973项目连发两篇J. Virol获病毒研究新进展

　　

生物通报道：中国科学院水生生物研究所桂建芳研究组主要研究方向为鱼类发育遗传学和遗传育种工程，承担了多项973，国家自然科学基金项目，近期这一研究组与其它研究组合作，完成了鱼类抗病毒协同作用，以及一种噬藻体的全基因组结构分析，相关成果均发表在病毒学Journal of Virology杂志上。

噬藻体是侵染蓝藻的病毒。噬藻体与宿主的相互作用以及对蓝藻高度专一的致死性感染，可调节控制水华蓝藻的密度与种群结构，减少蓝藻水华的形成与危害，有助于蓝藻细胞溶解物参与地球生物化学循环。作为一种潜在控制有害蓝藻水华的生物效应因子，噬藻体成为水生病毒学和水环境科学研究的热点对象。

桂建芳研究组与张奇亚研究组合作，共同阐明了首株无尾噬藻体PaV-LD(Planktothrix agardhii Virus from Lake Dong -hu) 全基因组结构及其编码的藻胆体降解蛋白(NblA)的生物学功能。

PaV-LD是从武汉东湖水样中培养分离的水华丝状蓝藻噬藻体，其基因组为双链DNA，共有95299个碱基对，含有142个推导基因。质谱分析从PaV-LD中鉴定出13个分子量大小范围在 27KDa 至 172KDa 的主要结构蛋白，但缺少已知噬藻(菌)体必备的几种尾结构蛋白。

系统进化分析揭示，PaV-LD主要衣壳蛋白结构与已知有尾噬藻(菌)体也大不相同，为一个新的独立分枝；与有尾噬藻体比较，电镜观察也确信PaV-LD为无尾噬藻体。重要的是，PaV-LD基因组中的ORF 022L基因是一个蓝藻NblA同源基因，而该基因通常只存在于蓝藻细胞中，是藻胆体蛋白降解的关键基因。通过基因克隆、免疫印迹、光谱测定和电镜观察等综合分析，证实了PaV-LD的ORF 022L确实能编码具有蓝藻NblA功能的蛋白。PaV-LD ORF 022L表达，能使蓝藻培养物由蓝绿色变成浅黄色，细胞中藻胆体蛋白含量显著下降，藻的类囊体结构遭受破坏或完全消失。

这一新型无尾噬藻体全基因组的解析及其存在的藻胆体降解蛋白基因生物学功能的诠释，为了解噬藻体的遗传多样性、噬藻体与宿主和环境的相互作用，以及噬藻体功能基因的控藻机理等研究新开启了一扇门。

另外桂建芳研究组还与张义兵研究组合作，证明了串联存在于鲫囊胚培养细胞基因组中的PKR与鱼类特有的PKZ都属于干扰素诱导的eIF2a激酶，在病毒感染时通过磷酸化底物eIF2a协同发挥抗病毒作用。

哺乳类eIF2a激酶家族有四个成员：PKR、PERK、HRI和GCN2，分别响应不同的细胞胁迫环境。当病毒感染细胞时，PKR被激活，然后磷酸化底物eIF2a阻断蛋白合成起始，抑制病毒复制。鱼类eIF2a激酶基因首先获得鉴定的不是PKR，而是由中国科学院水生生物研究所桂建芳研究员主持的鱼类发育遗传学与细胞工程学科组鉴定的新基因PKR-like，即PKZ。2008年，该实验室证明鱼类也存在PKR的直向同源基因。

这项研究成果不仅证明了鱼类PKZ是一个新的脊椎动物eIF2a激酶，而且回答了PKZ和PKR共存于鱼类基因组的生物学意义在于协同响应病毒入侵并发挥抗病毒功能。此外，PKZ是在脊椎动物中发现的第三个具有Z-DNA结合结构域的蛋白。因此，同PKR是一个dsRNA的识别受体一样，PKZ有可能作为一个新的Z-DNA/Z-RNA识别受体发挥某种功能。

（生物通：万纹）

原文摘要：

A Novel Cyanophage with Cyanobacterial Non-bleaching Protein A Gene in the Genome

A cyanophage PaV-LD has been isolated from harmful filamentous cyanobacterum Planktothrix agardhii in a shallow freshwater Lake Donghu. Here, we present its genomic organization and major structural proteins. The genome is 95,299 bp linear double stranded DNA and contains 142 potential genes. Blast searches revealed 29 proteins of known function in cyanophages, cyanobacteria or bacteria. Thirteen major structural proteins ranging in size from 27KDa to 172KDa were identified by SDS-PAGE and mass-spectrometric analysis. The genome lacks major genes that are necessary to the tail structure, and the tailless PaV-LD has been confirmed by a comparative electron microscope observation with other tail cyanophages and phages. Phylogenetic analysis of the major capsid proteins also reveals an independent branch of PaV-LD that is quite different from other known tail cyanophages and phages. Moreover, the unique genome carries a non-bleaching Protein A (NblA) gene (ORF 022L), which presents in all phycobilisome-containing organisms and mediates phycobilisome degradation. Western blot detection confirmed that 022L was expressed after PaV-LD infection in their host filamentous cyanobacterium. And, its appearance was companied by a significant decline of phycocyanobilin content and a color change of the cyanobacterial cells from blue-green to yellow-green. The biological function of PaV-LD NblA was further confirmed by expression in a model cyanobacterium via an integration platform, by spectroscopic analysis and electron microscopy observation. The data indicate that PaV-LD is an exceptional cyanophage of filamentous cyanobacteria and the novel cyanophage will also provide us a new vision for the cyanophage-host interactions.

Cooperative Roles of Fish Protein Kinase Containing Z-DNA Binding Domains and Double-Stranded RNA-Dependent Protein Kinase in Interferon-Mediated Antiviral Response.

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). In fish species, in addition to PKR, there exists a PKR-like protein kinase containing Z-DNA binding domains (PKZ). However, the antiviral role of fish PKZ and the functional relationship between fish PKZ and PKR remain unknown. Here we confirmed the coexpression of fish PKZ and PKR proteins in Carassius auratus blastula embryonic (CAB) cells and identified them as two typical interferon (IFN)-inducible eIF2α kinases, both of which displayed an ability to inhibit virus replication. Strikingly, fish IFN or all kinds of IFN stimuli activated PKZ and PKR to phosphorylated eIF2α. Overexpression of both fish kinases together conferred much more significant inhibition of virus replication than overexpression of either protein, whereas morpholino knockdown of both made fish cells more vulnerable to virus infection than knockdown of either. The antiviral ability of fish PKZ was weaker than fish PKR, which correlated with its lower ability to phosphorylate eIF2α than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2α independently on fish PKR and vice versa. These results suggest that fish PKZ and PKR play a nonredundant but cooperative role in IFN antiviral response.