Functional characterization of heptad repeat 1 and 2 mutants of the spike protein of severe acute respiratory syndrome coronavirus.
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 alpha-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-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.
Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China.
Woan-Eng
WE
Chan
Chin-Kai
CK
Chuang
Shiou-Hwei
SH
Yeh
Mau-Sun
MS
Chang
Steve S-L
SS
Chen
D019943:Amino Acid Substitution; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008561:Membrane Fusion; D008958:Models, Molecular; D008970:Molecular Weight; D011233:Precipitin Tests; D011392:Proline / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000378:metabolism
D014760:Viral Fusion Proteins; D011392:Proline; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase