Mapping of the coronavirus membrane protein domains involved in interaction with the spike protein

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) prote in can be demonstrated by coimmunoprecipitation and by immunofluorescence c olocalization, 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 appear ed that the 25-residue luminally exposed amino-terminal domain of the M pro tein is not important for M-S interaction. A 15-residue deletion, the inser tion of a His tag, and replacement of the ectodomain by that of another cor onavirus M protein did not affect the ability of the Mi protein to associat e with the S protein. However, complex formation was sensitive to changes i n the transmembrane domains of this triple-spanning protein. Deletion of ei ther the first two or the last two transmembrane domains, known not to affe ct the topology of the protein, led to a considerable decrease in complex f ormation, but association was not completely abrogated. Various effects of changes in the part of the bl protein that is located at the cytoplasmic fa ce of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deleti ons in the amphipathic domain severely affected M-S interaction. Interestin gly, 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 t he ability of the protein to engage in virus particle formation (C. A. M. d e Haan, L. Kuo, P. S. Masters, H, Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998). Apparently, the structural requirements of the M prote in for virus particle assembly differ from the requirements for the formati on of M-S complexes.