Variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion

The prototype JHM strain of murine hepatitis virus (MHV) is an enveloped, R NA-containing,a coronavirus that has been selected in vivo for extreme neur ovirulence, This virus encodes spike (S) glycoproteins that are extraordina rily effective mediators of intercellular membrane fusion, unique in their ability to initiate fusion even without prior interaction with the primary MHV receptor, a murine carcinoembryonic antigen-related cell adhesion molec ule (CEACAM). In considering the possible role of this hyperactive membrane fusion activity in neurovirulence, we discovered that the growth of JHM in tissue culture selected for variants that had lost murine CEACAM-independe nt fusion activity. Among the collection of variants, mutations were identi fied in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein, Each mutation was separately introduce d into cDNA encoding the prototype JHM spike, and the set of cDNAs was expr essed using vaccinia virus vectors. The variant spikes were similar to that of JHM in their assembly into oligomers, their proteolysis into S1 and S2 cleavage products, their transport to cell surfaces, and their affinity for a soluble form of murine CEACAM. However, these tissue culture-adapted spi kes were significantly stabilized as S1-S2 heteromers, and their entirely C EACAM-dependent fusion activity was delayed or reduced relative to prototyp e JHM spikes. The mutations that we have identified therefore point to regi ons of the S protein that specifically regulate the membrane fusion reactio n. We suggest that cultured cells, unlike certain in vivo environments, sel ect for S proteins with delayed, CEACAM-dependent fusion activities that ma y increase the likelihood of virus internalization prior to the irreversibl e uncoating process.