CypA Binding Research Articles

Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

Interplay among Conformation, Intramolecular Hydrogen Bonds, and Chameleonicity in the Membrane Permeability and Cyclophilin A Binding of Macrocyclic Peptide Cyclosporin O Derivatives.

A macrocyclic peptide scaffold with well-established structure-property relationship is desirable for tackling undruggable targets. Here, we adopted a natural macrocycle, cyclosporin O (CsO) and its derivatives (CP1-3), and evaluated the impact of conformation on membrane permeability, cyclophilin A (CypA) binding, and the pharmacokinetic (PK) profile. In nonpolar media, CsO showed a similar conformation to cyclosporin A (CsA), a well-known chameleonic macrocycle, but less chameleonic behavior in a polar environment. The weak chameleonicity of CsO resulted in decreased membrane permeability; however, the more rigid conformation of CsO was not detrimental to its PK profile. CsO exhibited a higher plasma concentration than CsA, which resulted from minimal CypA binding and lower accumulation in red blood cells and moderate oral bioavailability (F = 12%). Our study aids understanding of CsO, a macrocyclic peptide that is less explored than CsA but with greater potential for diversity generation and rational design.

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis-Trans Isomerization and the Chameleonic Roles of Calcium.

Falling outside of Lipinski's rule of five, macrocyclic drugs have accessed unique binding sites of their target receptors unreachable by traditional small molecules. Cyclosporin(e) A (CycA), an extensively studied macrocyclic natural product, is an immunosuppressant with undesirable side effects such as electrolytic imbalances. In this work, a comprehensive view on the conformational landscape of CycA, its interactions with Ca2+, and host-guest interactions with cyclophilin A (CypA) is reported through exhaustive analyses that combine ion-mobility spectrometry-mass spectrometry (IMS-MS), nuclear magnetic resonance (NMR) spectroscopy, distance-geometry modeling, and NMR-driven molecular dynamics. Our IMS-MS data show that CycA can adopt extremely compact conformations with significantly smaller collisional cross sections than the closed conformation observed in CDCl3. To adopt these conformations, the macrocyclic ring has to twist and bend via cis-trans isomerization of backbone amides, and thus, we termed this family of structures the "bent" conformation. Furthermore, NMR measurements indicate that the closed conformation exists at 19% in CD3OD/H2O and 55% in CD3CN. However, upon interacting with Ca2+, in addition to the bent and previously reported closed conformations of free CycA, the CycA:Ca2+ complex is open and has all-trans peptide bonds. Previous NMR studies using calcium perchlorate reported only the closed conformation of CycA (which contains one cis peptide bond). Here, calcium chloride, a more biologically relevant salt, was used, and interestingly, it helps converting the cis-MeLeu9-MeLeu10 peptide bond into a trans bond. Last, we were able to capture the native binding of CycA and CypA to give forth evidence that IMS-MS is able to probe the solution-phase structures of the complexes and that the Ca2+:CycA complex may play an essential role in the binding of CycA to CypA.

TRIM5\u03b1 self-assembly and compartmentalization of the HIV-1 viral capsid

The tripartite-motif protein, TRIM5α, is an innate immune sensor that potently restricts retrovirus infection by binding to human immunodeficiency virus capsids. Higher-ordered oligomerization of this protein forms hexagonally patterned structures that wrap around the viral capsid, despite an anomalously low affinity for the capsid protein (CA). Several studies suggest TRIM5α oligomerizes into a lattice with a symmetry and spacing that matches the underlying capsid, to compensate for the weak affinity, yet little is known about how these lattices form. Using a combination of computational simulations and electron cryo-tomography imaging, we reveal the dynamical mechanisms by which these lattices self-assemble. Constrained diffusion allows the lattice to reorganize, whereas defects form on highly curved capsid surfaces to alleviate strain and lattice symmetry mismatches. Statistical analysis localizes the TRIM5α binding interface at or near the CypA binding loop of CA. These simulations elucidate the molecular-scale mechanisms of viral capsid cellular compartmentalization by TRIM5α.

