Direct Inhibition of Tombusvirus Plus-Strand RNA Synthesis by a Dominant Negative Mutant of a Host Metabolic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, in Yeast and Plants
Direct Inhibition of Tombusvirus Plus-Strand RNA Synthesis by a Dominant Negative Mutant of a Host Metabolic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, in Yeast and Plants
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DOI：
10.1128/jvi.00666-11
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发表时间：
2011-09-01
影响因子：
5.4
通讯作者：
Nagy, Peter D.
中科院分区：
文献类型：
--
作者：
Huang, Tyng-Shyan;Nagy, Peter D.
正链RNA病毒的复制依赖于许多细胞因子。甘油醛-3-磷酸脱氢酶（GAPDH）是番茄丛矮病毒（TBSV）复制酶复合体中的一个重要代谢酶，影响病毒RNA的不对称合成。为了进一步理解GAPDH在TBSV复制中的作用，我们使用了基于重组p33和p92（pol）病毒复制蛋白和无细胞酵母提取物的体外TBSV复制测定。我们发现，将纯化的重组GAPDH添加到从GAPDH耗尽的酵母制备的细胞提取物中导致增加的正链RNA合成和病毒RNA的不对称产生。我们的数据还表明，GAPDH与p92（pol）病毒复制蛋白相互作用，这可能有助于GAPDH在酵母模型宿主中招募到病毒复制酶复合物中。此外，我们还鉴定了GAPDH的显性负突变体，其在体外抑制RNA合成和RNA募集。此外，该突变体还表现出强烈的抑制在酵母和病毒感染的本塞姆氏烟草（Nicotiana benthamiana）中的烟草花叶病毒积累。总体而言，所获得的数据支持的模型，增选GAPDH发挥直接作用，在TBSV复制通过刺激正链合成的病毒复制酶。
The replication of plus-strand RNA viruses depends on many cellular factors. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an abundant metabolic enzyme that is recruited to the replicase complex of Tomato bushy stunt virus (TBSV) and affects asymmetric viral RNA synthesis. To further our understanding on the role of GAPDH in TBSV replication, we used an in vitro TBSV replication assay based on recombinant p33 and p92(pol) viral replication proteins and cell-free yeast extract. We found that the addition of purified recombinant GAPDH to the cell extract prepared from GAPDH-depleted yeast results in increased plus-strand RNA synthesis and asymmetric production of viral RNAs. Our data also demonstrate that GAPDH interacts with p92(pol) viral replication protein, which may facilitate the recruitment of GAPDH into the viral replicase complex in the yeast model host. In addition, we have identified a dominant negative mutant of GAPDH, which inhibits RNA synthesis and RNA recruitment in vitro. Moreover, this mutant also exhibits strong suppression of tombusvirus accumulation in yeast and in virus-infected Nicotiana benthamiana. Overall, the obtained data support the model that the co-opted GAPDH plays a direct role in TBSV replication by stimulating plus-strand synthesis by the viral replicase.