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Journal of Virology, December 2002, p. 12712-12722, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12712-12722.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Novel Mutations That Control the Sphingolipid and Cholesterol Dependence of the Semliki Forest Virus Fusion Protein

Prodyot K. Chatterjee, Christina H. Eng,{dagger} and Margaret Kielian*

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

Received 19 June 2002/ Accepted 16 September 2002

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction mediated by the E1 membrane protein. Efficient SFV-membrane fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants, srf-4 and srf-5, selected for growth in cholesterol-depleted cells. Like the previously isolated srf-3 mutant (E1 proline 226 to serine), the phenotypes of the srf-4 and srf-5 mutants were conferred by single-amino-acid changes in the E1 protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like srf-3, srf-4 and srf-5 show striking increases in the cholesterol independence of growth, infection, membrane fusion, and exit. Unexpectedly, and unlike srf-3, srf-4 and srf-5 showed highly efficient fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both srf-4 and srf-5 formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the srf-3 mutant. All three srf mutations lie in the same domain of E1, but the srf-4 and srf-5 mutations are spatially separated from srf-3. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the srf-4 and srf-5 mutations identify novel regions of E1 that are distinct from the fusion peptide and srf-3 region and modulate the requirements for both sphingolipid and cholesterol in virus-membrane fusion.

* 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.

{dagger} Present address: The Integrated Program in Cellular, Molecular and Biophysical Studies, College of Physicians and Surgeons, Columbia University, New York, NY 10032.

Journal of Virology, December 2002, p. 12712-12722, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.12712-12722.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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