertation is to investigate the catalytic interaction between cyclophilin A and HIV-1 capsid in order to gain insight into the biological role of cyclophilin A during HIV-1 replication.; NMR exchange experiments have been used here to detect and quantify cyclophilin A catalyzed cis/trans isomerization of Gly-Pro90 within capsid, providing the first, direct experimental evidence of a catalytic interaction between cyclophilin A and capsid (Chapter 1). In order to address whether this catalytic interaction is biologically relevant in vivo, a series of conservative cyclophilin A active site mutants have been designed and characterized with the intention of dissecting cyclophilin A binding from catalysis during the HIV-1 replication cycle (Chapter 2). The W121Y mutant exhibits reduced catalysis and efficient binding, and thus is a promising candidate for investigating the role of cyclophilin A catalysis using in vivo HIV-1 replication assays.; 15N backbone dynamics have been used to investigate the catalytic interaction between cyclophilin A and capsid at atomic resolution. The experimental approach was first validated for characterization of cyclophilin A during catalysis of the Suc-Ala-Phe-Pro-Phe-pNA peptide (Chapter 3), and subsequently for cyclophilin A during catalysis of capsid (Chapter 4). Although similar regions within cyclophilin A exhibit conformational exchange during catalysis of both substrates, the rates of conformational exchange are ∼6 fold slower during catalysis of capsid. Interestingly, cyclophilin A residues involved in catalysis already exhibit conformational exchange in the unliganded state, suggesting these residues are ‘poised’ for substrate turnover.; The interaction between cyclophilin A and the isolated N-terminal domain of capsid is investigated in chapters 1, 2 and 4. It has been hypothesized that during the HIV-1 replication cycle, maturation dependent conformational changes occur within capsid that alter the cyclophilin A/capsid interaction, including the formation of additional cyclophilin A binding sites within the C-terminal domain of capsid. NMR titration and 2D exchange experiments have been used to demonstrate that cyclophilin A binding and catalysis of Gly-Pro 222* within the N-terminal of capsid is unaltered in the presence of the C-terminal domain, suggesting Gly-Pro222 is the preferential cyclophilin A binding and catalytic site.; *Several different capsid constructs are used in chapter 5, and therefore sequence numbers refer to the Gag precursor protein. Gly-Pro 222 in chapter 5 is the same as Gly-Pro90 in chapters 1, 2 and 4.