Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression.
Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression.
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杆状病毒鱼精蛋白样蛋白 P6.9 的翻译后修饰及其过度磷酸化对病毒极晚期基因超表达的意义。
DOI：
10.1128/jvi.00333-15
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发表时间：
2015
影响因子：
5.4
通讯作者：
Yang Kai
中科院分区：
文献类型：
--
作者：
Li Ao;Zhao Haizhou;Lai Qingying;Huang Zhihong;Yuan Meijin;Yang Kai
许多病毒利用病毒或细胞染色质机制进行有效感染。杆状病毒编码一种保守的鱼精蛋白样蛋白，P6.9。这种蛋白质在感染过程中的各种病毒生理过程中发挥重要作用。然而，P6.9调节转录的机制仍然未知。在这项研究中，7个磷酸化的物种P6.9被解析的Sf 9细胞感染的杆状病毒型物种苜蓿银纹夜蛾多核型多角体病毒（AcMNPV）。质谱鉴定了22个磷酸化和10个甲基化位点，但没有乙酰化位点的P6.9。免疫荧光显示P6.9和病毒编码的丝氨酸/苏氨酸激酶PK 1在感染细胞中表现出相似的分布模式，免疫共沉淀证实了它们之间的相互作用。uponpk 1缺失、核衣壳组装和多角体形成中断，病毒极晚期基因转录下调。有趣的是，我们发现3种磷酸化程度最高的P6.9在转染pk 1缺失突变体的Sf 9细胞中消失，这表明PK 1参与了P6.9的过度磷酸化。质谱分析表明P6.9的7个Ser/Thr和5个Arg残基的磷酸化是PK 1依赖的。将7个Ser/Thr残基替换为Ala后，P6.9磷酸化模式与pk 1缺失突变体相似。重要的是，病毒极晚期基因转录水平的降低和病毒感染性是一致的。我们的研究结果表明，P6.9的过度磷酸化是一个先决条件的最大超表达的杆状病毒非常晚的基因，并提供了第一个实验的杆状病毒鱼精蛋白样蛋白和相关的蛋白激酶在epigenetics.IMPORTANCEDiverse翻译后修饰（PTM）的组蛋白的功能的见解构成一个代码，创建结合平台，招募转录因子来调节基因的表达。许多病毒还利用宿主或病毒诱导的染色质机制来促进有效感染。杆状病毒编码一种鱼精蛋白样蛋白P6.9，它是感染周期中各种过程所必需的。目前，P6.9的PTM位点及其调控因子尚不清楚。在这里，我们发现，P6.9可以分为非磷酸化，hypophosphorylated，hyperphosphorylated物种和病毒编码的丝氨酸/苏氨酸激酶，PK 1，是必不可少的P6.9过度磷酸化。在P6.9上发现了丰富的PTM位点，其中7个Ser/Thr磷酸化位点是PK 1依赖的。这些Ser/Thr位点的突变降低了非常晚的病毒基因转录和病毒感染性，表明PK 1介导的P6.9过度磷酸化有助于病毒增殖。这些数据表明，一个代码存在于复杂的PTM病毒鱼精蛋白样蛋白，并参与病毒基因转录。
Many viruses utilize viral or cellular chromatin machinery for efficient infection. Baculoviruses encode a conserved protamine-like protein, P6.9. This protein plays essential roles in various viral physiological processes during infection. However, the mechanism by which P6.9 regulates transcription remains unknown. In this study, 7 phosphorylated species of P6.9 were resolved in Sf9 cells infected with the baculovirus type species Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Mass spectrometry identified 22 phosphorylation and 10 methylation sites but no acetylation sites in P6.9. Immunofluorescence demonstrated that the P6.9 and virus-encoded serine/threonine kinase PK1 exhibited similar distribution patterns in infected cells, and coimmunoprecipitation confirmed the interaction between them. Uponpk1deletion, nucleocapsid assembly and polyhedron formation were interrupted and the transcription of viral very late genes was downregulated. Interestingly, we found that the 3 most phosphorylated P6.9 species vanished from Sf9 cells transfected with thepk1deletion mutant, suggesting that PK1 is involved in the hyperphosphorylation of P6.9. Mass spectrometry suggested that the phosphorylation of the 7 Ser/Thr and 5 Arg residues in P6.9 was PK1 dependent. Replacement of the 7 Ser/Thr residues with Ala resulted in a P6.9 phosphorylation pattern similar to that of thepk1deletion mutant. Importantly, the decreases in the transcription level of viral very late genes and viral infectivity were consistent. Our findings reveal that P6.9 hyperphosphorylation is a precondition for the maximal hyperexpression of baculovirus very late genes and provide the first experimental insights into the function of the baculovirus protamine-like protein and the related protein kinase in epigenetics.IMPORTANCEDiverse posttranslational modifications (PTMs) of histones constitute a code that creates binding platforms that recruit transcription factors to regulate gene expression. Many viruses also utilize host- or virus-induced chromatin machinery to promote efficient infections. Baculoviruses encode a protamine-like protein, P6.9, which is required for a variety of processes in the infection cycle. Currently, P6.9's PTM sites and its regulating factors remain unknown. Here, we found that P6.9 could be categorized as unphosphorylated, hypophosphorylated, and hyperphosphorylated species and that a virus-encoded serine/threonine kinase, PK1, was essential for P6.9 hyperphosphorylation. Abundant PTM sites on P6.9 were identified, among which 7 Ser/Thr phosphorylated sites were PK1 dependent. Mutation of these Ser/Thr sites reduced very late viral gene transcription and viral infectivity, indicating that the PK1-mediated P6.9 hyperphosphorylation contributes to viral proliferation. These data suggest that a code exists in the sophisticated PTM of viral protamine-like proteins and participates in viral gene transcription.