Prasanth K Reddisiva, Barajas Daniel, Nagy Peter D
Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, USA.
Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, USA
J Virol. 2015 Mar;89(5):2750-63. doi: 10.1128/JVI.02620-14. Epub 2014 Dec 24.
RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs.
RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination.
RNA病毒会利用大量影响病毒复制的细胞蛋白，在某些情况下还会影响病毒基因重组。RNA重组有助于病毒在与宿主抗病毒反应的进化军备竞赛中以及使病毒适应新宿主。番茄丛矮病毒属病毒和酵母模型宿主被用于鉴定影响RNA病毒复制和RNA重组的细胞因子。在本研究中，我们研究了蛋白酶体中保守的Rpn11p金属蛋白酶亚基在酿酒酵母和植物中番茄丛矮病毒复制和重组中的作用，该亚基负责蛋白酶体底物的去泛素化和降解。Rpn11p的缺失或突变会导致病毒RNA重组体快速形成，同时基于细胞提取物的酵母或体外实验中病毒RNA复制水平降低。Rpn11p与病毒复制蛋白相互作用，并被招募到病毒复制酶复合物（VRC）中。对多功能Rpn11p的分析表明，Rpn11p的主要作用是充当“媒人”，将病毒p92（pol）复制蛋白和DDX3样Ded1p/RH20 DEAD盒解旋酶带入VRC。Ded1p的过表达可以通过减少番茄丛矮病毒重组来弥补rpn11突变酵母中观察到的缺陷。这表明Rpn11p可以通过促进细胞Ded1p解旋酶（一种强大的病毒重组抑制剂）被招募到VRC中来抑制番茄丛矮病毒重组。总体而言，这项工作表明，被利用的Rpn11p不仅参与功能性蛋白酶体的组装，还在番茄丛矮病毒VRC的正确组装中发挥作用。
由于基于突变和RNA重组的基因变化，RNA病毒进化迅速。病毒基因重组有助于病毒在与宿主抗病毒反应的进化军备竞赛中，并促进病毒适应新宿主。细胞因子会影响病毒RNA重组，尽管宿主在病毒进化中的作用仍未得到充分研究。在本研究中，我们使用植物RNA病毒番茄丛矮病毒，在酵母模型宿主、植物和体外实验中研究了一种细胞蛋白酶体蛋白Rpn11在番茄丛矮病毒重组中的作用。我们发现细胞Rpn11被用于番茄丛矮病毒复制，并且Rpn11在促进病毒复制方面具有蛋白酶体非依赖性功能。当Rpn11水平被敲低或表达突变的Rpn11时，番茄丛矮病毒RNA会经历快速的病毒重组和进化。综上所述，结果表明，被利用的细胞Rpn11通过调节病毒复制和基因重组，是番茄丛矮病毒的关键宿主因子。
