Role of basic residues in the proteolytic activation of Sendai virus fusion glycoprotein.
Cleavage activation of the Sendai virus (Fushimi strain) fusion (F) protein was analyzed by site-directed mutagenesis of the amino acids proximal to the highly conserved fusion peptide. In addition, the functional properties of the wild-type and mutant proteins were examined to determine their ability to elicit the formation of syncytia when co-expressed with the hemagglutinin-neuraminidase (HN) glycoprotein. Viral genes were expressed from recombinant T7 transcription vectors (pT7/T3 plasmids) containing F or HN genes, after transfection into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). The wild-type F protein sequence (112VPQSRF) which contains a monobasic cleavage activation site was altered to include a tribasic, 112VPRKRF (mB3), or a pentabasic sequence, 112RRRKRF (mB5) adjacent to the fusion peptide. Although addition of basic residues to the normal protein sequence resulted in enhanced cleavage activation of the mB3 and mB5 proteins, only the mB5 protein was able to induce syncytia formation in CV-1 or HeLa T4 cells. Further analysis by the introduction of acidic residues upstream of the cleavage activation site was performed to determine whether increased hydrophilicity of the surrounding residues might contribute to cleavage activation. The mutants examined, mAcB1 (104NDDEENAGVPQSRF), mAcB3 (104NDDEENAGVPRKRF), and mAcB5 (104NDDEENAGRRRKRF) all contained DEE in replacement for the wild-type TTQ sequence (104NDTTQNAGVPQSRF). Analysis showed that only mAcB3 was efficiently cleaved by the endogenous cellular proteases, while mAcB1 was minimally cleaved, and mAcB5 not at all. Consequently, only the mAcB3 mutant was able to support fusion of CV-1 or HeLa T4 cells when co-expressed with HN.
Heminaway
B R
BR
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195, USA.
Yang
Y
Y
Tanaka
Y
Y
Panin
M
M
Huang
Y T
YT
Galinski
M S
MS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral; D015726:Giant Cells / Q000821:virology; D015707:HN Protein / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010222:Parainfluenza Virus 1, Human / Q000254:growth & development / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D015707:HN Protein; D011498:Protein Precursors; D014760:Viral Fusion Proteins