Abstract
BackgroundCyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.ResultsCharacterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.ConclusionsOnly N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP35RIW motif of N-terminal Vpr.
Highlights
Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, the mechanism through which CypA modulates HIV-1 infectivity still remains unclear
In addition to the extensively studied interaction between CypA and HIV1 capsid, that is crucial for viral replication [12,13], CypA was reported to be significant for the de novo synthesis of viral protein R (Vpr), as the Vpr-mediated cell cycle arrest in HIV-1 infected T cells appeared to be eliminated in the absence of CypA activity [9]
Characterization of sVpr1-20, sVpr25-40 and sVpr21-40 by nuclear magnetic resonace (NMR) spectroscopy Previous studies [7,19,24,25] have shown that Vpr has a relatively random structure in aqueous solution at pH 7, but has a propensity for three well-defined a-helical structural domains in aqueous solution at lower pH or in the presence of organic co-solvent (TFE-d2 or CD3CN), where the extent of secondary structure is dependent on the hydrophobicity of the solvent
Summary
Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The 96 amino acid virion-associated multifunctional viral protein R (Vpr) [1,2] is encoded by primate lentiviruses, the human immunodeficiency viruses, types 1 and 2 (HIV-1/HIV-2), and simian immunodeficiency viruses (SIV). In particular Pro-35 exhibited a relatively high proportion of the cis isomer under these solvent conditions (15% cis isomer content) This suggested prolyl cis/trans isomerisation may be important for the folding of the molecule. At pH 7 Vpr has a relatively random structure in aqueous solution but assumes a folded structure in a hydrophobic membranous environment [7,19] This fact together with observation of considerable amounts of CypA in virions [20] prompted a study, using surface plasmon resonance (SPR) spectroscopy, of the interaction of Vpr with the prolyl cis/trans isomerase CypA [9]. The nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined
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