Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound
Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound
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DOI：
10.1128/jvi.00545-06
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发表时间：
2006-09-01
影响因子：
5.4
通讯作者：
Ishikawa, Masayuki
中科院分区：
文献类型：
--
作者：
Nishikiori, Masaki;Dohi, Koji;Ishikawa, Masayuki
液泡耗尽、番茄花叶病毒 (ToMV) 感染的植物原生质体的提取物含有 RNA 依赖性 RNA 聚合酶 (RdRp)，该酶利用内源模板以与体内观察到的模式相似的模式合成 ToMV 相关的正链 RNA。尽管事实上只有一小部分 ToMV 130-和 180-kDa 复制蛋白与膜相关，但 RdRp 活性完全与膜相关。基因组大小的负链 RNA 模板与膜相关，并且对微球菌核酸酶具有抗性，除非用去污剂处理。非膜结合复制蛋白不表现出 RdRp 活性，即使存在 ToMV RNA 也是如此。虽然非膜结合复制蛋白在用 Triton X-100 处理后仍然可溶，但同样的处理使膜结合复制蛋白处于低速离心时沉淀的形式。另一方面，去污剂溶血磷脂酰胆碱（LPC）有效地溶解了膜结合的复制蛋白。 LPC处理后，内源模板依赖性RdRp活性降低，取而代之的是外源ToMV RNA模板依赖性RdRp活性。该活性以及病毒 130-kDa 蛋白和宿主蛋白 Hsp70、真核翻译延伸因子 1A (eEF1A)、TOM1 和 TOM2A 与来自 LPC 溶解膜的 FLAG 标记病毒 180-kDa 蛋白共纯化。相比之下，Hsp70 和仅少量 130 kDa 蛋白以及 eEF1A 与 FLAG 标记的非膜结合 180 kDa 蛋白共纯化。这些结果表明，病毒复制蛋白与含有 TOM1 和 TOM2A 的细胞内膜相关，并且这种关联对于 RdRp 活性很重要。病毒复制蛋白的自关联以及它们与其他宿主蛋白的关联对于 RdRp 活性也可能很重要。
Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the nonmembrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidyleholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.