| Co-Investigator(Kenkyū-buntansha) |
HOTTA Hak Kobe Univeristy, Graduate School of Medicine, Department of Microbiology, Professor, 大学院・医学系研究科, 教授 (40116249)
OKUNO Yoshinobu Osaka Prefectural Institute of Public Health, Virology Division, Director, 公衆衛生部, ウイルス課長 (30112064)
NAKAGAW Naoko Osaka Prefectural Institute of Public Health, Virology Division, Senior Researcher (10280835)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
(1) Mapping of the functional sites on the C protein.
1) C(del10-15) mutan t: Preparing a mutant with a deletion at the position 10-15 of the C protein, we showed that amino acids at 10-15 were indispensable for inhibition of STAT1 stabilization, resistance against the anti-viral state of the host cells, suppression of apoptosis and expression of pathogenicity. These amino acids, however, were not involved in phosphorylation of STAT1, inhibition of the trans activation of ISGs as well as suppression of viral RNA polymerase activity.
2) C170Ser(MVC11) mutant : A single point mutation at C170Phe→Ser demonstrated that C170 plays an important role in suppression of viral RNA polymerase activity, inhibition of STAT 1 stability and phosphorylation, inhibition of transact!vation of ISGs, suppression of apoptosis, resistance against anti-viral state of the host cells and expression of viral virulence. The Phe, however, had no relation with the formation of virus particles.
(2) Mechanism ofattenua
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tion of the C protein mutant.
1) Interferon sensitivity : When MVC11 was infected to interferon α/β receptor (IFNR)-knock-out mice (A129), mice exhibited only slight decrease of body weight. The result demonstrated that MVC11 was attenuated in A129 to the same degree as in ordinary mice possessing IFNR, suggesting that interferon sensitivity alone does not explain the attenuation of MVC11.
2) Effect of death of infected cells : MVC11, an attenuated virus with a mutation in the C protein, induced necrosis as well as apoptosis. Death of the infected cells caused interruption of progeny virus production afterwards, and resulted in attenuation of viral pathogenicity. On the other hand, parental pathogenic virus Ml did not demonstrate significant cytopathic effect and released progeny virus continuously for a long time after infection. In the presence of caspase inhibitors which suppressed apoptosis but not necrosis, MVC11-infected cells died rapidly. These results suggested that necrosis plays an important role in causing death to attenuated virus-infected cells.
(3) Mechanism ofapoptosis-induction by MVC11.
Using the specific inhibitors of each caspase, we demonstrated that activation of caspase 8 occurred during apoptosis induced by MVC11. On the other hand, it was shown that caspase 9 had less relation to MVC11-induced apoptosis. FADD molecule was not involved in the activation of caspase 8.
(4) Localization of C170Ser.
C170Ser, when expressed independently from other MVC11 proteins, strongly interacted with cytoskeleton. The phenomenon might have some relation with induction of apoptosis by C170Ser. Less
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