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 Ile residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an ␣-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lowermolecular-weight S L form, while the wild type and all other mutants produced similar levels of both the S L form and the slower-migrating, higher-molecular-weight S H form. The wild-type S L form was processed to the S H form, whereas the S L form of the 913P mutant was inefficiently converted to the S H 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 S H form into the virions as effectively as did the wild-type S H 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.The severe acute respiratory syndrome coronavirus (SARSCoV), the etiologic agent of the outbreak of atypical pneumonia known as SARS, is a membrane-enveloped virus which encodes a 29,727-nucleotide, polyadenylated, positive-strand RNA. The genome of this virus contains five major open reading frames that encode the replicase polyprotein: the spike (S), envelope (E), and membrane (M) glycoproteins and the nucleocapsid (N) protein in the same order and of approximately the same sizes as those of other CoVs (34,39). In CoVs, the structural proteins, S, E, M, and N, play crucial roles during host cell entry and virion morphogenesis, assembly, and budding. The S protein is incorporated into the viral envelope by interaction with the M protein and forms large spikes on virion surfaces, and mature virions are released from smooth vesicles (20).The S protein of CoVs, which is oligomerized in the endoplasmic reticulum, is N glycosylated and has an apparent molecular mass of 150 to 180 kDa (reviewed in reference 28). The S1 domain is required for binding to specific cellular receptors, thus defining the host range of the virus. Angiotensin-converting enzyme 2 (ACE2) has been shown to be a physiologically relevant, functional receptor for the SARS-CoV (31). SARSCoV S protein-mediated viral infection can be effectively blocked by either a soluble form of ACE2, anti-ACE2 antibodies, or an antibody recognizing the ACE2-binding domain of the S protein (31, 44). The S2 domain forms the membraneanchored stalk region and mediates the fusion between the viral an...