Molecular insights into the function of the viral RNA silencing suppressor HCPro
Molecular insights into the function of the viral RNA silencing suppressor HCPro
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DOI：
10.1111/tpj.13088
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发表时间：
2016-01-01
期刊：
影响因子：
7.2
通讯作者：
Makinen, Kristiina
中科院分区：
文献类型：
--
作者：
Ivanov, Konstantin I.;Eskelin, Katri;Makinen, Kristiina
马铃薯Y病毒辅助组分蛋白酶（HCPro）是一种抗病毒RNA沉默的抑制剂，但其作用机制尚未完全了解。在这项研究中，我们使用亲和纯化结合质谱法来鉴定马铃薯Y病毒感染的植物细胞中HCPro的结合伙伴。这种方法导致各种HCPro相互作用，包括蛋氨酸循环的两个关键酶，S-腺苷-L-蛋氨酸合酶和S-腺苷-L-同型半胱氨酸水解酶的鉴定。这一发现，连同酶活性和基因敲除实验的结果，表明了一种机制，其中HCPro复合物含有病毒和宿主蛋白，通过局部破坏甲硫氨酸循环来抑制抗病毒RNA沉默。本研究中鉴定的另一组HCPro相互作用物包括核糖体蛋白。核糖体的免疫亲和纯化表明，HCPro是与病毒感染的细胞中的核糖体。此外，我们表明，HCPro和ARGONAUTE 1（AGO 1），RNA诱导的沉默复合物（RISC）的核心组成部分，相互作用，都与植物中的核糖体。这些结果，连同AGO 1与核糖体的关联是RISC介导的翻译抑制的标志的事实，表明HCPro作用的第二种机制，其中含有HCPro的核糖体相关的多蛋白复合物通过与AGO 1的相互作用减轻病毒RNA的翻译抑制。
Potyviral helper component proteinase (HCPro) is a well-characterized suppressor of antiviral RNA silencing, but its mechanism of action is not yet fully understood. In this study, we used affinity purification coupled with mass spectrometry to identify binding partners of HCPro in potyvirus-infected plant cells. This approach led to identification of various HCPro interactors, including two key enzymes of the methionine cycle, S-adenosyl-L-methionine synthase and S-adenosyl-L-homocysteine hydrolase. This finding, together with the results of enzymatic activity and gene knockdown experiments, suggests a mechanism in which HCPro complexes containing viral and host proteins act to suppress antiviral RNA silencing through local disruption of the methionine cycle. Another group of HCPro interactors identified in this study comprised ribosomal proteins. Immunoaffinity purification of ribosomes demonstrated that HCPro is associated with ribosomes in virus-infected cells. Furthermore, we show that HCPro and ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC), interact with each other and are both associated with ribosomes in planta. These results, together with the fact that AGO1 association with ribosomes is a hallmark of RISC-mediated translational repression, suggest a second mechanism of HCPro action, whereby ribosome-associated multiprotein complexes containing HCPro relieve viral RNA translational repression through interaction with AGO1.