CORONAVIRUS M-PROTEINS ACCUMULATE IN THE GOLGI-COMPLEX BEYOND THE SITE OF VIRION BUDDING

The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane prote in M (previously called E1). We tested this by analyzing the site of b udding of four different coronaviruses in relation to the intracellula r localization of their M proteins. Mouse hepatitis virus (MHV) and in fectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infe ctious peritonitis virus (Fl[PV) and transmissible gastroenteritis vir us (TGEV) grown in CrFK cells, all budded exclusively into smooth-wall ed, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called b udding compartment previously identified for MHV. Indirect immunofluor escence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense label ing in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and me dial Golgi cisternae. The corresponding M proteins of the four viruses , when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase an alysis of metabolically labeled cells showed that in each case the maj ority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectro n microscopy localized recombinant MHV M and IBV M to the same membran es as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demons trate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intr insic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.