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.     

Covalent anthocyanin-flavone dimer from leaves of Oxalis triangularis

The anthocyanin-flavone C-glycoside, (malvidin 3-O-(6(II)-O-alpha-rhamnopyranosyl(AIV)-beta-glucopyranoside(AII))-5-O-beta-glucopyranoside(AIII)) (apigenin 6-C-(2(II)-O-beta-glucopyranosyl(FIII)-beta-glucopyranoside(FII))) malonate(AV) (A(IV)-4-->A(V)-1, F(III)-6-->A(V)-3) (1), has been isolated from leaves of Oxalis triangularis A. St.-Hil. In the 1D (1)H NMR spectrum of 1 dissolved in CD(3)OD-CF(3)CO(2)D (95:5), MTFA, recorded 45 min after sample preparation, this covalently linked dimer occurred mainly as flavylium cation (38%) and two equilibrium forms assigned to be quinonoidal bases (54%), whereas only minor amounts of the hemiacetal forms were present. After five days storage at 300 K, the hemiacetals (39%) and flavylium cation (38%) constituted the main forms of 1. More simple anthocyanins are normally considered to be on the flavylium cation form in acidified deuterated methanol. The cross-peaks observed in NOESY NMR spectra of 1 indicated the presence of vertical 'pi-pi' stacking between the B-ring of the flavone unit and the A-ring of each of the two forms assigned to be quinonoidal bases. It was not possible to discriminate between inter- or intramolecular association mechanisms. The equilibria between the various forms of 1 were studied by two-dimensional NOESY and ROESY NMR spectroscopy. 2D HSQC-TOCSY NMR spectroscopy was among the methods used for characterization of the various forms.     

Isolation and identification of flavonoids, including flavone rotamers, from the herbal drug 'Crataegi folium cum flore' (hawthorn)

Twelve flavonoids, including seven flavones, four flavonols and one flavanone, were isolated from methanolic extract of the herbal drug 'Crataegi folium cum flore' (hawthorn leaves and flowers) by a combination of CC (over Amberlite XAD-7 and Sephadex LH-20) and preparative HPLC. Their structures, including that of the novel flavonol 8-methoxykaempferol 3-O-(6"-malonyl-beta-glucopyranoside), were elucidated by homo- and heteronuclear NMR and electrospray/MS. The 1H- and 13C-NMR of all compounds, including rotameric pairs of five flavone C-glycosides, were assigned. The presence and relative proportion of each rotamer was shown by various NMR experiments, including two-dimensional nuclear Overhauser and exchange spectroscopy, to depend on solvent, linkage position and structure of the C-glycosyl substituent.     

Acylated anthocyanins from leaves of Oxalis triangularis

The novel anthocyanins, malvidin 3-O-(6-O-(4-O-malonyl-alpha-rhamnopyranosyl)-beta-glucopyranoside)-5-O-beta-glucopyranoside (2), malvidin 3-O-(6-O-alpha-rhamnopyranosyl-beta-glucopyranoside)-5-O-(6-O-malonyl-beta-glucopyranoside) (3), malvidin 3-O-(6-O-(4-O-malonyl-alpha-rhamnopyranosyl)-beta-glucopyranoside)-5-O-(6-O-malonyl-beta-glucopyranoside) (4), malvidin 3-O-(6-O-(4-O-malonyl-alpha-rhamnopyranosyl)-beta-glucopyranoside) (5) and malvidin 3-O-(6-O-(Z)-p-coumaroyl-beta-glucopyranoside)-5-O-beta-glucopyranoside (6), in addition to the 3-O-(6-O-alpha-rhamnopyranosyl-beta-glucopyranoside)-5-O-beta-glucopyranoside (1) and the 3-O-(6-O-(E)-p-coumaroyl-beta-glucopyranoside)-5-O-beta-glucopyranoside (7) of malvidin have been isolated from purple leaves of Oxalis triangularis A. St.-Hil. In pigments 2, 4 and 5 a malonyl unit is linked to the rhamnose 4-position, which has not been reported previously for any anthocyanin before. The identifications were mainly based on 2D NMR spectroscopy and electrospray MS.     

