Xu Kai, 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. 2017 Mar 13;91(7). doi: 10.1128/JVI.01984-16. Print 2017 Apr 1.
Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterol Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication.
源自各种细胞器的膜结构对于正链RNA病毒的复制至关重要。尽管宿主蛋白在RNA病毒复制中的重要作用已被认识了十年，但细胞脂质在病毒复制中同样重要的功能直到最近才受到充分关注。先前在模式宿主酵母中对番茄丛矮病毒（TBSV）的研究揭示了磷脂酰乙醇胺和固醇在病毒复制中的重要作用。为了进一步了解固醇在番茄丛矮病毒复制中的作用，在这项研究中，我们发现TBSV的p33和p92复制蛋白能够结合固醇。p33与固醇的结合得到了p33两个跨膜结构域内胆固醇识别/相互作用氨基酸共有序列（CRAC）和类CARC序列的支持。CRAC和CARC序列内关键Y氨基酸的诱变阻断了TBSV在酵母和植物细胞中的复制。我们还发现，在从复制TBSV的酵母和植物细胞中获得的耐去污剂膜（DRM）组分中固醇含量丰富。这些DRM能够支持内源性和外源性模板上的病毒RNA合成。脂质组学方法显示，复制TBSV的酵母和植物细胞中固醇水平没有升高。这些数据支持这样一种观点，即TBSV复制蛋白与酵母和植物细胞中富含固醇的耐去污剂膜相关联。总之，本研究获得的结果以及先前发表的结果支持病毒复制蛋白周围固醇的局部富集对TBSV复制至关重要。病毒感染的一个有趣方面是它们依赖于高效的亚细胞组装平台来进行复制、病毒粒子组装或通过从受感染细胞中出芽实现病毒释放。这些组装平台可能涉及富含固醇的膜微区，这是各种病毒利用的异质且高度动态的纳米级结构。在这里，我们证明TBSV的p33和p92复制蛋白能够结合固醇。对参与固醇结合的CRAC和CARC序列内的p33进行诱变分析表明，p33结合固醇的能力与在酵母和植物细胞中支持TBSV复制的能力之间存在重要联系。总之，这些结果进一步强化了细胞固醇在病毒复制过程中作为前病毒脂质必不可少的模型。
