CORONAVIRUS PARTICLE ASSEMBLY - PRIMARY STRUCTURE REQUIREMENTS OF THEMEMBRANE-PROTEIN

Coronavirus-like particles morphologically similar to normal virions a re assembled when genes encoding the viral membrane proteins M and E a re coexpressed in eukaryotic cells. Using this envelope assembly assay , we have studied the primary sequence requirements for particle forma tion of the mouse hepatitis virus (MEN) M protein, the major protein o f the coronavirion membrane. Our results show that each of the differe nt domains of the protein is important. Mutations (deletions, insertio ns, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles, Strikingly, the extreme c arboxy-terminal residue is crucial. Deletion of this single residue ab olished particle assembly almost completely; most substitutions were s trongly inhibitory. Site-directed mutations in the carboxy terminus of hi were also incorporated into the MHV genome by targeted recombinati on. The results supported a critical role for this domain of M in vira l assembly, although the M carboxy terminus was more tolerant of alter ation in the complete virion than in virus-like particles, likely beca use of the stabilization of virions by additional intermolecular inter actions. interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were fou nd to inhibit the budding of assembly-competent M in a concentration-d ependent manner. However, assembly-competent M was able to rescue asse mbly-incompetent M when the latter was present in low amounts. These o bservations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assem bly.