| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Measles virus (MV) C protein is a small and basic nonstructural protein, but its function is not well understood. First, we generated a recombinant wild-type MV lacking the C protein, called wtMV(C-), by using a reverse genetics system. wtMV(C-) grew normally in B95a cells, but not in 293/hSLAM cells. EGFP-expressing wtMV(C-) consistently induced late-stage cell rounding and cell death, suggesting that the C protein can prevent cell death and is required for long-term MV infection. When cynomolgus monkeys were infected with wtMV(C-) or wtMV, the number of MV-infected cells in the thymus was >1,000-fold smaller for wtMV(C-) than for wtMV. Immunohistochemical analyses showed strong expression of an MV antigen in the spleen, lymph nodes, tonsils, and larynx of a cynomolgus monkey infected with wtMV(C-). These data indicate that the MV C protein is necessary for efficient MV replication both in vitro and in cynomolgus monkeys.
Second, we have found that a FLAG-tagged wild-type MV C protein expressed from cDNA was accumulated exclusively in the nucleus. Mutation analysis revealed that ^<41>PPARKRRQ^<48>, belonging to the classical nuclear localization signal was important for nuclear localization. Analysis of the amino acid sequence of C protein revealed that it has a nuclear export signal (NES)-like sequence, ^<76>LEKAMTTLKL^<85>. Addition of the putative NES to the EGFP resulted in the translocation of EGFP to the cytoplasm. The Rev(1. 4)-EGFP nuclear export assay showed that this putative NES has a CRM1-dependent NES activity. C-EGFP accumulated in HeLa nuclei could be translocated to NIH3T3 nuclei in heterokaryon assays. In MV-infected cells, C-EGFP was accumulated in the nuclei in early phase but in the cytoplasm in late phase. These results indicated that the putative NES is functional and that C protein has the ability to shuttle between the nucleus and the cytoplasm.
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