ROLES IN CELL-TO-CELL FUSION OF 2 CONSERVED HYDROPHOBIC REGIONS IN THE MURINE CORONAVIRUS SPIKE PROTEIN

The spike (S) protein of coronavirus, mouse hepatitis virus (MHV), med iates attachment and fusion during viral entry and cell-to-cell fusion later in infection. By analogy with other viral proteins that induce cell fusion the MHV S protein would be expected to have a hydrophobic stretch of amino acids that serves as a fusion peptide. Sequence analy sis suggests that the S protein falls within the group of fusion prote ins having internal rather than N-terminal fusion peptides. Based on t he features of known Viral fusion peptides, we identified two regions (PEP1 and PEP2) of MHV-A59 S2 as possible fusion peptides. Site-direct ed mutagenesis and an in vitro cell-to-cell fusion assay were used to evaluate the roles of PEP1 and PEP2, as well as a third previously ide ntified putative fusion domain (PEP3) in membrane fusion. Substitution of bulky hydrophobic residues with charged residues within PEP1 affec ts the fusion activity of the S protein without affecting processing a nd surface expression. Similar substitutions within PEP2 result in a f usion-negative phenotype; however, these mutant S proteins also exhibi t defects in protein processing and surface expression which likely ex plain the loss of the ability to induce fusion. Thus PEP1 remains a ca ndidate fusion peptide, while PEP2 may play a significant role in the overall structure or oligomerization of the S protein. PEP3 is an unli kely putative fusion peptide since it is not conserved among coronavir uses and nonconservative amino acid substitutions in PEP3 have minimal effects on cell-to-cell fusion. (C) 1998 Academic Press.