Noel Eric A, Kang Ming, Adamec Jiri, Van Etten James L, Oyler George A
Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska, USA School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, USA.
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, USA.
J Virol. 2014 Dec;88(23):13798-810. doi: 10.1128/JVI.02109-14. Epub 2014 Sep 24.
The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins.
Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in the manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses.
泛素-蛋白酶体系统是许多病毒的攻击目标，这些病毒已经进化出重定向宿主泛素化机制的策略。绿藻病毒属的成员被认为与更广泛的相关病毒群——核质大DNA病毒（NCLDV）有着共同的祖先谱系。绿藻病毒编码一种Skp1同源物和锚蛋白重复序列（ANK）蛋白。几种绿藻病毒编码的ANK重复序列包含细胞F-盒或相关NCLDV脊索痘病毒PRANC（C端锚蛋白的痘病毒蛋白重复序列）结构域的特征性C端结构域。这些观察结果表明，Skp1和ANK重复蛋白的这种独特组合可能形成类似于细胞Skp1-Cul1-F-盒（SCF）泛素连接酶复合物的复合物。我们从原型绿藻病毒草履虫小球藻病毒-1（PBCV-1）中鉴定出两种ANK蛋白，它们作为病毒编码的Skp1、蛋白A682L和A607R的结合伴侣发挥作用。这些ANK蛋白具有一个C端Skp1相互作用基序，其功能类似于细胞F-盒结构域。ANK蛋白A682L的C端基序与来自广泛不同物种的Skp1蛋白结合。使用连续结构域缺失构建体的酵母双杂交分析证实了PBCV-1 A682L中Skp1相互作用基序的C端定位。ANK蛋白A607R代表一个ANK家族，在所有41种已测序绿藻病毒中都有一个成员。对这些相关的ANK和病毒Skp蛋白进行的全面系统发育分析表明，它们具有针对宿主藻类或共同祖先遗传特征的配对功能。在这里，我们展示了三种绿藻病毒类型的病毒编码的ANK和Skp1蛋白的相应家族簇之间的蛋白质-蛋白质相互作用。总的来说，我们的结果表明，绿藻病毒已经进化出互补的Skp1和ANK蛋白，它们模仿细胞SCF相关蛋白。
病毒已经进化出引导泛素化事件的方式，以创造有利于其复制的环境。如本文所述，大型绿藻病毒编码参与SCF泛素连接酶复合物的几个成分，包括一种病毒Skp1同源物。关于绿藻病毒如何操纵其宿主藻类泛素化系统的研究将为病毒蛋白模拟、底物识别以及控制选择性蛋白降解的关键相互作用结构域提供见解。这些发现也可能进一步加深对其他大型DNA病毒（如痘病毒）进化的理解，据报道痘病毒与绿藻病毒共享相同的单系谱系。