Cyclophilin A Prevents HIV-1 Restriction in Lymphocytes by Blocking Human TRIM5α Binding to the Viral Core

Disruption of cyclophilin A (CypA)-capsid interactions affects HIV-1 replication in human lymphocytes. To understand this mechanism, we utilize human Jurkat cells, peripheral blood mononuclear cells (PBMCs), and CD4+ Tcells. Our results show that inhibition of HIV-1 infection caused by disrupting CypA-capsid interactions is dependent on human tripartite motif 5α (TRIM5αhu), showing that TRIM5αhu restricts HIV-1 in CD4+ Tcells. Accordingly, depletion of TRIM5αhu in CD4+ Tcells rescues HIV-1 that fail to interact with CypA, such as HIV-1-P90A. We found that TRIM5αhu binds to the HIV-1 core. Disruption of CypA-capsid interactions fail to affect HIV-1-A92E/G94D infection, correlating with the loss of TRIM5αhu binding to HIV-1-A92E/G94D cores. Disruption of CypA-capsid interactions in primary cells has a greater inhibitory effect on HIV-1 when compared to Jurkat cells. Consistent with TRIM5α restriction, disruption of CypA-capsid interactions in CD4+ Tcells inhibits reverse transcription. Overall, our results reveal that CypA binding to the core protects HIV-1 from TRIM5αhu restriction.

Abstract 4508: Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex-mediated signaling, mammary tumorigenesis, and metastasis

Abstract Prolactin (PRL) and its receptor (PRLr) have been implicated in the development and progression of human breast cancer. PRL activates its receptor and induces activation of proximal Janus kinase 2 (Jak2). Jak2 associates with PRLr, phosphorylates c-terminus of the PRLr, which leads to Stat5 recruitment and activation. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase (PPI), which is constitutively bound to the PRLr that catalyze the cis-trans interconversion of proline imide bonds. Treatment with Cyclosporine (CsA) inhibited CypA binding to the PRLr and blocked PRLr-driven activation of Jak2/Stat5. Recently, Waters et al., utilizing Fluorescence Resonance Energy Transfer (FRET) with transfectants expressing CFP and YFP-tagged forms of the growth hormone receptor (GHR) showed that GHR activation induced a rotational movement in C-terminus of the GHR, resulting in a loss of baseline FRET signal. Like the GHR, PRL stimulation of transfectants expressing CFP/YFP-tagged PRLr constructs resulted in a loss of FRET efficiency. In contrast, treatment with NIM811 (a non-immunosuppressive form of CsA) or siRNA knockdown of CypA resulted in a return of FRET signal in the presence of PRL. These studies reveal that ligand stimulation of the PRLr results in a conformational change as measured by FRET signal of the receptor that is reversed by CypA inhibition or knockdown, implicating CypA as the mediator of this conformational change and ligand-induced signaling. To further assess the consequences of CypA inhibition or knockdown on the PRLr/Jak2 mediated signaling/functions, analyses of phospho-tyrosine residues that are believed to be important for interactions/signaling were investigated in breast cancer cells. It was found that NIM811 inhibition or CypA shRNA knockdown significantly reduced prolactin-stimulated phosphorylation of PRLr/Jak2 intermediates in ER+/PR+ T47D cells in a time dependent manner. A microarray analysis revealed that NIM811 inhibited approximately 66% of the top 50 PRL induced genes. NIM811 inhibited ER-/+, and HER2+ breast cancer cell proliferation, survival, migration and anchorage-independent growth. Subsequent pre-clinical testing of NIM811 in relevant mouse mammary cancer models has found that NIM811 treatment of a TNBC xenograft inhibited primary tumor growth, outgrowth of macro-metastasis and induced central tumor necrosis. Furthermore, loss of CypA (by constitutive genetic deletion) in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. Overall, CypA modulates conformational change in the C-terminus of the PRLr through its PPI activity, and alters PRLr/Jak2 complex signaling/functions in breast cancer and mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention as a chemo-preventive and as an inhibitor of metastasis. Citation Format: Shawn Hakim, Shannon E. Hedrick, Justin M. Craig, Charles V. Clevenger. Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex-mediated signaling, mammary tumorigenesis, and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4508.

Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A

BackgroundEfficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme’s active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016).ResultsWe performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1.ConclusionsThe amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA.

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells

BackgroundRetroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC.ResultsIn this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation.ConclusionsWe showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

Modeling global changes induced by local perturbations to the HIV-1 capsid

The HIV-1 capsid is a conical protein shell made up of hexamers and pentamers of the capsid protein. The capsid houses the viral genome and replication machinery, and its opening, or uncoating, within the host cell marks a critical step in the HIV-1 lifecycle. Binding of host factors such as TRIM5α and cyclophilin A (CypA) can alter the capsid’s stability, accelerating or delaying the onset of uncoating and disrupting infectivity. We employ coarse-grained computational modeling to investigate the effects of point mutations and host factor binding on HIV-1 capsid stability. We find that the largest fluctuations occur in the low-curvature regions of the capsid, and that its structural dynamics are affected by perturbations at the inter-hexamer interfaces and near the CypA binding loop, suggesting roles for these features in capsid stability. Our models show that linking capsid proteins across hexamers attenuates vibration in the low-curvature regions of the capsid, but that linking within hexamers does not. These results indicate a possible mechanism through which CypA binding alters capsid stability and highlight the utility of coarse-grained network modeling for understanding capsid mechanics.

Dynamic allostery governs cyclophilin A–HIV capsid interplay

Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone (1)H-(15)N and (1)H-(13)C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1's escape from CypA dependence.

Mutations in CypA Binding Region of HIV-1 Capsid Affect Capsid Stability and Viral Replication in Primary Macrophages.

Mutations in the cyclophilin A (CypA) binding region in the HIV-1 capsid affect their dependency on the known HIV-1 cofactor CypA and allow escape from the HIV-1 restriction factor Trim5α in human and simian cells. Here we study the effect of these mutations in the CypA binding region of capsid on cofactor binding, capsid destabilization, and viral replication in primary cells. We showed that the viral capsid with mutations in the CypA binding region (CypA-BR) interacted efficiently with CypA, but had an increased stability upon infection as compared to the wild-type capsid. Interestingly, the wild-type virus was able to infect monocyte-derived macrophages (MDM) more efficiently as compared to the CypA-BR mutant variant. The lower infectivity of the CypA-BR mutant virus in MDM was associated with lower levels of reverse transcription products. Similar to the wild-type virus, the CypA-BR mutant variant was unable to induce a strong innate response in primary macrophages. These data demonstrate that mutations in the CypA binding site of the capsid resulted in higher capsid stability and hampered infectivity in macrophages.

The highly polymorphic cyclophilin A-binding loop in HIV-1 capsid modulates viral resistance to MxB.

BackgroundThe human myxovirus-resistance protein B (MxB, also called Mx2) was recently reported to inhibit HIV-1 infection by impeding the nuclear import and integration of viral DNA. However, it is currently unknown whether there exist MxB-resistant HIV-1 strains in the infected individuals. Answer to this question should address whether MxB exerts an inhibitory pressure on HIV-1 in vivo and whether HIV-1 has evolved to evade MxB inhibition.FindingsWe have examined ten transmitted founder (T/F) HIV-1 strains for their sensitivity to MxB inhibition by infecting CD4+ T cell lines SupT1 and PM1 that were stably transduced to express MxB. Two T/F stains, CH040.c and RHPA.c, were found resistant and this resistance phenotype was mapped to the amino acid positions 87 and 208 in viral capsid. The H87Q mutation is located in the cyclophilin A (CypA) binding loop and has a prevalence of 21% in HIV-1 sequences registered in HIV database. This finding prompted us to test other frequent amino acid variants in the CypA-binding region and the results revealed MxB-resistant mutations at amino acid positions 86, 87, 88 and 92 in capsid. All these mutations diminished the interaction of HIV-1 capsid with CypA.ConclusionsOur results demonstrate the existence of MxB-resistant T/F HIV-1 strains. The high prevalence of MxB-resistant mutations in the CypA-binding loop indicates the significant selective pressure of MxB on HIV-1 replication in vivo especially given that this viral resistance mechanism operates at expense of losing CypA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0129-1) contains supplementary material, which is available to authorized users.

