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. 1999 Sep;73(9):7441-52.
doi: 10.1128/JVI.73.9.7441-7452.1999.

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

C A de Haan  1 , M Smeets, F Vernooij, H Vennema, P J Rottier
Affiliations

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

C A de Haan et al. J Virol. 1999 Sep.

Abstract

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 immunofluorescence 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 first 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. Interestingly, 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. 72:6838-6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.

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Figures

FIG. 1
FIG. 1
Overview of mutant M proteins. A schematic representation of the structure of the M protein, with the three transmembrane domains (a, b, and c) indicated, is shown above each set of mutants. Amino acid sequences of the amino-terminal and carboxy-terminal domains and mutations in these domains are shown in panels A and D, respectively. Mutants with deletions in the transmembrane region or in the amphipathic domain are shown in panels B and C, respectively. Gaps represent deletions; the deleted amino acids are indicated. The ability of the different M proteins to interact with the S protein is indicated for each mutant at the right. The coimmunoprecipitation of M and S proteins with anti-S antibodies was taken as a measure of M-S interaction. The semiquantitative scores ++, +, +/−, and − indicate efficient, moderately efficient, inefficient, and undetectable M-S interaction, respectively. The abilities of the different M proteins to support VLP assembly, based on published (4) and unpublished results, are also indicated. The scores + and − indicate whether or not VLPs are synthesized when an M protein is coexpressed with the E protein.
FIG. 2
FIG. 2
Demonstration of WT M-S complexes. WT M and S genes were expressed in OST7-1 cells, alone or in combination, by using the MVA-T7pol expression system. Cells were labeled for 1 h, and this was followed by a 2-h chase. Cell lysates were prepared and subjected to immunoprecipitation with either the anti-MHV serum (αMHV), the anti-MC serum (αMC), the monoclonal anti-MN antibody (αMN), or the monoclonal anti-S antibody (αS), and the precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a control for the double expression (d), lysates of cells singly expressing M or S were pooled and subsequently processed similarly for immunoprecipitation (p). The positions of the S and M proteins are indicated on the left, while the molecular mass marker is indicated on the right.
FIG. 3
FIG. 3
The amino-terminal domain of M is not important for M-S interaction. Expression of M and S genes was performed as described in the legend to Fig. 2. The different M genes tested are indicated on the left. Only the relevant parts of the polyacrylamide gels are shown.
FIG. 4
FIG. 4
The transmembrane domains of M are necessary for efficient interaction. M and S genes were expressed as described in the legend to Fig. 2.
FIG. 5
FIG. 5
Effect of mutations in the amphipathic domain on M-S interaction. M and S genes were expressed as described in the legend to Fig. 2.
FIG. 6
FIG. 6
Effect of mutations in the hydrophilic tail on M-S interaction. M and S genes were expressed as described in the legend to Fig. 2.
FIG. 7
FIG. 7
Localization of coexpressed M and S proteins. The gene encoding the S protein was expressed in BHK-21 cells, by using the MVA-T7pol expression system, alone (A) or in combination with the gene encoding WT M (B and C), mutant ΔC (D and E), or mutant Y211G (F and G). At 5 h p.i., cells were treated with cycloheximide for 3 h to block protein synthesis. The cells were fixed at 8 h p.i. and processed for double labeling with the monoclonal anti-S antibody A3.10 (αS; A, C, E, and G) and the peptide serum specific for the carboxy-terminal tail of the M protein (αMC; B, D, and F).
FIG. 8
FIG. 8
Localization of S protein coexpressed with M mutants having truncations of the hydrophilic tail. M and S genes were expressed as described in the legend to Fig. 7. Cells were processed for double labeling with either the monoclonal antibody to the amino terminus of M (αMN; B, F, and J) or the monoclonal antibody to S (αS; D, H, and L) and the rabbit serum against the resident Golgi protein α-mannosidase II (A, C, E, G, I, and K).
FIG. 9
FIG. 9
Coimmunoprecipitation of S protein with the control protein EAV M+9A. EAV M+9A and S genes were expressed as described in the legend to Fig. 2. Immunoprecipitations were performed with the monoclonal antibody to the amino terminus of MHV M (αMN), the monoclonal antibody to S (αS), and the antipeptide serum specific for EAV M (αMEAV).
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