Folding of the mouse hepatitis virus spike protein and its association with the membrane protein

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Abstract
Coronaviruses are assembled by budding into pre-Golgi membranes. Using different approaches we have demonstrated that the spike (S) protein and the membrane (M) protein of mouse hepatitis virus (MHV) associate to form large complexes. Newly synthesized M was found in these complexes almost immediately after its synthesis, whereas the S protein started to appear in heterocomplexes after 10–20 min. This is consistent with the slow rate of folding of S and with the observation that folding of S preceeds its association with M. While the folding of S involves the formation of multiple disulfide bonds, folding of M is disulfide-independent. This contrast was reflected by the differential sensitivity of the two proteins to reduction with dithiothreitol (DTT). Addition of DTT to the culture medium of MHV-infected cells drastically impaired the folding of S, but not of M. Consequently, the S protein was unable to interact with M. Under these conditions, S stayed in the ER while M was transported efficiently beyond the site of budding to the Golgi complex. We conclude that the association of S with M is an essential step in the formation of the viral envelope and in the accumulation of both proteins at the site of virus assembly.