Functional analysis of Sendai virus C protein

(1)C蛋白上功能位点的定位。1）C（del 10 -15）突变体t：制备在C蛋白的位置10-15处具有缺失的突变体，我们表明10-15处的氨基酸对于抑制STAT 1稳定、抵抗宿主细胞的抗病毒状态、抑制细胞凋亡和表达致病性是必不可少的。2）C170 Ser（MVC 11）突变体：C170 Phe →Ser的单点突变表明，C170在抑制病毒RNA聚合酶活性、抑制STAT 1稳定性和磷酸化、抑制ISGs的反式激活以及抑制病毒RNA聚合酶活性等方面发挥重要作用。ISG的表达、细胞凋亡的抑制、对宿主细胞的抗病毒状态的抗性和病毒毒力的表达。而苯丙氨酸则与病毒粒子的形成无关。(2)衰减机理 关于我们 干扰素敏感性：当MVC 11感染干扰素α/β受体（IFNR）敲除小鼠（A129）时，小鼠体重仅轻微下降。结果表明，MVC 11在A129中的减毒程度与在具有IFNR的普通小鼠中的减毒程度相同，这表明干扰素敏感性单独不能解释MVC 11的减毒。2）感染细胞死亡的影响：MVC 11是一种在C蛋白中具有突变的减毒病毒，诱导坏死以及凋亡。感染细胞的死亡导致子代病毒生产中断，导致病毒致病性减弱。另一方面，亲本致病性病毒Ml没有表现出显著的细胞病变效应，并且在感染后长时间持续释放子代病毒。在存在抑制凋亡但不抑制坏死的半胱天冬酶抑制剂的情况下，MVC 11感染的细胞迅速死亡。这些结果表明，坏死在导致减毒病毒感染细胞死亡方面发挥着重要作用。(3)MVC 11诱导细胞凋亡的机制。利用caspase 8的特异性抑制剂，我们证实了在MVC 11诱导细胞凋亡的过程中，caspase 8被激活。另一方面，显示caspase 9与MVC 11诱导的细胞凋亡关系较小。FADD分子不参与caspase 8的激活。(4)C170 Ser的定位，C170 Ser，当独立于其他MVC 11蛋白表达时，强烈地与细胞骨架相互作用。这可能与C170 Ser诱导细胞凋亡有关。少

(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 … More 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