Cyclophilin A allows the allosteric regulation of a structural motif in the disordered domain 2 of NS5A and thereby fine-tunes HCV RNA replication.

Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues. Whether and how proline conformation regulates the functional aspects of IDPs remains an open question, however. Here, we studied the disordered domain 2 of nonstructural protein 5A (NS5A-D2) of hepatitis C virus (HCV). NS5A-D2 comprises a short structural motif (PW-turn) embedded in a proline-rich sequence, whose interaction with the human prolyl isomerase cyclophilin A (CypA) is essential for viral RNA replication. Using NMR, we show here that the PW-turn motif exists in a conformational equilibrium between folded and disordered states. We found that the fraction of conformers in the NS5A-D2 ensemble that adopt the structured motif is allosterically modulated both by the cis/trans isomerization of the surrounding prolines that are CypA substrates and by substitutions conferring resistance to cyclophilin inhibitor. Moreover, we noted that this fraction is directly correlated with HCV RNA replication efficiency. We conclude that CypA can fine-tune the dynamic ensemble of the disordered NS5A-D2, thereby regulating viral RNA replication efficiency.

Different mechanisms of hepatitis C virus RNA polymerase activation by cyclophilin A and B in vitro.

BACKGROUND: Cyclophilins (CyPs) are cellular proteins that are essential to hepatitis C virus (HCV) replication. Since cyclosporine A was discovered to inhibit HCV infection, the CyP pathway contributing to HCV replication is a potential attractive stratagem for controlling HCV infection. Among them, CyPA is accepted to interact with HCV nonstructural protein (NS) 5A, although interaction of CyPB and NS5B, an RNA-dependent RNA polymerase (RdRp), was proposed first. METHODS: CyPA, CyPB, and HCV RdRp were expressed in bacteria and purified using combination column chromatography. HCV RdRp activity was analyzed in vitro with purified CyPA and CyPB. RESULTS: CyPA at a high concentration (50× higher than that of RdRp) but not at low concentration activated HCV RdRp. CyPB had an allosteric effect on genotype 1b RdRp activation. CyPB showed genotype specificity and activated genotype 1b and J6CF (2a) RdRps but not genotype 1a or JFH1 (2a) RdRps. CyPA activated RdRps of genotypes 1a, 1b, and 2a. CyPB may also support HCV genotype 1b replication within the infected cells, although its knockdown effect on HCV 1b replicon activity was controversial in earlier reports. CONCLUSIONS: CyPA activated HCV RdRp at the early stages of transcription, including template RNA binding. CyPB also activated genotype 1b RdRp. However, their activation mechanisms are different. GENERAL SIGNIFICANCE: These data suggest that both CyPA and CyPB are excellent targets for the treatment of HCV 1b, which shows the greatest resistance to interferon and ribavirin combination therapy.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein from Piriformospora indica.

Cyclophilins are widely distributed both in eukaryotes and prokaryotes and have a primary role as peptidyl-prolyl cis-trans isomerases (PPIases). This study focuses on the cloning, expression, purification and crystallization of a salinity-stress-induced cyclophilin A (CypA) homologue from the symbiotic fungus Piriformospora indica. Crystallization experiments in the presence of 56 mM sodium phosphate monobasic monohydrate, 1.34 M potassium phosphate dibasic pH 8.2 yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 121.15, b = 144.12, c = 110.63 Å. The crystals diffracted to a resolution limit of 2.0 Å. Analysis of the diffraction data indicated the presence of three molecules of the protein per asymmetric unit (V(M) = 4.48 Å(3) Da(-1), 72.6% solvent content).

Streamlined, automated protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) using AKTAxpress.

We developed streamlined, automated purification protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) from Escherichia coli, using the AKTAxpress chromatography system. The automated 2-step (cation exchange and size exclusion) purification protocol for untagged hCypA results in final purity and yields of 93% and approximately 5 mg L(-1) of original cell culture, respectively, in under 12h, including all primary sample processing and column equilibration steps. The novel automated 4-step (anion exchange, desalt, heparin-affinity and size exclusion, in linear sequence) purification protocol for untagged hPCNA results in final purity and yields of 87% and approximately 4 mg L(-1) of original cell culture, respectively, in under 24h, including all primary sample processing and column equilibration steps. This saves in excess of four full working days when compared to the traditional protocol, producing protein with similar final yield, purity and activity. Furthermore, it limits a time-dependent protein aggregation, a problem with the traditional protocol that results in a loss of final yield. Both automated protocols were developed to use generic commercially available pre-packed columns and automatically prepared minimal buffers, designed to eliminate user and system variations, maximize run reproducibility, standardize yield and purity between batches, increase throughput and reduce user input to a minimum. Both protocols represent robust generic methods for the automated production of untagged hCypA and hPCNA.

Analyses of the toxic properties of recombinant human Cyclophilin A in mice.

Cyclophilin A (CypA), an 18 kDa multi-functional protein with cis-trans isomerase activity, is both a ligand for cyclosporine A and a proinflammatory factor. CypA is also a chemoattractant for hemopoietic stem cells and progenitors of different lineages, and can mediate regenerative processes in an organism. Accumulated experimental data have suggested there are practical applications for this protein in the treatment of several diseases (i.e. neutralization of cyclosporine A side effects, etc.). However, the range of CypA safe doses as well as its toxic effects remain unknown. The study here investigated the acute toxicity of a single intraperitoneal (IP) or subcutaneous (SC) dosing of recombinant human CypA (rhCypA) in both female and male mice and its effect on gene expression of acute phase proteins (APP) in the female mice after IP treatment. The results showed that toxicity of rhCypA was most evident in female and male mice dosed IP with 750 mg/kg, and manifested as kidney injury and increased granulocyte/lymphocyte ratios in the blood. Enhanced expression of Sаа1 and Sаа2 genes was induced with doses of 0.1-2 mg/mouse of rhCypA. Injection of the maximal dose (750 mg/kg) significantly stimulated expression of all the APP genes studied.

[Multiple isoforms of cyclophilin A associated with human immunodeficiency virus type 1].

It is well-known that a peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA) is incorporated into Human immunodeficiency virus type 1 (HIV-1) particle. The proteome analysis of the purified HIV-1 strain LAV-1 (HIV-1(LAV-1)) reveals that three isoforms of CyPA with an isoelectric point (pI) of 6.00, 6.40, and 6.53 are inside the viral membrane and another isoform with a pI of 6.88 is outside the viral membrane; and that the CyPA isoform with a pI of 6.53 is N-acetylated. The mechanisms that permit the redistribution of CyPA with a pI of 6.88 on the viral surface have not yet been clarified, but it penetrates the viral membrane after budding.

[Expression, purification and characterization of recombinant human cyclophilin A].

Plasmid-derived expression of the human CyPA in E. coli would make it possible to obtain ample protein quantities and to avoid difficult task of obtaining human tissues for protein purification. The cDNA encoding human CyPA from MT4 lymph cell line has been cloned and an expression vector (pET11/CyPA) has been constructed under control of the T7 promoter for efficient expression in E. coli. The recombinant CyPA is produced at 41% of total soluble cell protein, and showed the peptidyl-prodyl cis-trans isomerase activity.

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A.

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.