Evolved variants of the membrane protein can partially replace the envelope protein in murine coronavirus assembly.

The coronavirus small envelope (E) protein plays a crucial, but poorly defined, role in the assembly of virions. To investigate E protein function, we previously generated E gene point mutants of mouse hepatitis virus (MHV) that were defective in growth and assembled virions with anomalous morphologies. We subsequently constructed an E gene deletion (ΔE) mutant that was only minimally viable. The ΔE virus formed tiny plaques and reached optimal infectious titers many orders of magnitude below those of wild-type virus. We have now characterized highly aberrant viral transcription patterns that developed in some stocks of the ΔE mutant. Extensive analysis of three independent stocks revealed that, in each, a faster-growing virus harboring a genomic duplication had been selected. Remarkably, the net result of each duplication was the creation of a variant version of the membrane protein (M) gene that was situated upstream of the native copy of the M gene. Each different variant M gene encoded an expressed protein (M*) containing a truncated endodomain. Reconstruction of one variant M gene in a ΔE background showed that expression of the M* protein markedly enhanced the growth of the ΔE mutant and that the M* protein was incorporated into assembled virions. These findings suggest that M* proteins were repeatedly selected as surrogates for the E protein and that one role of E is to mediate interactions between transmembrane domains of M protein monomers. Our results provide a demonstration of the capability of coronaviruses to evolve new gene functions through recombination.