Cyclophilin a Stabilizes the Mature HIV-1 Capsid through a Novel Non-Canonical Binding Site

Host cell factor cyclophilin A (CypA) plays an important role in modulating HIV-1 capsid function. Several other host cell proteins, including TRIMCyp and NUP358, contain a CypA domain that interacts directly with viral capsid. The binding of the host protein CypA to the viral capsid is important for infection, yet the structural effects of this binding have not been identified. We determined the cryoEM structure of CypA in complex with an HIV-1 capsid assembly at 8 A resolution. The density map displays a non-random, selective binding of CypA along the most curved helical direction, forming a bridge directly above the CA CTD-CTD dimer interface cross the adjacent CA hexamers. CryoEM structure-based modeling and large scale all-atoms molecular dynamics simulations reveal unexpectedly that one CypA molecule simultaneously interacts with two CA molecules through a non-canonical novel interface. The individual residuals critical for the interactions were further identified by solution and solid state NMR and confirmed by mutagenesis studies. Our combined cryoEM, computational and NMR studies provide mechanistic insights into the functional role of CypA in modulating capsid uncoating and viral infectivity, and our structure further highlights the novel CypA and CA interface as an attractive therapeutic target for pharmacological intervention.

Contribution of glutamine residues in the helix 4-5 loop to capsid-capsid interactions in simian immunodeficiency virus of macaques.

Following retrovirus entry, the viral capsid (CA) disassembles into its component capsid proteins. The rate of this uncoating process, which is regulated by CA-CA interactions and by the association of the capsid with host cell factors like cyclophilin A (CypA), can influence the efficiency of reverse transcription. Inspection of the CA sequences of lentiviruses reveals that several species of simian immunodeficiency viruses (SIVs) have lost the glycine-proline motif in the helix 4-5 loop important for CypA binding; instead, the helix 4-5 loop in these SIVs exhibits an increase in the number of glutamine residues. In this study, we investigated the role of these glutamine residues in SIVmac239 replication. Changes in these residues, particularly glutamine 89 and glutamine 92, resulted in a decreased efficiency of core condensation, decreased stability of the capsids in infected cells, and blocks to reverse transcription. In some cases, coexpression of two different CA mutants produced chimeric virions that exhibited higher infectivity than either parental mutant virus. For this complementation of infectivity, glutamine 89 was apparently required on one of the complementing pair of mutants and glutamine 92 on the other. Modeling suggests that glutamines 89 and 92 are located on the distal face of hexameric capsid spokes and thus are well positioned to contribute to interhexamer interactions. Requirements to evade host restriction factors like TRIMCyp may drive some SIV lineages to evolve means other than CypA binding to stabilize the capsid. One solution used by several SIV strains consists of glutamine-based bonding. The retroviral capsid is an assembly of individual capsid proteins that surrounds the viral RNA. After a retrovirus enters a cell, the capsid must disassemble, or uncoat, at a proper rate. The interactions among capsid proteins contribute to this rate of uncoating. We found that some simian immunodeficiency viruses use arrays of glutamine residues, which can form hydrogen bonds efficiently, to keep their capsids stable. This strategy may allow these viruses to forego the use of capsid-stabilizing factors from the host cell, some of which have antiviral activity.

