To understand the roles of heptad repeat 1 (HR1) and HR2 of the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) in virus-cell interactions, the conserved Leu or He residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an ฮฑ-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-weight SL form, while the wild type and all other mutants produced similar levels of both the SL form and the slower-migrating, higher-molecular-weight S H form. The wild-type SL form was processed to the S H form, whereas the SL form of the 913P mutant was inefficiently converted to the SH form after biosynthesis. None of these mutations affected cell surface expression or binding to its cognate ACE2 receptor. In a human immunodeficiency virus type 1/SARS S coexpression study, all mutants except the 913P mutant incorporated the SH form into the virions as effectively as did the wild-type SH form. The mutation at Ile-1151 did not affect membrane fusion or viral entry. The impaired viral entry of the 927P, 941P, 955P, and 1165P mutants was due to their inability to mediate membrane fusion, whereas the defect in viral entry of the 1179P mutant occurred not at the stage of membrane fusion but rather at a postfusion stage. Our study demonstrates the functional importance of HR1 and HR2 of the SARS-CoV spike protein in membrane fusion and viral entry. Copyright ยฉ 2006, American Society for Microbiology.