Species delimitation in northern European water scavenger beetles of the genus Hydrobius (Coleoptera,Hydrophilidae)

The chiefly Holarctic Hydrobius species complex (Coleoptera, Hydrophilidae) currently consists of Hydrobius arcticus Kuwert, 1890, and three morphological variants of Hydrobius fuscipes (Linnaeus, 1758): var. fuscipes, var. rottenbergii and var. subrotundus in northern Europe. Here molecular and morphological data are used to test the species boundaries in this species complex. Three gene segments (COI, H3 and ITS2) were sequenced and analyzed with Bayesian methods to infer phylogenetic relationships. The Generalized Mixed Yule Coalescent (GMYC) model and two versions of the Bayesian species delimitation method BPP, with or without an a priori defined guide tree (v2.2 & v3.0), were used to evaluate species limits. External and male genital characters of primarily Fennoscandian specimens were measured and statistically analyzed to test for significant differences in quantitative morphological characters. The four morphotypes formed separate genetic clusters on gene trees and were delimited as separate species by GMYC and by both versions of BPP, despite specimens of Hydrobius fuscipes var. fuscipes and Hydrobius fuscipes var. subrotundus being sympatric. Hydrobius arcticus and Hydrobius fuscipes var. rottenbergii could only be separated genetically with ITS2, and were delimited statistically with GMYC on ITS2 and with BPP on the combined data. In addition, six or seven potentially cryptic species of the Hydrobius fuscipes complex from regions outside northern Europe were delimited genetically. Although some overlap was found, the mean values of six male genital characters were significantly different between the morphotypes (p < 0.001). Morphological characters previously presumed to be diagnostic were less reliable to separate Hydrobius fuscipes var. fuscipes from Hydrobius fuscipes var. subrotundus, but characters in the literature for Hydrobius arcticus and Hydrobius fuscipes var. rottenbergii were diagnostic. Overall, morphological and molecular evidence strongly suggest that Hydrobius arcticus and the three morphological variants of Hydrobius fuscipes are separate species and Hydrobius rottenbergii Gerhardt, 1872, stat. n. and Hydrobius subrotundus Stephens, 1829, stat. n. are elevated to valid species. An identification key to northern European species of Hydrobius is provided.     

Novel aminoalkaloids from European mistletoe (Viscum album L.)

The European white-berry mistletoe (Viscum album L.) has remained an important medicinal plant for millennia. Preparations of the plant have found application in the treatment of cancer and the anticancer activity of mistletoe extracts has been ascribed to the presence of lectins, viscotoxins and alkaloids. However, the alkaloids of this species have hitherto remained unidentified because of their claimed extreme lability. Here we report on the isolation and characterisation of the novel aminoalkaloids 4,5,4′-trihydroxy-3,3′-iminodibenzoic acid (1) and 4,5,4′,5′-tetrahydroxy-3,3′-iminodibenzoic acid (2) from V. album L. The compounds define a novel group of aminoalkaloids and are the first novel alkaloids ever identified in any mistletoe species. The structures were established using a combination of several 2D NMR spectroscopic techniques and high resolution mass spectrometry.     

Sexual maturity cycle and spawning of Greenland halibut Reinhardtius hippoglossoides in the Davis Strait

Female sexual maturation cycle and the main spawning time of Greenland halibut Reinhardtius hippoglossoides in the Davis Strait were studied through regularly collected samples during 1 year starting in spring 2003. Samples were collected from the southern slope of the Davis Strait Ridge between Canada and Greenland in the depth range 1000–1500 m. Female sexual maturation was described using different approaches: gonado‐somatic index, visual macroscopic maturity stage index, histological microscopic maturity index and oocyte diameter measurements. A significant increase in the gonado‐somatic index was seen from September onwards until February with a maximum estimated value of 18%. The proportion of mature fish increased from December until March. At the same time, the proportion of females with a low gonado‐somatic index also increased from February, indicating that spawning had occurred and females were recovering. Oocyte diameter distribution revealed a leading cohort development during autumn through to December to February. A coupling between sexual maturity and fish condition was seen for females in maturing condition indicating a steady build up of stored energy in the liver from June to November.     

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.     

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.     

Dimeric anthocyanins from strawberry (Fragaria ananassa) consisting of pelargonidin 3-glucoside covalently linked to four flavan-3-ols

Flavanol-anthocyanin complexes were isolated by successive use of Amberlite XAD-7 chromatography, Sephadex LH-20 gel filtration and preparative HPLC from acidified, methanolic extract of strawberries (Fragaria ananassa Dutch.). These purple minor pigments were characterized by UV-Vis spectroscopy, 1D and 2D NMR techniques, and electrospray mass spectrometry to be catechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (1), epicatechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (2), afzelechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (3) and epiafzelechin(4alpha --> 8)pelargonidin 3-O-beta-glucopyranoside (4). The stereochemistry at the 3- and 4-positions of the flavan-3-ol units was based on assumption of R-configuration at C-2. Each of the four pigments occurred in the NMR solvent as a pair of rotamers. Proved by cross-peaks in the 1H-1H NOESY NMR spectra of 1, 2 and 4, the two conformations within each rotameric pair were in equilibrium with each other. Even though 1 and 2 are based on a different aglycone, their structures may be similar to tentatively identified pigments, which have been assumed to contribute to the colour of red wines.