Abstract
Coronavirus membrane (M) protein can form virus-like particles (VLPs) when coexpressed with nucleocapsid (N) or envelope (E) proteins, suggesting a pivotal role for M in virion assembly. Here we demonstrate the self-assembly and release of severe acute respiratory syndrome coronavirus (SARS-CoV) M protein in medium in the form of membrane-enveloped vesicles with densities lower than those of VLPs formed by M plus N. Although efficient N-N interactions require the presence of RNA, we found that M-M interactions were RNA-independent. SARS-CoV M was observed in both the Golgi area and plasma membranes of a variety of cells. Blocking M glycosylation does not appear to significantly affect M plasma membrane labeling intensity, M-containing vesicle release, or VLP formation. Results from a genetic analysis indicate involvement of the third transmembrane domain of M in plasma membrane-targeting signal. Fusion proteins containing M amino-terminal 50 residues encompassing the first transmembrane domain were found to be sufficient for membrane binding, multimerization, and Golgi retention. Surprisingly, we found that fusion proteins lacking all three transmembrane domains were still capable of membrane binding, Golgi retention, and interacting with M. The data suggest that multiple SARS-CoV M regions are involved in M self-assembly and subcellular localization.