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Journal of Virology, March 2001, p. 2792-2802, Vol. 75, No. 6
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.6.2792-2802.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Variations in Disparate Regions of the Murine Coronavirus Spike Protein Impact the Initiation of Membrane Fusion

Dawn K. Krueger, Sean M. Kelly, Daniel N. Lewicki, Rosanna Ruffolo, and Thomas M. Gallagher*

Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153

Received 7 July 2000/Accepted 18 December 2000

The prototype JHM strain of murine hepatitis virus (MHV) is an enveloped, RNA-containing coronavirus that has been selected in vivo for extreme neurovirulence. This virus encodes spike (S) glycoproteins that are extraordinarily effective mediators of intercellular membrane fusion, unique in their ability to initiate fusion even without prior interaction with the primary MHV receptor, a murine carcinoembryonic antigen-related cell adhesion molecule (CEACAM). In considering the possible role of this hyperactive membrane fusion activity in neurovirulence, we discovered that the growth of JHM in tissue culture selected for variants that had lost murine CEACAM-independent fusion activity. Among the collection of variants, mutations were identified in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein. Each mutation was separately introduced into cDNA encoding the prototype JHM spike, and the set of cDNAs was expressed using vaccinia virus vectors. The variant spikes were similar to that of JHM in their assembly into oligomers, their proteolysis into S1 and S2 cleavage products, their transport to cell surfaces, and their affinity for a soluble form of murine CEACAM. However, these tissue culture-adapted spikes were significantly stabilized as S1-S2 heteromers, and their entirely CEACAM-dependent fusion activity was delayed or reduced relative to prototype JHM spikes. The mutations that we have identified therefore point to regions of the S protein that specifically regulate the membrane fusion reaction. We suggest that cultured cells, unlike certain in vivo environments, select for S proteins with delayed, CEACAM-dependent fusion activities that may increase the likelihood of virus internalization prior to the irreversible uncoating process.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Loyola University Medical Center, 2160 South First Ave., Maywood, IL 60153. Phone: (708) 216-4850. Fax: (708) 216-9574. E-mail: tgallag{at}luc.edu.

Journal of Virology, March 2001, p. 2792-2802, Vol. 75, No. 6
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.6.2792-2802.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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