Amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-to-cell fusion.
The murine coronavirus spike (S) protein contains a leucine zipper domain which is highly conserved among coronaviruses. To assess the role of this leucine zipper domain in S-induced cell-to-cell fusion, the six heptadic leucine and isoleucine residues were replaced with alanine by site-directed mutagenesis. The mutant S proteins were analyzed for cell-to-cell membrane fusion activity as well as for progress through the glycoprotein maturation process, including intracellular glycosylation, oligomerization, and cell surface expression. Single-alanine-substitution mutations had minimal, if any, effects on S-induced cell-to-cell fusion. Significant reduction in fusion activity was observed, however, when two of the four middle heptadic leucine or isoleucine residues were replaced with alanine. Double alanine substitutions that involved either of the two end heptadic leucine residues did not significantly affect fusion. All double-substitution mutant S proteins displayed levels of endoglycosidase H resistance and cell surface expression similar to those of the wild-type S. However, fusion-defective double-alanine-substitution mutants exhibited defects in S oligomerization. These results indicate that the leucine zipper domain plays a role in S-induced cell-to-cell fusion and that the ability of S to induce fusion may be dependent on the oligomeric structure of S.
Luo
Z
Z
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA.
Matthews
A M
AM
Weiss
S R
SR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D017934:Coronavirus / Q000502:physiology*; D016350:Leucine Zippers / Q000235:genetics; D008562:Membrane Glycoproteins / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics*
D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus