The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

Background

Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear.

Results

Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues ⁷⁵GCRHSRIGVTRQRRAR⁹⁰, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr^75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA.

Conclusions

For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.     

HIV-1 p6—Another viral interaction partner to the host cellular protein cyclophilin A

The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D ¹H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl-prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme-substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.     

Induction of G2 arrest and binding to cyclophilin A are independent phenotypes of human immunodeficiency virus type 1 Vpr

Cyclophilin A (CypA) is a member of a family of cellular proteins that share a peptidyl prolyl cis-trans isomerase (PPIase) activity. CypA was previously reported to be required for the biochemical stability and function (specifically, induction of G2 arrest) of the human immunodeficiency virus type 1 (HIV-1) protein R (Vpr). In the present study, we examine the role of the Vpr-CypA interaction on Vpr-induced G2 arrest. We find that Vpr coimmunoprecipitates with CypA and that this interaction is disrupted by substitution of proline-35 of Vpr as well as incubation with the CypA inhibitor cyclosporine A (CsA). Surprisingly, the presence of CypA or its binding to Vpr is dispensable for the ability of Vpr to induce G2 arrest. Vpr expression in CypA-/- cells leads to induction of G2 arrest in a manner that is indistinguishable from that in CypA+ cells. CsA abolished CypA-Vpr binding but had no effect on induction of G2 arrest or Vpr steady-state levels. In view of these results, we propose that the interaction with CypA is independent of the ability of Vpr to induce cell cycle arrest. The interaction between Vpr and CypA is intriguing, and further studies should examine its potential effects on other functions of Vpr.     

Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41

Background

The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W)-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clément, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816–48826, 2002). In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae.

Results

Our data clearly shows that integrity of N-terminal α-helix I (17–33) and α-helix III (53–83) is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides.

Conclusions

Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes α-helix I (17–33) and α-helix III (53–83). Furthermore, the mechanism(s) underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells.     

HIV-1 p6-Another viral interaction partner to the host cellular protein cyclophilin A

The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D (1)H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl-prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme-substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.     

Intricacies of interactions,dynamics and solvent effects in enzyme catalysis: a computational perspective on cyclophilin A

The essential role of enzymes in biological processes has continually ignited sparks of interest in their mechanism of action. Fully understanding the mechanism of enzymes has broad implications in protein engineering and drug design. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerisation by cyclophilin A (CypA) has been the target of intense research. CypA serves as a tractable model system, because it reversibly catalyses the rotation around peptidyl–prolyl bonds without any bond breakage or formation. Here, we discuss the results of recent computational approaches used to study the mechanism of CypA. We highlight the critical role of enzyme and substrate conformational dynamics in the developing interactions as the substrate approaches the transition state that results in an astonishing enhancement of isomerisation rate. The rate of isomerisation is affected by the intricate coupling between the dynamics of the substrate, enzyme and solvent. CypA binds its substrates via conformational selection, where rearrangements of key active site residues are necessary for substrate recognition. The conformational plasticity of the active site allows the enzyme to accommodate the most favourable interactions with the transition state that can be exploited for structure-based drug design.     

Identification of human immunodeficiency virus type 1 (HIV-1) gp120-binding sites on scavenger receptor cysteine rich 1 (SRCR1) domain of gp340

Background

gp340, a member of scavenger receptor cysteine rich family encoded by Deleted in Malignant Brain Tumors 1 (DMBT1), is an important component in innate immune defense. The first scavenger receptor cysteine rich domain (SRCR1) of gp340 has been shown to inhibit HIV-1 infection through binding to the N-terminal flank of the V3 loop of HIV-1 gp120.

Results

Through homology modeling and docking analysis of SRCR1 to a gp120-CD4-X5 antibody complex, we identified three loop regions containing polar or acidic residues that directly interacted with gp120. To confirm the docking prediction, a series of over-lapping peptides covering the SRCR1 sequence were synthesized and analyzed by gp120-peptide binding assay. Five peptides coincide with three loop regions showed the relative high binding index. An alanine substitution scan revealed that Asp34, Asp35, Asn96 and Glu101 in two peptides with the highest binding index are the critical residues in SRCR1 interaction with gp120.

Conclusion

We pinpointed the vital gp120-binding regions in SRCR1 and narrowed down the amino acids which play critical roles in contacting with gp120.     

Mechanism of Action of Cyclophilin A Explored by Metadynamics Simulations

Trans/cis prolyl isomerisation is involved in several biological processes, including the development of numerous diseases. In the HIV-1 capsid protein (CA), such a process takes place in the uncoating and recruitment of the virion and is catalyzed by cyclophilin A (CypA). Here, we use metadynamics simulations to investigate the isomerization of CA's model substrate HAGPIA in water and in its target protein CypA. Our results allow us to propose a novel mechanistic hypothesis, which is finally consistent with all of the available molecular biology data.     

Proline 35 of human immunodeficiency virus type 1 (HIV-1) Vpr regulates the integrity of the N-terminal helix and the incorporation of Vpr into virus particles and supports the replication of R5-tropic HIV-1 in human lymphoid tissue ex vivo

Mutational analysis of the four conserved proline residues in human immunodeficiency virus type 1 (HIV-1) Vpr reveals that only Pro-35 is required for efficient replication of R5-tropic, but not of X4-tropic, viruses in human lymphoid tissue (HLT) cultivated ex vivo. While Vpr-mediated apoptosis and G(2) cell cycle arrest, as well as the expression and subcellular localization of Vpr, were independent, the capacity for encapsidation of Vpr into budding virions was dependent on Pro-35. (1)H nuclear magnetic resonance data suggest that mutation of Pro-35 causes a conformational change in the hydrophobic core of the molecule, whose integrity is required for the encapsidation of Vpr, and thus, Pro-35 supports the replication of R5-tropic HIV-1 in HLT.     

Catalysis and binding of cyclophilin A with different HIV-1 capsid constructs

The prolyl isomerase cyclophilin A (CypA) is required for efficient HIV-1 replication and is incorporated into virions through a binding interaction at the Gly-Pro(222) bond located within the capsid domain of the HIV-1 Gag precursor polyprotein (Pr(gag)). It has recently been shown that CypA efficiently catalyzes the cis/trans isomerization of Gly-Pro(222) within the isolated N-terminal domain of capsid (CA(N)). To address the proposal that CypA interacts with Gly-Pro sequences in the C-terminal domain of a mature capsid, the interaction between CypA and the natively folded, full-length capsid protein (CA(FL)) has been investigated here using nuclear magnetic resonance spectroscopy. In addition, a fragment of the Pr(gag) protein encoding the full-matrix protein and the N-terminal domain of capsid (MA-CA(N)) has been used to probe the catalytic interaction between CypA and an immature form of the capsid. The results discussed herein strongly suggest that Gly-Pro(222) located within the N-terminal domain of the capsid is the preferential site for CypA binding and catalysis and that catalysis of Gly-Pro(222) is unaffected by maturational processing at the N-terminus of the capsid.     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

trans-Complementation rescue of cyclophilin A-deficient viruses reveals that the requirement for cyclophilin A in human immunodeficiency virus type 1 replication is independent of its isomerase activity

Human immunodeficiency virus type 1 (HIV-1) requires the incorporation of cyclophilin A (CypA) for replication. CypA is packaged by binding to the capsid (CA) region of Gag. This interaction is disrupted by cyclosporine (CsA). Preventing CypA incorporation, either by mutations in the binding region of CA or by the presence of CsA, abrogates virus infectivity. Given that CypA possesses an isomerase activity, it has been proposed that CypA acts as an uncoating factor by destabilizing the shell of CA that surrounds the viral genome. However, because the same domain of CypA is responsible for both its isomerase activity and its capacity to be packaged, it has been challenging to determine if isomerase activity is required for HIV-1 replication. To address this issue, we fused CypA to viral protein R (Vpr), creating a Vpr-CypA chimera. Because Vpr is packaged via the p6 region of Gag, this approach bypasses the interaction with CA and allows CypA incorporation even in the presence of CsA. Using this system, we found that Vpr-CypA rescues the infectivity of viruses lacking CypA, either produced in the presence of CsA or mutated in the CypA packaging signal of CA. Furthermore, a Vpr-CypA mutant which has no isomerase activity and no capacity to bind to CA also rescues HIV-1 replication. Thus, this study demonstrates that the isomerase activity of CypA is not required for HIV-1 replication and suggests that the interaction of the catalytic site of CypA with CA serves no other function than to incorporate CypA into viruses.     

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.     

Crystal structure of Diedel, a marker of the immune response of Drosophila melanogaster

Background

The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines.

Methodology/Principal Findings

We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 Å resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel β-sheet covered by an α-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families.

Conclusion/Significance

Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small family of proteins present in drosophilids, aphids and DNA viruses infecting lepidopterans. Diedel is an extracellular protein composed of two sub-domains. Two special structural features (hydrophobic surface patch and cis/trans conformation for proline 52) may indicate a putative interaction site, and support an extra cellular signaling function for Diedel, which is in accordance with its proposed role as negative regulator of the JAK/STAT signaling pathway.     

PP2A1 binding, cell transducing and apoptotic properties of Vpr(77-92): a new functional domain of HIV-1 Vpr proteins

Background

The hallmark of HIV-1 pathogenesis is the progressive CD4⁺ T cell depletion and high propensity of CD4⁺ T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr_71-82 mitochondriotoxic domain containing the conserved sequence _71-HFRIGCRHSRIG_-82 with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A₁ is involved in the induction of G2 arrest by HIV-1 Vpr.

Principal Findings

Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A₁ binding sequence from Vpr_77–92 is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR_83–85 and T89A substitutions in the Vpr_77–92 sequence prevent PP2A₁ binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr_71–82 domain, has no effect on the biological properties of the Vpr_77–92 domain.

Conclusion

Together our data provide evidence for the first time that the Vpr_77–92 sequence delineates a biological active domain of Vpr with PP2A₁ binding and pro-apopototic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr_77–92 domain in full length Vpr protein and also in entire HIV provirus.     

Structural insights into the catalytic mechanism of cyclophilin A

Cyclophilins constitute a ubiquitous protein family whose functions include protein folding, transport and signaling. They possess both sequence-specific binding and proline cis-trans isomerase activities, as exemplified by the interaction between cyclophilin A (CypA) and the HIV-1 CA protein. Here, we report crystal structures of CypA in complex with HIV-1 CA protein variants that bind preferentially with the substrate proline residue in either the cis or the trans conformation. Cis- and trans-Pro substrates are accommodated within the enzyme active site by rearrangement of their N-terminal residues and with minimal distortions in the path of the main chain. CypA Arg55 guanidinium group probably facilitates catalysis by anchoring the substrate proline oxygen and stabilizing sp3 hybridization of the proline nitrogen in the transition state.