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Journal of Virology, October 1998, p. 7885-7894, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Analysis of Constructed E Gene Mutants of Mouse Hepatitis Virus Confirms a Pivotal Role for E Protein in Coronavirus Assembly

Françoise Fischer,1,dagger Carola F. Stegen,2,Dagger Paul S. Masters,1,3,* and William A. Samsonoff1,3

Departments of Biomedical Sciences1 and Biological Sciences,2 State University of New York at Albany, and Wadsworth Center for Laboratories and Research, New York State Department of Health,3 Albany, New York 12201

Received 20 April 1998/Accepted 8 July 1998

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

* Corresponding author. Mailing address: David Axelrod Institute, Wadsworth Center, NYSDOH, New Scotland Ave., P.O. Box 22002, Albany, NY 12201-2002. Phone: (518) 474-1283. Fax: (518) 473-1326. E-mail: masters{at}wadsworth.org.

dagger Present address: LaboRétro, INSERM U412, ENS, 46 allee d'Italie, 69364 LYON cedex 07, France.

Dagger Present address: Physiologisch-chemisches Institut, Universitaet Tuebingen, Germany.

Journal of Virology, October 1998, p. 7885-7894, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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