Host Protein Cyclophilin Research Articles

Self-Assembly of Fluorescent HIV Capsid Spheres for Detection of Capsid Binders.

The human immunodeficiency virus (HIV) capsid is a cone-shaped capsule formed from the viral capsid protein (CA), which is arranged into a lattice of hexamers and pentamers. The capsid comprises multiple binding interfaces for the recruitment of host proteins and macromolecules used by the virus to establish infection. Here, we coassembled CA proteins engineered for pentamer cross-linking and fluorescence labeling, into spherical particles. The CA spheres, which resemble the pentamer-rich structure of the end caps of the native HIV capsid, were immobilized onto surfaces as biorecognition elements for fluorescence microscopy-based quantification of host protein binding. The capsid-binding host protein cyclophilin A (CypA) is bound to CA spheres with the same affinity as CA tubes but at a higher CypA/CA stoichiometry, suggesting that the level of recruitment of CypA to the HIV capsid is dependent on curvature.

Shielding the HIV-1 capsid.

The host protein cyclophilin A binds to the HIV-1 capsid and its role in infection has long been enigmatic. A new study shows that this interaction protects the virus from inhibition by TRIM5α in physiologically relevant primary human cells.

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.

Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells.

Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process.

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.

The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import

BackgroundHIV-1 is inhibited early after entry into cells expressing some simian orthologues of the tripartite motif protein family member TRIM5α. Mutants of the human orthologue (TRIM5αhu) can also provide protection against HIV-1. The host protein cyclophilin A (CypA) binds incoming HIV-1 capsid (CA) proteins and enhances early stages of HIV-1 replication by unknown mechanisms. On the other hand, the CA-CypA interaction is known to increase HIV-1 susceptibility to restriction by TRIM5α. Previously, the mutation V86M in the CypA-binding loop of HIV-1 CA was found to be selected upon serial passaging of HIV-1 in cells expressing Rhesus macaque TRIM5α (TRIM5αrh). The objectives of this study were (i) to analyze whether V86M CA allows HIV-1 to escape mutants of TRIM5αhu, and (ii) to characterize the role of CypA in the resistance to TRIM5α conferred by V86M.ResultsWe find that in single-cycle HIV-1 vector transduction experiments, V86M confers partial resistance against R332G-R335G TRIM5αhu and other TRIM5αhu variable 1 region mutants previously isolated in mutagenic screens. However, V86M HIV-1 does not seem to be resistant to R332G-R335G TRIM5αhu in a spreading infection context. Strikingly, restriction of V86M HIV-1 vectors by TRIM5αhu mutants is mostly insensitive to the presence of CypA in infected cells. NMR experiments reveal that V86M alters CypA interactions with, and isomerisation of CA. On the other hand, V86M does not affect the CypA-mediated enhancement of HIV-1 replication in permissive human cells. Finally, qPCR experiments show that V86M increases HIV-1 transport to the nucleus of cells expressing restrictive TRIM5α.ConclusionsOur study shows that V86M de-couples the two functions associated with CA-CypA binding, i.e. the enhancement of restriction by TRIM5α and the enhancement of HIV-1 replication in permissive human cells. V86M enhances the early stages of HIV-1 replication in restrictive cells by improving nuclear import. In summary, our data suggest that HIV-1 escapes restriction by TRIM5α through the selective disruption of CypA-dependent, TRIM5α-mediated inhibition of nuclear import. However, V86M does not seem to relieve restriction of a spreading HIV-1 infection by TRIM5αhu mutants, underscoring context-specific restriction mechanisms.

The Host Proteins Transportin SR2/TNPO3 and Cyclophilin A Exert Opposing Effects on HIV-1 Uncoating

Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells.

Retroviral Restriction Factors TRIM5α: Therapeutic Strategy to Inhibit HIV-1 Replication

Tripartite motif protein 5-alpha (TRIM5α) is a cytoplasmic protein that efficiently recognizes the incoming capsid (CA) protein of retroviruses and potently inhibits virus infection in a species-specific manner. Through directly recognizing and interacting with HIV CA, TRIM5α is capable of disrupting the ordered process of viral uncoating, eventually interfering with HIV-1 reverse transcription and virus replication. TRIM5α protein contains four domains: RING domain, B-box 2 domain, coiled-coil domain, and B30.2 domain (SPRY) domain. All of the domains are necessary for efficient retrovirus restriction and the B30.2 domain has been shown to be the determinant of the specificity of restriction. Species-specific innate resistance against viral infections offers novel avenues for antiviral therapeutics. Various mutants of TRIM5α have been described which differently affect the HIV-1 reverse transcription process. This makes the establishment of new and improved models for HIV replication and AIDS pathogenesis by monitoring endogenous TRIM5α an attractive approach. TRIM5α-mediated restriction is modulated by the host protein Cyclophilin A (Cyp A) which could effectively interact with the CA of HIV-1. Here we will review the structure and roles of TRIM5α protein, the interaction between Cyp A and TRIM5α, as well as gene therapy strategies associated with TRIM5α to inhibit HIV-1 infection.

Small-Molecule Inhibition of Human Immunodeficiency Virus Type 1 Infection by Virus Capsid Destabilization

Human immunodeficiency virus type 1 (HIV-1) infection is dependent on the proper disassembly of the viral capsid, or "uncoating," in target cells. The HIV-1 capsid consists of a conical multimeric complex of the viral capsid protein (CA) arranged in a hexagonal lattice. Mutations in CA that destabilize the viral capsid result in impaired infection owing to defects in reverse transcription in target cells. We describe here the mechanism of action of a small molecule HIV-1 inhibitor, PF-3450074 (PF74), which targets CA. PF74 acts at an early stage of HIV-1 infection and inhibits reverse transcription in target cells. We show that PF74 binds specifically to HIV-1 particles, and substitutions in CA that confer resistance to the compound prevent binding. A single point mutation in CA that stabilizes the HIV-1 core also conferred strong resistance to the virus without inhibiting compound binding. Treatment of HIV-1 particles or purified cores with PF74 destabilized the viral capsid in vitro. Furthermore, the compound induced the rapid dissolution of the HIV-1 capsid in target cells. PF74 antiviral activity was promoted by binding of the host protein cyclophilin A to the HIV-1 capsid, and PF74 and cyclosporine exhibited mutual antagonism. Our data suggest that PF74 triggers premature HIV-1 uncoating in target cells, thereby mimicking the activity of the retrovirus restriction factor TRIM5α. This study highlights uncoating as a step in the HIV-1 life cycle that is susceptible to small molecule intervention.

Determinants of cyclophilin A-dependent TRIM5α restriction against HIV-1

TRIM5α is a host protein that can bind to incoming retroviral capsid (CA) and inhibit retroviruses in a species-specific manner. The CA protein of HIV-1 also interacts with high affinity to the host protein cyclophilin A (CypA). This binding has been shown to positively affect some early stage of the viral life cycle in human cells. However, the CypA/CA interaction also renders HIV-1 more susceptible to rhesus TRIM5α (rhTRIM5α) restriction. We find that the ability of old world monkey TRIM5α genes to restrict HIV-1 in a CypA-dependent manner is widespread. On the other hand, we find that simian immunodeficiency viruses from tantalus monkeys (SIVagmTAN), is unlike HIV-1 in that CypA does not enhance the rhTRIM5α restriction against the virus even though the CA of this virus, like HIV-1, does bind CypA. Mapping of the determinants for this phenotype by swapping regions on CA between SIVagmTAN and HIV-1 showed that when SIVagmTAN contains loops between helices 4/5 (4–5 loop) and 6/7 (6–7 loop) from HIV-1 CA, it becomes susceptible to the CypA-enhanced rhTRIM5α restriction. Surprisingly, when SIVagmTAN contains either loop from HIV-1 CA, it gains sensitivity to TRIM5α from species which originally have no effect on the wild-type virus. Moreover, we find that CypA/CA interaction occurs early after viral entry but the CypA-enhanced restriction mostly acts on the stage after reverse transcription.

Nef enhances HIV-1 infectivity via association with the virus assembly complex

The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle.

Cyclophilin A interacts with diverse lentiviral capsids

BackgroundThe capsid (CA) protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA). This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay.ResultsWe find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA.ConclusionThese results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.

The Cysteine Residues of HIV-1 Capsid Regulate Oligomerization and Cyclophilin A-Induced Changes

Assembly of the HIV-1 virus involves, in part, strong interactions between the capsid (CA) domains of the Gag polyprotein. During maturation, the core of HIV-1 virions undergoes profound morphological changes due primarily to proteolysis of the CA domain from other Gag domains which may allow for more efficient disassembly of the viral core in the early stages of infection. The host protein cyclophilin A (CypA), a cis-trans prolyl isomerase, in some way seems to assist in this assembly/disassembly process. Using an unproteolyzed construct of CA, we show that binding of CypA induces a large-scale conformational change in CA that is independent of its cis-trans prolyl isomerase activity. This change appears to be mediated by Cys-198 of CA since mutation to Ala renders CypA unable to induce this change and alters the kinetics and stability of protein cores that may ultimately result in inefficient disassembly of viral cores. Alternately, mutation of the second CA Cys (C218A) allows for CypA-induced conformational changes but alters the kinetics and morphology of the protein cores that may ultimately result in inefficient assembly of viral cores. These studies show the importance of the CA Cys residues in mediating the contacts needed for viral assembly and disassembly.

The pH dependence of HIV-1 capsid assembly and its interaction with cyclophilin A

Immature HIV-1 virions have spherical cores which become conical due to cleavage of the capsid domain of Gag. Here, we have used an immature form of capsid and show by electron microscopy, atomic force microscopy and single angle light scattering that it aggregates to spherical cores resembling immature virions at high ionic strengths and at pH values above 6. Dynamic angle light scattering of the dissociated protein shows structural changes that promote oligomerization above pH 6. We then examined the role of the required host protein cyclophilin A on assembly. Cyclophilin A is incorporated into virions at a 1:10 cyclophilin A/capsid ratio. We find that although cyclophilin A does not affect the oligomerization rate or stability of immature capsid cores, it does bind strongly to immature capsid at physiological stoichiometry above pH 6. This association serves as an entry route of cyclophilin A into HIV-1 virions.