Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core

The difference in membrane (M) protein compositions between the transmissib le gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cen do topology (with the amino terminus exposed to the virus surface and the c arboxy terminus inside the virus particle) and a Nexo-Cexo topology (with b oth the amino and carboxy termini exposed to the virion surface). The exist ence of a population of M molecules adopting a Nexo-Cexo topology in the vi rion envelope was demonstrated by (i) immunopurification of S-35-labeled TG EV virions using monoclonal antibodies (MAbs) specific for the M protein ca rboxy terminus (this immunopurification was inhibited only by deletion muta nt M proteins that maintained an intact carboxy terminus), (ii) direct bind ing of M-specific MAbs to the virus surface, and (iii) mass spectrometry an alysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated wi th the cores, and one-third was lost during core purification. MAbs specifi c for the M protein carboxy terminus were bound to native virions through t he M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purifica tion. All of the M protein was susceptible to N-glycosidase IT treatment of the native virions, which indicates that all the M protein molecules are e xposed to the virus surface. Cores purified from glycosidase-treated virion s included M protein molecules that completely or partially lost the carboh ydrate moiety, which strongly suggests that the M protein found in the core s was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is pr oposed. According to this working model, the TGEV virion consists of an int ernal core, made of the nucleocapsid and the carboxy terminus of the M prot ein, and the envelope, containing the spike (S) protein, the envelope (E) p rotein, and the M protein in two conformations. The two-thirds of the molec ules that are in a Nexo-Cendo conformation (with their carboxy termini embe dded within the virus core) interact with the internal core, and the remain ing third of the molecules, whose carboxy termini are in a Nexo-Cexo confor mation, are lost during virus core purification.