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Journal of Virology, June 2006, p. 5897-5907, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.00008-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of an N-Terminal Trimeric Coiled-Coil Core within Arenavirus Glycoprotein 2 Permits Assignment to Class I Viral Fusion Proteins

Bruno Eschli,1* Katharina Quirin,2 Alexander Wepf,1 Jacqueline Weber,1 Rolf Zinkernagel,1 and Hans Hengartner1

Institute of Experimental Immunology, University Hospital Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland,1 Institute of Biochemistry, Swiss Federal Institute of Technology, ETH Hoenggerberg, CH-8093 Zürich, Switzerland2

Received 3 January 2006/ Accepted 28 March 2006

The lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) consists of the transmembrane subunit GP-2 and the receptor binding subunit GP-1. Both are synthesized as one precursor protein and stay noncovalently attached after cleavage. In this study, we determined the oligomeric state of the LCMV GP and expressed it in two different conformations suitable for structural analysis. Sequence analysis of GP-2 identified a trimeric heptad repeat pattern containing an N-terminal {alpha}-helix. An {alpha}-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second {alpha}-helical peptide corresponding to a predicted C-terminal {alpha}-helix within GP-2 did not oligomerize. Refolding of the complete GP-2 ectodomain revealed trimeric all-alpha complexes probably representing the six-helix bundle state that is considered a hallmark of class I viral fusion proteins. Based on these results, we generated a construct consisting of the complete uncleavable LCMV GP ectodomain fused C-terminally to the trimeric motif of fibritin. Gel filtration analysis of the secreted fusion protein identified two complexes of ~230 and ~440 kDa. Both complexes bound to a set of conformational and linear antibodies. Cross-linking confirmed the 230-kDa complex to be a trimer. The 440-kDa complexes were found to represent disulfide-linked pairs of trimers, since partial reduction converted them to a complex species migrating at 250 kDa. By electron microscopy, the 230-kDa complexes appeared as single spherical particles and showed no signs of rosette formation. Our results clearly demonstrate that the arenavirus GP is a trimer and must be considered a member of the class I viral fusion protein family.

* Corresponding author. Mailing address: Institute of Experimental Immunology, University Hospital Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland. Phone: 41 (0) 44 255 2734. Fax: 41 (0) 44 255 4420. E-mail: eschlib{at}student.ethz.ch.

Journal of Virology, June 2006, p. 5897-5907, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.00008-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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