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Journal of Virology, November 2009, p. 11298-11306, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01147-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

E1 Mutants Identify a Critical Region in the Trimer Interface of the Semliki Forest Virus Fusion Protein{triangledown}

Catherine Y. Liu and Margaret Kielian*

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

Received 4 June 2009/ Accepted 10 August 2009

The alphavirus Semliki Forest virus (SFV) uses a membrane fusion reaction to infect host cells. Fusion of the virus and cell membranes is triggered by low pH in the endosome and is mediated by the viral membrane protein E1. During fusion, E1 inserts into the target membrane, trimerizes, and refolds into a hairpin conformation. Formation of the E1 homotrimer is critical to membrane fusion, but the mechanism of trimerization is not understood. The crystal structure of the postfusion E1 trimer shows that an aspartate residue, D188, is positioned in the central core trimer interface. D188 is conserved in all reported alphavirus E1 sequences. We tested the contribution of this amino acid to trimerization and fusion by replacing D188 with alanine (D188A) or lysine (D188K) in an SFV infectious clone. These mutations were predicted to disrupt specific interactions at this position and/or change their pH dependence. Our results indicated that the D188K mutation blocked SFV fusion and infection. At low pH, D188K E1 inserted into target membranes but was trapped as a target membrane-inserted monomer that did not efficiently form the stable core trimer. In contrast, the D188A mutant was infectious, although trimerization and fusion required a lower pH. While there are extensive contacts between E1 subunits in the homotrimer, the D188K mutant identifies an important "hot spot" for protein-protein interactions within the core trimer.

* 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: margaret.kielian{at}einstein.yu.edu

{triangledown} Published ahead of print on 19 August 2009.

Journal of Virology, November 2009, p. 11298-11306, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01147-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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