Study of the interaction of GB virus C/Hepatitis G virus fusion peptides belonging to the E2 protein with phospholipid Langmuir monolayers.

In order to determine the ability of 1,2-dipalmitoyl phosphatidylcholine (DPPC) and 1,2-dioleoyl phosphatidylglycerol (DOPG) to host peptide sequences belonging to the E2 protein of GBV virus C/Hepatitis G virus, the behaviour of Langmuir monolayers formed by these phospholipids and E2 (12-26), E2 (354-363) and E2 (chimeric) peptide sequences was analysed from data of surface pressure (π) versus area per molecule (A) isotherms, compression modulus (Cs-1), excess Gibbs energy of mixing (ΔGexc) and total Gibbs energy of mixing (ΔGmix). Three different behaviours were observed. Mixed films of E2 (12-26) with DPPC or DOPC showed negative values for the excess thermodynamic functions, and thus attractive interactions between mixed films components are greater than in ideal films. Mixtures of E2 (354-363) with DPPC or DOPG, exhibited positive values of excess functions, evidencing weaker interactions in the mixed films in relation to those of pure components. Finally, positive and negative excess functions were observed in E2 (chimeric)/DPPC or DOPG mixed films, depending on their composition. In short, the interaction between the phospholipids used in this work as models of cell membranes and E2 peptides varies with the type of phospholipid and the nature of the peptide (size, bulky, hydrophobicity and electric charge).

Interaction of GB virus C/hepatitis G virus synthetic peptides with lipid langmuir monolayers and large unilamellar vesicles.

In this paper, we aimed to continue the previous study undertaken with one segment of E1 protein belonging to the GB virus C/hepatitis G virus (GBV-C/HGV), specifically between the 53-66 amino acids and their palmitoyl derivative peptide. The sequence selection has been made on the basis of different prediction algorithms of hydrophobicity and antigenicity. Their interactions between two different in vitro membrane models, lipid Langmuir monolayers and vesicles of different lipidic composition, have been evaluated. For this purpose, different lipids, varying the charge and the unsaturations of the hydrocarbon chain 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPG) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG), have been selected. Miscibility and peptides/lipids interactions have been analyzed on the basis of surface pressure (pi)-mean molecular area (A) isotherms, which have been recorded for pure and mixed monolayers of different composition spread at the air/water interface. Furthermore, E1(53-66) sequence and PalmE1(53-66) have been labeled with a fluorescent group, succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD succinimide), in order to study their behavior in the presence of vesicles. The obtained results are consistent with the existence of electrostatic (attractive) intermolecular interactions between the two positive net charges of the peptides and the polar heads of negative-charged lipids. However, both the lipidic membrane fluidity and the palmitic chain linked to the native peptide play an important role in the balance between the electrostatic forces established at the interface and the hydrophobic ones established inside the membrane. The fluorescence assays have demonstrated that electrostatic forces clearly predominate over the hydrophobic interactions only when the native sequence is retained at the polar interface of DPPG and DOPG vesicles. However, the palmitic tail linked to the peptide helped its penetration in the hydrophobic environment of the membrane, and this process was favored by decreasing the membrane fluidity.

Behaviour of a peptide sequence from the GB virus C/hepatitis G virus E2 protein in Langmuir monolayers: its interaction with phospholipid membrane models.

The GB virus C/hepatitis G virus (GBV C/HGV) is a Flaviviridae member that despite its non pathogenicity, has become of great interest given that it could inhibit the replication of the human immunodeficiency virus (HIV). Therefore, a better knowledge of the virus peptides involved in the cellular membrane fusion mechanism has become our aim. The selected peptide, named E2(347-363), corresponds to the GBV-C/HGV E2 protein and has been synthesized in order to study its interaction with in vitro membrane models. Two phospholipids, varying the charge and the unsaturations of the hydrocarbon chain have been chosen: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG). For our porpoise, we have used the Langmuir monolayer technique and Brewster angle microscopy (BAM) to gain deeper insight into the peptide/lipid interactions. The results obtained allow us to argue in favour of considering E2(347-363) a success candidate for developing further experiments in order to determine its potential role in the GBV C/HGV virus/cell membrane fusion process.

Perturbations induced by synthetic peptides from hepatitis G virus structural proteins in lipid model membranes: a fluorescent approach.

The name HGV/GBV-C remains as an acronym for hepatitis G virus (HGV) and GB virus-C (GBV-C), strain variants of this enveloped RNA virus independently but simultaneously discovered in 1995. Nowadays there is no evidence that it causes hepatitis in humans either during initial infection or after long-term carriage, but it has been recently related with HIV regarding the inhibition of progression to AIDS. The overall genomic organization of HGV/GBV-C is similar to that of hepatitis C virus (HCV) and other members of the Flavivirus family in Hepacivirus genus. Although a stretch of conserved, hydrophobic amino acids within the envelop glycoprotein of HCV has been proposed as the virus fusion peptide, the mode of entry of GBV-C/HGV into target cells is at present unknown. In the present work, sequences derived from the structural E2-protein of HGV/GBV-C have been selected by means of semiempirical methods and then synthesized manually following solid-phase methodologies. Their ability to induce perturbations in model membranes has been analysed by measuring the penetration of such peptides in lipid monolayers and by a series of experiments based on tryptophan peptide fluorescence emission spectra. Besides, release of vesicular contents to the medium was monitored by the ANTS/DPX assay. The membrane destabilization properties of these peptides was found very related with the length of the sequence.

Study in mono and bilayers of the interaction of hepatitis G virus (GBV-C/HGV) synthetic antigen E2(99-118) with cell membrane phospholipids.

The interaction of the hepatitis G synthetic peptide E2(99-118) with cell membrane phospholipids of different characteristics such as dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) was studied by Langmuir isotherms. Epifluorescence microscopy and Atomic force microscopy (AFM) was also used to study interactions with DPPC. Compression isotherms of DPPC/E2(99-118) and DPPG/E2(99-118) mixed monolayers showed negative deviation from ideallity consistent with the existence of attractive interactions. The incorporation of the peptide in DPPC monolayer was also confirmed in epifluorescence microscopy and AFM studies. The peptide retarded the formation of DPPC domains and did not let the phospholipid get organized. No important differences in the interactions with DPPC (neutral) or DPPG (anionic) were found, thus suggesting that electrostatics forces do not have a predominant influence in these interactions.