Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity

Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.     

Antifungal mechanism of the combination of Cinnamomum verum and Pelargonium graveolens essential oils with fluconazole against pathogenic Candida strains

The present study aimed to investigate the anti-Candida activity of ten essential oils (EOs) and to evaluate their potential synergism with conventional drugs. The effect on secreted aspartic protease (SAP) activity and the mechanism of action were also explored. The antifungal properties of essential oils were investigated using standard micro-broth dilution assay. Only Cinnamomum verum, Thymus capitatus, Syzygium aromaticum, and Pelargonium graveolens exhibited a broad spectrum of activity against a variety of pathogenic Candida strains. Chemical composition of active essential oils was performed by gas chromatography-mass spectrometry (GC-MS). Synergistic effect was observed with the combinations C. verum/fluconazole and P. graveolens/fluconazole, with FIC value 0.37. Investigation of the mechanism of action revealed that C. verum EO reduced the quantity of ergosterol to 83%. A total inhibition was observed for the combination C. verum/fluconazole. However, P. graveolens EO may disturb the permeability barrier of the fungal cell wall. An increase of MIC values of P. graveolens EO and the combination with fluconazole was observed with osmoprotectants (sorbitol and PEG6000). Furthermore, the combination with fluconazole may affect ergosterol biosynthesis and disturb fatty acid homeostasis in C. albicans cells as the quantity of ergosterol and oleic acid was reduced to 52.33 and 72%, respectively. The combination of P. graveolens and C. verum EOs with fluconazole inhibited 78.31 and 64.72% SAP activity, respectively. To our knowledge, this is the first report underlying the mechanism of action and the inhibitory effect of SAP activity of essential oils in synergy with fluconazole. Naturally occurring phytochemicals C. verum and P. graveolens could be effective candidate to enhance the efficacy of fluconazole-based therapy of C. albicans infections.     

SPC3, an anti-HIV peptide construct derived from the viral envelope,binds and enters HIV target cells

SPC₃ is a peptide construct (eight branches of the GPGRAF motif) derived from the consensus sequence present at the apex of the third variable domain of the human immunodeficiency virus (HIV) envelope (Env). It presents a potent anti-HIV activity and is currently tested in phase II clinical trials (FDA protocol 257A). Its mode of action remains unclear. It was thought that SPC₃ exerts its effect both during HIV interaction with CD4⁺ cells but also through interference either with a post-binding event or with Env processing. Accordingly, SPC₃ was supposed to be able to bind and to enter CD4⁺ cells. In this work, we addressed these points. SPC₃ was found to interact with CD4⁺ cell membrane with a K_0.5 value in the range of 500 nm . The binding of SPC₃ to CD4⁺ cells involves its interaction with a cell membrane associated protein which is pronase sensitive and different from CD4. This interaction was similar from 2 to 37°C. The maximum binding occurred at acidic pH whereas the interaction was inhibited in alkaline conditions. We observed also that SPC₃ was internalized rapidly into the cells—the maximal intracell amount was reached within 30 min—where it remained stable for at least 24 h. Altogether, these data suggest that SPC₃ can exert its antiviral activity via interference with events occurring at the cell surface but also into the target cell. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Evolutionary information helps understand distinctive features of the angiotensin II receptors AT1 and AT2 in amniota

In vertebrates, the octopeptide angiotensin II (AngII) is an important in vivo regulator of the cardiovascular system. It acts mainly through two G protein-coupled receptors, AT1 and AT2. To better understand distinctive features of these receptors, we carried out a phylogenetic analysis that revealed a mirror evolution of AT1 and AT2, each one split into two clades, separating fish from terrestrial receptors. It also revealed that hallmark mutations occurred at, or near, the sodium binding site in both AT1 and AT2. Electrostatics computations and molecular dynamics simulations support maintained sodium binding to human AT1 with slow ingress from the extracellular side and an electrostatic component of the binding free energy around -3kT, to be compared to around -2kT for human AT2 and the δ opioid receptor. Comparison of the sodium binding modes in wild type and mutated AT1 and AT2 from humans and eels indicates that the allosteric control by sodium in both AT1 and AT2 evolved during the transition from fish to amniota. The unusual S7.46N mutation in AT1 is mirrored by a L3.36M mutation in AT2. In the presence of sodium, the N7.46 pattern in amniota AT1 stabilizes the inward orientation of N3.35 in the apo receptor, which should contribute to efficient N3.35 driven biased signaling. The M3.36 pattern in amniota AT2 favours the outward orientation of N3.35 and the receptor promiscuity. Both mutations have physiological consequences for the regulation of the renin-angiotensin system.     

Inoculation of the attenuated Coxsackievirus B3 Sabin3-like strain induces a protection against virulent CVB3 Nancy and CVB4 E2 strains in Swiss mice by both oral and intraperitoneal routes

We have previously addressed the question of whether the attenuating mutations of domain V of the Poliovirus IRES were specific for a given genomic context or whether they could be extrapolated to a genomic related virus, the Coxsackievirus B3 (CVB3). Accordingly, we have described that Sabin3-like mutation (U⁴⁷³→C) introduced in the CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we assessed the protection provided by the Sabin3-like mutant against CVB3 infection. For this purpose, we analyzed, in vivo, the Sabin3-like phenotype in Swiss mice inoculated with CVB3 and CVB4 E2 prototype strains either by oral or intraperitoneal (i.p) routes and explored the capacity of this mutant to act as a vaccine vector after the challenge. The Sabin3-like RNA was detected by semi-nested PCR in different organs: heart, pancreas and intestine at 10 days post-inoculation with both oral and i.p routes. Additionally, we did not observe any histological alterations in heart and intestine tissues. RNA was detected in the different organs of all mice immunized with the Sabin3-like strain and challenged with either CVB3 or CVB4 E2 by oral route at 7 days post-challenge. In contrast, no histological alteration of heart or pancreas tissues was observed after challenge with both wild-strains. Interestingly, the detection of viral RNA in heart, pancreas and intestine of mice immunized by i.p route was negative at 7 days post-challenge with CVB3 and CVB4 E2, and mice were protected from myocarditis and pancreatitis.     

The RNA structure alignment ontology

Multiple sequence alignments are powerful tools for understanding the structures, functions, and evolutionary histories of linear biological macromolecules (DNA, RNA, and proteins), and for finding homologs in sequence databases. We address several ontological issues related to RNA sequence alignments that are informed by structure. Multiple sequence alignments are usually shown as two-dimensional (2D) matrices, with rows representing individual sequences, and columns identifying nucleotides from different sequences that correspond structurally, functionally, and/or evolutionarily. However, the requirement that sequences and structures correspond nucleotide-by-nucleotide is unrealistic and hinders representation of important biological relationships. High-throughput sequencing efforts are also rapidly making 2D alignments unmanageable because of vertical and horizontal expansion as more sequences are added. Solving the shortcomings of traditional RNA sequence alignments requires explicit annotation of the meaning of each relationship within the alignment. We introduce the notion of “correspondence,” which is an equivalence relation between RNA elements in sets of sequences as the basis of an RNA alignment ontology. The purpose of this ontology is twofold: first, to enable the development of new representations of RNA data and of software tools that resolve the expansion problems with current RNA sequence alignments, and second, to facilitate the integration of sequence data with secondary and three-dimensional structural information, as well as other experimental information, to create simultaneously more accurate and more exploitable RNA alignments.