1999, 73(9):7441. J. Virol. and P. J. M. Rottier Cornelis A. M. de Haan, M. Smeets, F. Vernooij, H. Vennema with the Spike Protein Protein Domains Involved in Interaction Mapping of the Coronavirus Membrane http:/jvi.asm.org/content/73/9/7441 Updated information and services can be found at: These include: REFERENCES http:/jvi.asm.org/content/73/9/7441#ref-list-1at: This article cites 32 articles, 21 of which can be accessed free CONTENT ALERTS morearticles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http:/journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http:/journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to: on April 4, 2014 by USC Norris Medical Libraryhttp:/jvi.asm.org/Downloaded from on April 4, 2014 by USC Norris Medical Libraryhttp:/jvi.asm.org/Downloaded from JOURNAL OFVIROLOGY, 0022-538X/99/$04.0010 Sept. 1999, p. 74417452Vol. 73, No. 9 Copyright 1999, American Society for Microbiology. All Rights Reserved. Mapping of the Coronavirus Membrane Protein Domains Involved in Interaction with the Spike Protein CORNELIS A. M.DEHAAN, M. SMEETS, F. VERNOOIJ, H. VENNEMA,ANDP. J. M. ROTTIER* Institute of Virology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands Received 25 March 1999/Accepted 3 June 1999 The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofl uorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the fi rst two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Inter- estingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:68386850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the require- ments for the formation of M-S complexes. Enveloped viruses contain a nucleocapsid (NC) surrounded by a lipid bilayer which accommodates the viral membrane proteins. This envelope is formed by budding of the NC through cellular membranes. For most viruses, the viral enve- lope proteins are incorporated effi ciently while host proteins are excluded. The specifi city of the virus assembly process is determined by interactions between the viral membrane pro- teins and with NC or matrix proteins. Coronaviruses, positive-strand RNA viruses, acquire their envelope by budding of the helical NC into the intermediate compartment between the endoplasmic reticulum (ER) and the Golgi complex (11, 12, 35). The coronavirus envelope con- tains three or four viral proteins. The membrane (M) glyco- protein is the most abundant envelope protein. It is a triple- spanning membrane protein with a short amino-terminal domain on the outside of the virus (or in the lumen of intra- cellular organelles) and a long carboxy-terminal domain on the inside (or in the cytoplasm) (reviewed by Rottier 27). The spike (S) glycoprotein, trimers of which form the virion peplomers, is another major structural protein. It is involved in binding of virions to the host cell and in virus-cell and cell-cell fusion (reviewed by Cavanagh 3). Some, but not all, corona- viruses contain a third major envelope protein, the hemagglu- tinin esterase (HE) (reviewed by Brian et al. 2). Finally, the small envelope (E) protein is a minor, poorly characterized but essential structural component (7, 30, 37). Lateral interactions between the coronav