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MOLECULAR UNDERSTANDING OF MEMBRANE EVENTS LEADING TO RETROVIRUS ENTRY INTO HOST CELLS AND VIRAL PROPAGATION

Objective


Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of simian immunodeficiency virus (SIV) gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptide can induce lipid mixing of small unilamellar vesicles (SUV) of various compositions at pH 7.4 and 37 C large unilamellar vesicles (LUV) were shown to undergo fusion, provided they contained phosphatidylethanolamine (PE) in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with dioleoylphosphatidylcholine (DOPC) SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. Evidence is given that this oblique orientation is a prerequisite to the fusion process.

Attenuated total reflection Fourier transform infrared spectroscopy (FTIR) of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary stucture of the protein. The analysis of the amide 1 band of deuterated samples by Fourier self deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the alpha-helix and beta-sheet structure content with a standard deviation of 8.6% when X-ray structures are taken as a reference. It is also shown that the orientation of the different secondary structures resolved by the Fourier self deconvolution curve fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid protein ratio can be accurately and quickly determined from the infrared spectrum.

The complete amino acid sequence of viral fusion proteins has been analyzed by the Eisenberg procedure. The region surrounding the cleavage site contains a highly hydrophilic region immediately followed by a membrane like region. Since the effective cleavage between these 2 domains seems required to expose the fusogenic domain (located at the N-terminal sequence of the transmembrane like region) which is assumed to interact with the lipid membrane of the host cell analysis has been focused on the conformation and mode of insertion of this membrane like domain in a lipid monolayer. It was inserted as an alpha-helical structure into a dipalmitoylphosphatidylcholine (DPPC) monolayer and its orientation at the lipid/water interface was determined using a theoretical analysis procedure allowing the assembly of membrane components. For each viral protein sequence these N-terminal helical segments oriented obliquely with respect to the lipid/water interface. This rather unusual orientation is envisaged as a prerequisite to membrane destabilization and fusogenic activity.

The fusion domain of simian immunodeficiency virus (SIV) envelope glycoproteins is a hydrophobic region located at the amino terminal extremity of the transmembrane protein (gp32). Assuming an alpha helical structure for the SIV fusogenic domain of gp32 in lipid environment, theoretical studies have predicted that the fusion peptide would insert obliquely in the lipid bilayer. This oblique insertion could be an initial step of the fusion process by disorganizing locally the structure of the lipid bilayer. This hypothesis has been tested by selectively mutagenizing the SIV gp160 expressed via a vaccinia virus vector, to alter the theoretical angle of insertion of the fusion peptide. The fusogenic activity of the wild type and mutant glycoproteins was tested after infection of T4 lymphocytic cell lines by the recombinant vaccinia virus, and measure of syncytia formation. Mutations that modified the oblique orientation reduced the fusogenic activity. In contrast, mutations that conserve the oblique orientation did not alter the fusogenic properties. The results support the hypothesis that oblique orientations is important for fusogenic activity.

Modified bovine leukemia virus (BLV) glycoproteins were expressed by using vaccinia virus recombinants, and their fusogenic capacities were examined by a syncytia formation assay. This analysis indicates that both BLV envelope glycoproteins gp51 and gp30 are necessary for cell fusion, and insertion of the N-terminal segment of gp30 (fusion peptide) into the lipid bilayer in an oblique of orientation, as predicted by computer conformational analysis, results in fusogenic capacities higher than insertion in a perpendicular or parallel orientation. It was also found that replacement of the BLV fusion peptide with its simian immunodeficiency virus counterpart does not modify the fusogenic capacity of the BLV glycoprotein.
The detailled molecular mechanism responsible for retrovirus-host cell fusion and viral transmission mediated by fusion of infected with uninfected cells is still obscure. From an identification of the membrane components and an understanding of the molecular mechanism responsible for fusion, new methodologies could be elabored on a rational basis in order to inhibit infection. It is the purpose of this multidisciplinary project which brings together highly
specialized know-hows existing in European laboratories.
Our research plan can be summarized as follows:
- Identification of the peptide domains and lipid components responsible for the fusion activity
- Rational design of membrane structures inhibiting cytopathogenicity - Induction of an immune response against epitopes involved in the fusion steps.
This investigation will concern HIV 1 and HIV 2 (Human
Immunodeficiency Virus) and SIV (Simian Immunodeficiency Virus)

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Universite Libre de Bruxelles
Address
Avenue Franklin Roosevelt 50
Bruxelles
Belgium

Participants (4)

Danish Biotechnology Center for Neuropeptide Research
Denmark
Address

2200 København N
GSF - FORSCHUNGSZENTRUM FUER UMWELT UND GESUNDHEIT GMBH
Germany
Address
Ingolstaedter Landstrasse 1
85764 Neuherberg
THE UNIVERSITY OF THE BASQUE COUNTRY
Spain
Address
1397,Barrio Sarriena S/n
48940 Leioa
UNIVERSIDADE DE LISBOA
Portugal
Address
Campo Grande
1699 Lisboa