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Journal of Virology, February 2002, p. 1194-1205, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1194-1205.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Dissection of the Semliki Forest Virus Homotrimer Reveals Two Functionally Distinct Regions of the Fusion Protein

Don L. Gibbons and Margaret Kielian*

Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461

Received 31 July 2001/ Accepted 23 October 2001

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by the acidic pH of endosomes. In response to low pH, the E1 proteins on the virus membrane undergo a series of conformational changes, resulting in the formation of a stable E1 homotrimer. Little is known about the structural basis of either the E1 conformational changes or the resulting homotrimer or about the mechanism of action of the homotrimer in fusion. Here, the E1 homotrimer was formed in vitro from either virus or soluble E1 ectodomain and then probed by various perturbants, proteases, or glycosidase. The preformed homotrimer was extremely stable to moderately harsh conditions and proteases. By contrast, mild reducing conditions selectively disrupted the N-terminal region of trimeric E1, making it accessible to proteolytic cleavage and producing E1 fragments that retained trimer interactions. Trypsin digestion produced a fragment missing a portion of the N terminus just proximal to the putative fusion peptide. Digestion with elastase produced several fragments with cleavage sites between residues 78 and 102, resulting in the loss of the putative fusion peptide and the release of membrane-bound E1 ectodomain as a soluble trimer. Elastase also cleaved the homotrimer within an E1 loop located near the fusion peptide in the native E1 structure. Mass spectrometry was used to map the C termini of several differentially produced and fully functional E1 ectodomains. Together, our data identify two separate regions of the SFV E1 ectodomain, one responsible for target membrane association and one necessary for trimer interactions.

* Corresponding author. Mailing address: Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-3638. Fax: (718) 430-8574. E-mail: kielian{at}aecom.yu.edu.

Journal of Virology, February 2002, p. 1194-1205, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1194-1205.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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