Cleavage of poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: Another mechanism for host protein synthesis shutoff?
Cleavage of poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: Another mechanism for host protein synthesis shutoff?
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DOI：
10.1128/jvi.73.1.709-717.1999
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发表时间：
1999-01-01
影响因子：
5.4
通讯作者：
Rhoads, RE
中科院分区：
文献类型：
--
作者：
Kerekatte, V;Keiper, BD;Rhoads, RE
鼻病毒、口蹄疫病毒和肠道病毒群的小核糖核酸病毒感染细胞，导致宿主蛋白质合成中断，但允许病毒蛋白质合成进行。尽管相当多的证据表明，这种关闭是由序列特异的病毒蛋白酶(柯萨奇病毒中的2A酶)对真核细胞翻译起始因子eIF4G的切割所介导的，但一些实验观察与这一观点不同。因此，其他细胞蛋白的切割可能有助于宿主蛋白合成的关闭和病毒蛋白合成的刺激。最近的证据表明，高度保守的70 kDa细胞质Poly(A)结合蛋白(PABP)直接参与翻译起始。我们现在发现，在柯萨奇病毒感染HeLa细胞的过程中，PABP也被蛋白水解性切割。随着时间的推移，PABP的裂解与宿主翻译的关闭和病毒蛋白质合成的开始之间的相关性比eIF4G的裂解更好。对纯化的兔PABP和重组人PABP的体外实验以及对非洲爪哇卵母细胞和重组非洲爪哇PABP的体内实验表明，这种切割是由2A酶直接催化的。N-末端和C-末端测序表明，人PABP在(482)VANTSTQTM下箭头GPRPAAAAAA(500)处唯一发生切割，将四个N-末端RNA识别基序(80%)与C-末端同源二聚结构域(20%)分开。PABP的N端裂解产物在恢复PABP依赖的兔网织红细胞裂解物翻译系统方面不如全长PABP有效。这些结果表明，PABP的裂解可能是微小核糖核酸病毒改变蛋白质合成速度和光谱的另一种机制。
Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at (482)VANTSTQTM down arrow GPRPAAAAAA(500), separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.