| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Rabies virus nucleoprotein (N) is an RNA-binding protrein, and phosphorylation of the protein is highly conserved among the every virus isolate, and might be involved in the RNA-binding activity. In this study project, we investigated possible role and mechanism of the phosphorylation, and obtained several findings as follows.
1) Among several monoclonal antibodies (mAb) against the N protein we have prepared, we found (1) mAb #5-2-26 which recognizes only the phosphorylated form of N protein and (2) mAb #1-7-11 and others which recognize the conformations epitopes. Using these mAbs, we investigated the process of modification and maturation of the N protein.
2) From the experiments with an mAb which recognized the epitope formed on the phosphorylatred form of N protein, it was suggested that the N protein is not phosphorylated before it is involved in the encapsidation process.
3) The N protein is associated with P protein before its involvement in the encapsidation of viral RNA, and the
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association was disrupted in the presence of deoxycholate. Dissociated N protein was not recognized anymore bv any of the mAbs, suggesting that the absence of the epitope on the free N protein is not due to masking of the epitope site by P protein binding, but probably be due to the conformational change (s) of the N protein which occurs during or after the encapsidation.
4) The phosphorylation-deficient mutant N protein could encapsidate the viral RNA and formed the RNP, suggesting that the phosphorylation occurs during or after the encapsidation of viral RNA.
5) From the studies with the mutants and mAb #1-7-11, N proteins of the RNP have undergone conformational change (s), for which the phosphorylation is not required.
6) The conformational change (s) of N protein, which might occur due to its association with viral RNA seems to be reversible, and recover its original configuration after its release from the nucleocapsid.
7) The rabies virus P protein also undergoes conformational change (s) when it associate with the nucleocapsid and it associates with nucleocapsid more tightly, while the P protein associated much less tightly with the nucleocapsid comprised of the phosphorylation-deficient N protein. Conformationa change of the P protein seemed to be dependent on the N protein phosphorylation and be involved in stronger association of L protein (catalytic subunit of the viral RNA polymerase) with the nucleocapsid. Less
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