Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, P. R. China.
Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA, United States of America.
PLoS Pathog. 2018 Apr 12;14(4):e1006988. doi: 10.1371/journal.ppat.1006988. eCollection 2018 Apr.
Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection.
正链 RNA 病毒 [(+)RNA 病毒]的复制发生在膜结合的病毒复制复合物 (VRC) 中。VRC 的形成需要病毒介导的对细胞脂质合成的操纵。在这里，我们报告了在缺乏 PAH1（pah1Δ）的酵母细胞中，显著增强了 Bromo mosaic virus (BMV) 的复制，并且细胞生长得到了很大改善。PAH1 编码 Pah1p（磷酸脂酸磷酸水解酶），它将磷酸脂酸 (PA) 转化为二酰基甘油，随后用于合成储存脂质三酰基甘油。Pah1p 的失活导致脂质组成发生改变，包括 PA、总磷脂、麦角固醇酯和游离脂肪酸的水平升高，以及核膜扩张。在 pah1Δ 细胞中，BMV 复制蛋白 1a 和双链 RNA 定位于扩展的核膜，形成的 VRC 数量显著增加，BMV 基因组复制增加了 2 倍与野生型细胞相比。在另一个缺乏 PAH1 和 DGK1（编码将二酰基甘油转化为 PA 的二酰基甘油激酶）的酵母突变体中，它具有正常的核膜，但保持与 pah1Δ 细胞相似的脂质组成变化，BMV 的复制效率与 pah1Δ 细胞一样高，这表明改变的脂质组成是增强 BMV 复制的原因。我们进一步表明，总磷脂水平的增加起着重要作用，因为增强的 BMV 复制需要主要膜磷脂磷脂酰胆碱的活性合成。此外，过表达磷脂酰胆碱合成基因 (CHO2) 促进了 BMV 的复制。相反，在酵母或 Nicotiana benthamiana 植物中过表达 PAH1 或植物 PAH1 同源物抑制了 BMV 的复制。与宿主争夺有限的资源，BMV 抑制了宿主的生长，在 pah1Δ 细胞中，这种情况明显缓解。我们的工作表明，Pah1p 促进了储存脂质的合成，从而抑制了磷脂的合成，这反过来又限制了病毒复制和病毒感染过程中的细胞生长。
