|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
1. I have already isolated a lot of monoclonal antibodies directed against the P or NP protein of hPIV-2. However, no MAbs specific for the V or L protein has been obtained. Therefore, I prepared anti-V or anti-L protein MAbs, and demonstrated the distribution of the V or L protein in virus-infected cells.
2. By Western immunoblotting with anti-P MAbs and P protein deletion mutants, the region essential for P-P interactions was determined. The P protein region encompassing aa 211-248 was required for proper folding and homotrimer which is mediated by predicted coiled-coils in this region.
3. By Western immunoblotting using anti-NP MAbs, the analysis of deletion mutants of the NP protein was performed to identify the region essential for NP-NP interaction and P protein-binding sites on the NP protein. The results indicate that the N-terminal 294 aa of the NP protein are all required for NP-Np self-assembly, and that two C-terminal parts of the P protein and another region. aa403-494, to the N-terminal part of the P protein.
4. By Western immunoblotting using anti-L MAbs, I demonstrated that the L protein directly binds to the P and NP proteins. Furthermore, the essential region for the L protein binding on the P or NP protein was determined. The region aa 278-353 on the P protein is required for binding to the L protein. And the region aa 403-494 on the NP protein is required for binding to the L protein.
5. By Northwestern blot assay, I demonstrated the regions on the P, V or NP protein which binds to RNA.