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, RNA-containing coronavirus that has been selected in vivo for extreme neurovirulence. This virus encodes spike (S) glycoproteins that are extraordinarily 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 molecule (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-independent fusion activity. Among the collection of variants, mutations were identified in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein. Each mutation was separately introduced into cDNA encoding the prototype JHM spike, and the set of cDNAs was expressed 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 spikes were significantly stabilized as S1-S2 heteromers, and their entirely CEACAM-dependent fusion activity was delayed or reduced relative to prototype JHM spikes. The mutations that we have identified therefore point to regions of the S protein that specifically regulate the membrane fusion reaction. We suggest that cultured cells, unlike certain in vivo environments, select for S proteins with delayed, CEACAM-dependent fusion activities that may increase the likelihood of virus internalization prior to the irreversible uncoating process.
Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153, USA.
D K
DK
Krueger
S M
SM
Kelly
D N
DN
Lewicki
R
R
Ruffolo
T M
TM
Gallagher
D000818:Animals; D015703:Antigens, CD; D002272:Carcinoembryonic Antigen / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D018352:Coronavirus Infections / Q000821:virology; D015967:Gene Expression Regulation, Viral; D014644:Genetic Variation; D015726:Giant Cells; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D006517:Murine hepatitis virus / Q000472:pathogenicity* / Q000502:physiology; D009154:Mutation; D011991:Receptors, Virus / Q000378:metabolism*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*
D015703:Antigens, CD; C069847:CD66 antigens; D002272:Carcinoembryonic Antigen; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011991:Receptors, Virus; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus