Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes

Budding of enveloped viruses has been shown to be driven by interactions between a nucleocapsid and a proteolipid membrane. By contrast, we here describe the assembly of viral envelopes independent of a nucleocapsid. Membrane particles containing coronaviral envelope proteins were assembled in and released from animal cells co-expressing these proteins' genes from transfected plasmids. Of the three viral membrane proteins only two were required for particle formation, the membrane glycoprotein (M) and the small envelope protein (E). The spike (S) protein was dispensable but was incorporated when present. Importantly, the nucleocapsid protein (N) was neither required not taken into the particles when present. The E protein, recently recognized to be a structural protein, was shown to be an integral membrane protein. The envelope vesicles were found by immunogold labelling and electron microscopy to form a homogeneous population of spherical particles indistinguishable from authentic coronavirions in size (approximately 100 nm in diameter) and shape. They were less dense than virions and sedimented slightly slower than virions in sucrose velocity gradients. The nucleocapsid-independent formation of apparently bona fide viral envelopes represents a novel mode of virus assembly.