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J Virol, August 1998, p. 6838-6850, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Coronavirus Particle Assembly: Primary Structure Requirements of the Membrane Protein

Cornelis A. M. de Haan,1 Lili Kuo,2 Paul S. Masters,2 Harry Vennema,1 and Peter J. M. Rottier1,*

Institute of Virology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The Netherlands,1 and Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 122012

Received 23 December 1997/Accepted 17 May 1998

Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.

* Corresponding author. Mailing address: Institute of Virology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands. Phone: 31-30-253246-2. Fax: 31-30-2536723. E-mail: P.Rottier{at}vetmic.dgk.ruu.nl.

J Virol, August 1998, p. 6838-6850, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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