Coronavirus Particle Assembly: Primary Structure Requirements of the Membrane Protein

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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 membraneproteins M and E are coexpressed in eukaryotic cells. Using thisenvelope assembly assay, we have studied the primary sequencerequirements 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 proteinis important. Mutations (deletions, insertions, point mutations)in the luminal domain, the transmembrane domains, the amphiphilicdomain, or the carboxy-terminal domain had effects on the assemblyof M into enveloped particles. Strikingly, the extreme carboxy-terminalresidue is crucial. Deletion of this single residue abolishedparticle assembly almost completely; most substitutions were stronglyinhibitory. Site-directed mutations in the carboxy terminus ofM were also incorporated into the MHV genome by targeted recombination.The results supported a critical role for this domain of M inviral assembly, although the M carboxy terminus was more tolerantof alteration in the complete virion than in virus-like particles,likely because of the stabilization of virions by additional intermolecularinteractions. Interestingly, glycosylation of M appeared not essentialfor assembly. Mutations in the luminal domain that abolished thenormal O glycosylation of the protein or created an N-glycosylatedform had no effect. Mutant M proteins unable to form virus-likeparticles were found to inhibit the budding of assembly-competentM in a concentration-dependent manner. However, assembly-competentM was able to rescue assembly-incompetent M when the latter waspresent in low amounts. These observations support the existenceof interactions between M molecules that are thought to be thedriving force in coronavirus envelope assembly.

^* Corresponding author. Mailing address: Institute of Virology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan1, 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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