Gene Expression Analysis of a Panel of Cell Lines That Differentially Restrict HIV-1 CA Mutants Infection in a Cyclophilin A-Dependent Manner

HIV-1 replication is dependent on binding of the viral capsid to the host protein cyclophilin A (CypA). Interference with cyclophilin A binding, either by mutations in the HIV-1 capsid protein (CA) or by the drug cyclosporine A (CsA), inhibits HIV-1 replication in cell culture. Resistance to CsA is conferred by A92E or G94D substitutions in CA. The mutant viruses are also dependent on CsA for their replication. Interestingly, infection of some cell lines by these mutants is enhanced by CsA, while infection of others is not affected by the drug. The cells are thus termed nonpermissive and permissive, respectively, for infection by CsA-dependent mutants. The mechanistic basis for the cell type dependence is not well understood, but has been hypothesized to result from a dominant-acting host factor that blocks HIV-1 infection by a mechanism that requires CypA binding to the viral capsid. In an effort to identify a CypA-dependent host restriction factor, we adopted a strategy involving comparative gene expression analysis in three permissive and three non-permissive cell types. We ranked the genes based on their relative overexpression in non-permissive cell types compared to the permissive cell types. Based on specific selection criteria, 26 candidate genes were selected and targeted using siRNA in nonpermissive (HeLa) cells. Depletion of none of the selected candidate genes led to the reversal of CsA-dependent phenotype of the A92E mutant. Our data suggest that none of the 26 genes tested is responsible for the dependence of the A92E mutant on CsA. Our study provides gene expression data that may be useful for future efforts to identify the putative CypA-dependent HIV-1 restriction factor and in studies of other cell-specific phenotypes.

Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA

BackgroundThe human peptidyl-prolyl isomerase Cyclophilin A (CypA) binds HIV-1 capsid (CA) and influences early steps in the HIV-1 replication cycle. The mechanism by which CypA regulates HIV-1 transduction efficiency is unknown. Disruption of CypA binding to CA, either by genetic means or by the competitive inhibitor cyclosporine A (CsA), reduces the efficiency of HIV-1 transduction in some cells but not in others. Transduction of certain cell types increases significantly when CypA binding to particular HIV-1 CA mutants, i.e., A92E, is prevented. Previous studies have suggested that this cell type-specific effect is due to a dominant-acting, CypA-dependent restriction factor.ResultsHere we investigated the mechanism by which CypA regulates HIV-1 transduction efficiency using 27 different human cell lines, 32 HeLa subclones, and several previously characterized HIV-1 CA mutants. Disruption of CypA binding to wild-type CA, or to any of the mutant CAs, caused a decrease in HIV-1 reverse transcription in all the cell lines analyzed here. This block to reverse transcription, though, did not correlate with cell type-specific effects on transduction efficiency. The level of 2-LTR circles, a marker for nuclear transport of the viral cDNA that results from reverse transcription, correlated closely with effects on infectivity. No correlation was observed between the cell type-specific effects on infectivity and the steady-state CypA protein levels in these cells. Instead, as indicated by a fate-of-capsid assay, CsA released the HIV-1 CA core from an apparent state of hyperstabilization, in a cell type-specific manner.ConclusionThese data demonstrate that, while CypA promotes reverse transcription under all conditions tested here, its effect on HIV-1 infectivity correlates more closely with effects on nuclear entry of the viral cDNA. The data also support the hypothesis that a cell-type specific CypA-dependent restriction factor blocks HIV-1 replication by delaying CA core uncoating and hindering nuclear entry.

