Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection
Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection
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DOI：
10.1105/tpc.012567
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发表时间：
2003-09-01
期刊：
影响因子：
11.6
通讯作者：
Mäkinen, K
中科院分区：
文献类型：
--
作者：
Ivanov, KI;Puustinen, P;Mäkinen, K
我们以前报道过，马铃薯病毒A（PVA）的衣壳蛋白（CP）在病毒感染的植物和体外磷酸化。在这项研究中，磷酸化PVA CP的酶被确定为蛋白激酶CK 2。从烟草中纯化了CK 2的α-催化亚基（CK 2 α），并使用凝胶内激酶测定和液相色谱-串联质谱进行了表征。克隆了烟草CK 2 α基因并在细菌细胞中表达。针对重组酶产生了特异性抗体，并用于证明PVA CP和CK 2 α在感染的烟草原生质体中的共定位。PVA CID中CK 2磷酸化的主要位点通过质谱分析、放射性磷酸肽测序和突变的组合鉴定为CK 2共有序列内的Thr-242。将影响CID中CK 2共有序列的氨基酸取代引入到用绿色荧光蛋白标记的PVA的全长感染性cDNA克隆中。对突变病毒的分析表明，它们在细胞间和长距离运动方面存在缺陷。使用体外试验，我们证明了CK 2磷酸化抑制PVA CP与RNA的结合，表明CK 2作用的分子机制。这些结果表明，磷酸化PVA CP的CK 2在病毒感染中起着重要的调节作用。
We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The alpha-catalytic subunit of CK2 (CK2alpha) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatography-tandem mass spectrometry. The tobacco CK2alpha gene was cloned and expressed in bacterial cells. Specific antibodies were raised against the recombinant enzyme and used to demonstrate the colocalization of PVA CP and CK2alpha in infected tobacco protoplasts. A major site of CK2 phosphorylation in PVA CID was identified by a combination of mass spectrometric analysis, radioactive phosphopeptide sequencing, and mutagenesis as Thr-242 within a CK2 consensus sequence. Amino acid substitutions that affect the CK2 consensus sequence in CID were introduced into a full-length infectious cDNA clone of PVA tagged with green fluorescent protein. Analysis of the mutant viruses showed that they were defective in cell-to-cell and long-distance movement. Using in vitro assays, we demonstrated that CK2 phosphorylation inhibited the binding of PVA CP to RNA, suggesting a molecular mechanism of CK2 action. These results suggest that the phosphorylation of PVA CP by CK2 plays an important regulatory role in virus infection.