Kovalev Nikolay, Inaba Jun-Ichi, Li Zhenghe, Nagy Peter D
Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, United States of America.
Institute of Biotechnology, State Key Laboratory of Rice Biology, Zhejiang University, Hangzhou, P. R. China.
PLoS Pathog. 2017 Jul 31;13(7):e1006520. doi: 10.1371/journal.ppat.1006520. eCollection 2017 Jul.
Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (endosomal sorting complexes required for transport I) or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs) used for in vitro assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in pex3Δ yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases.
在替代宿主酵母中重建抗病毒防御途径，作为一种细胞内探针，以加深我们对病毒-宿主相互作用以及被征用的宿主因子在形成膜结合病毒复制酶复合物以保护病毒RNA免受核糖核酸酶影响方面作用的理解。测定了仅由双组分DCR1和AGO1基因组成的酿酒酵母RNA干扰（RNAi）机制对野生型和突变型酵母中番茄丛矮病毒（TBSV）的抑制作用。我们发现，缺失被征用的ESCRT-I（转运所需的内体分选复合物I）或ESCRT-III因子会使TBSV复制对酵母中的RNAi机制更加敏感。此外，用于体外组装TBSV复制酶的无细胞提取物（CFE）中缺乏这些病毒促进细胞因子，导致在60分钟时间点时，dsRNA复制中间体被核糖核酸酶破坏，而野生型酵母的CFE能为dsRNA提供保护。此外，我们证明，参与在番茄病毒复制区室中富集固醇的被征用的氧固醇结合蛋白和膜接触位点，是保护病毒dsRNA所必需的。我们还表明，磷脂酰乙醇胺水平会影响抗RNAi复制区室的形成。在pex3Δ酵母中缺乏过氧化物酶体的情况下，TBSV会利用内质网（ER）膜，ER膜为TBSV dsRNA提供的抗RNAi或核糖核酸酶保护与野生型酵母中的过氧化物酶体膜一样好。总之，这些结果表明，番茄病毒利用被征用的蛋白质因子和篡夺的脂质构建保护性亚细胞环境，以抵御RNAi机制以及可能的其他细胞核糖核酸酶。
