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Journal of Virology, May 1999, p. 4272-4278, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Cholesterol Requirement for Sindbis Virus Entry and Exit and Characterization of a Spike Protein Region Involved in Cholesterol Dependence

Yanping E. Lu, Todd Cassese,dagger and Margaret Kielian*

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

Received 28 December 1998/Accepted 16 February 1999

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV, srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had the srf-3 amino acid sequence from E1 position 224 to 235 showed increases of ~100-fold in infection and ~250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGM exit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.

* 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: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Mass.

Journal of Virology, May 1999, p. 4272-4278, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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