Correlation of Naturally Occurring HIV-1 Resistance to DEB025 with Capsid Amino Acid Polymorphisms

DEB025 (alisporivir) is a synthetic cyclosporine with inhibitory activity against human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV). It binds to cyclophilin A (CypA) and blocks essential functions of CypA in the viral replication cycles of both viruses. DEB025 inhibits clinical HIV-1 isolates in vitro and decreases HIV-1 virus load in the majority of patients. HIV-1 isolates being naturally resistant to DEB025 have been detected in vitro and in nonresponder patients. By sequence analysis of their capsid protein (CA) region, two amino acid polymorphisms that correlated with DEB025 resistance were identified: H87Q and I91N, both located in the CypA-binding loop of the CA protein of HIV-1. The H87Q change was by far more abundant than I91N. Additional polymorphisms in the CypA-binding loop (positions 86, 91 and 96), as well as in the N-terminal loop of CA were detected in resistant isolates and are assumed to contribute to the degree of resistance. These amino acid changes may modulate the conformation of the CypA-binding loop of CA in such a way that binding and/or isomerase function of CypA are no longer necessary for virus replication. The resistant HIV-1 isolates thus are CypA-independent.

Subtype Specific Differences in NS5A Domain II Reveals Involvement of Proline at Position 310 in Cyclosporine Susceptibility of Hepatitis C Virus

Hepatitis C virus (HCV) is susceptible to cyclosporine (CsA) and other cyclophilin (CypA) inhibitors, but the genetic basis of susceptibility is controversial. Whether genetic variation in NS5A alters cell culture susceptibility of HCV to CypA inhibition is unclear. We constructed replicons containing NS5A chimeras from genotypes 1a, 2a and 4a to test how variation in carboxy terminal regions of NS5A altered the genotype 1b CsA susceptibility. All chimeric replicons including genotype 1b Con1LN-wt replicon exhibited some cell culture sensitivity to CsA with genotype 4a being most sensitive and 1a the least. The CypA binding pattern of truncated NS5A genotypes correlated with the susceptibility of these replicons to CsA. The Con1LN-wt replicon showed increased susceptibility towards CsA when proline at position 310P was mutated to either threonine or alanine. Furthermore, a 15 amino acid long peptide fused N terminally to GFP coding sequences confirmed involvement of proline at 310 in CypA binding. Our findings are consistent with CypA acting on multiple prolines outside of the previously identified CypA binding sites. These results suggest multiple specific genetic variants between genotype 1a and 1b in the C-terminus of NS5A alter the CsA susceptibility of replicons, and some variants may oppose the effects of others.

HCV NS5A and IRF9 compete for CypA binding

Cyclophilin A (CypA) is vital for HCV replication. Cyp inhibitors successfully decrease viral loads in HCV-infected patients. However, their mechanisms of action remain unknown. Since interferon (IFN) can also suppress HCV replication, we asked whether a link between CypA and the IFN response exists. We used cellular and recombinant pulldown approaches to investigate the possibility of a specific association of CypA with host ligands. We found for the first time that CypA binds to a major component of the IFN response - the IFN regulatory factor 9 (IRF9). IRF9 is the DNA-binding component of the transcriptional IFN-stimulated gene factor 3 (ISGF3). CypA binds directly to IRF9 via its peptidyl-prolyl isomerase (PPIase) pocket. Cyp inhibitors such as cyclosporine A (CsA) or non-immunosuppressive derivates such as alisporivir and SCY-635, prevent IRF9-CypA complex formation. CypA binds to the C-terminal IRF-association-domain (IAD), but not to the DNA-binding or linker domains of IRF9. Remarkably, CypA associates with the multimeric ISGF3 complex. We also obtained evidence that CypA neutralization enhances IFN-induced transcription. Interestingly, the hepatitis C virus (HCV) non-structural 5A (NS5A) protein, which is known to modulate the IFN response, competes with IRF9 for CypA binding and can prevent the formation of IRF9-CypA complexes. This study demonstrates for the first time that CypA binds specifically to a component of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, IRF9. This study also reveals a novel opportunity of HCV to modulate the IFN response via NS5A.

The Cyclophilin Inhibitor SCY-635 Disrupts Hepatitis C Virus NS5A-Cyclophilin A Complexes

The nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication both in vitro and in vivo and represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arose in vitro under SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA.