Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis

<div><p>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 <i>PAH1</i> (<i>pah1</i>Δ), the sole yeast ortholog of human <i>LIPIN</i> genes. <i>PAH1</i> 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 <i>pah1</i>Δ 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 <i>PAH1</i> and <i>DGK1</i> (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in <i>pah1</i>Δ cells, BMV replicated as efficiently as in <i>pah1</i>Δ 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 (<i>CHO2</i>) promoted BMV replication. Conversely, overexpression of <i>PAH1</i> or plant <i>PAH1</i> orthologs inhibited BMV replication in yeast or <i>Nicotiana benthamiana</i> plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in <i>pah1</i>Δ 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.</p>