The KxGxYR and DxE motifs in the C-tail of the Middle East respiratory syndrome coronavirus membrane protein are crucial for infectious virus assembly

The coronavirus’ (CoV) membrane (M) protein is the driving force during assembly, but this process remains poorly characterized. Previously, we described two motifs in the C-tail of the Middle East respiratory syndrome CoV (MERS-CoV) M protein involved in its endoplasmic reticulum (ER) exit ((211)DxE(213)) and trans-Golgi network (TGN) retention ((199)KxGxYR(204)). Here, their function in virus assembly was investigated by two different virus-like particle (VLP) assays and by mutating both motifs in an infectious MERS-CoV cDNA clone. It was shown that the (199)KxGxYR(204) motif was essential for VLP and infectious virus assembly. Moreover, the mislocalization of the M protein induced by mutation of this motif prevented M–E interaction. Hampering the ER export of M by mutating its (211)DxE(213) motif still allowed the formation of nucleocapsid-empty VLPs, but prevented the formation of fully assembled VLPs and infectious particles. Taken together, these data show that the MERS-CoV assembly process highly depends on the correct intracellular trafficking of its M protein, and hence that not only specific protein–protein interacting motifs but also correct subcellular localization of the M protein in infected cells is essential for virus formation and should be taken into consideration when studying the assembly process. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00018-023-05008-y.