|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1995 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Sendai virus contains two glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The F protein, which is proteolytically processed to F1 and F2, is absolutely required for fusion, but the role of the HN protein is less clear. The structure and the function of the F and HN proteins was examined by using site-directed mutagenesis and expression of the mutant proteins.
(1) In contrast to the wild type F protein, a mutant F,having a cleavage motif similar to that of virulent Newcastle disease virus F,could be cleaved by proteases present in COS cells. When mutant F and wild HN were coexpressed at the cell surface, syncytium formation was observed.
(2) The role of intramolecular disulfide bond in the F protein was examined. The two cysteine residues at position 70 of the F2 subunit and 199 of F1 subunit were changed to serine residues. Intracellular transport of the cysteine mutant F proteins to the cell surface was reduced and defective in cleavage. These results suggest that the disulfide bond is crucial to the correct folding of the F proteins.
(3) For the F protein, elimination of N-glycosylation in site 1 exhibited a great increase in fusion activity with HN protein, but elimination in other site leaded to a misfolded, aggregated molecule. For HN protein, elimination of N-glycosylation in sites of globular domain blocked transport out of the ER.
(4) The role of leucine zipper motif in transmembrane domain of HN protein was examined. The mutant HN protein containing all three leucine, isoleucine-to-alanine substitutions exhibited a decrease in fusion promoting activity.