Folding of the mouse hepatitis virus spike protein and its association with the membrane protein.
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.
Opstelten
D J
DJ
Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, The Netherlands.
de Groote
P
P
Horzinek
M C
MC
Rottier
P J
PJ
D001692:Biological Transport; D000086862:Coronavirus M Proteins; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism*; D006517:Murine hepatitis virus / Q000254:growth & development / Q000378:metabolism*; D010084:Oxidation-Reduction; D017510:Protein Folding; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000187:drug effects / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000378:metabolism*; D014779:Virus Replication
D000086862:Coronavirus M Proteins; D004220:Disulfides; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D004229:Dithiothreitol