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Coronaviruses are assembled by budding into smooth membranes of the intermediate ER-to-Golgi compartment. We have studied the association of the viral membrane glycoproteins M and S in the formation of the virion envelope. Using coimmunoprecipitation analysis we demonstrated that the M and S proteins of mouse hepatitis virus (MHV) interact specifically forming heteromultimeric complexes in infected cells. These could be detected only when the detergents used for their solubilization from cells or virions were carefully chosen: a combination of nonionic (NP-40) and ionic (deoxycholic acid) detergents proved to be optimal. Pulse-chase experiments revealed that newly made M and S proteins engaged in complex formation with different kinetics. Whereas the M protein appeared in complexes immediately after its synthesis, newly synthesized S protein did so only after a lag phase of > 20 min. Newly made M was incorporated into virus particles faster than S, which suggests that it associates with preexisting S molecules. Using the vaccinia virus T7-driven coexpression of M and S we also demonstrate formation of M/S complexes in the absence of other coronaviral proteins. Pulse-chase labelings and coimmunoprecipitation analyses revealed that M and S associate in pre-Golgi membranes because the unglycosylated form of M appeared in M/S complexes rapidly. Since no association of M and S was detected when protein export from the ER was blocked by brefeldin A, stable complexes most likely arise in the ER-to-Golgi intermediate compartment. Sucrose velocity gradient analysis showed the M/S complexes to be heterogeneous and of higher order, suggesting that they are maintained by homo- and heterotypic interactions. M/S complexes colocalized with alpha-mannosidase II, a resident Golgi protein. They acquired Golgi-specific oligosaccharide modifications but were not detected at the cell surface. Thus, the S protein, which on itself was transported to the plasma membrane, was retained in the Golgi complex by its association with the M protein. Because coronaviruses bud at pre-Golgi membranes, this result implies that the envelope glycoprotein complexes do not determine the site of budding. Yet, the self-association of the MHV envelope glycoproteins into higher order complexes is indicative of its role in the sorting of the viral membrane proteins and in driving the formation of the viral lipoprotein coat in virus assembly.