Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.
Author Summary All characterized eukaryotic positive-strand RNA [(+)RNA] viruses replicate their genomes using the viral replication complexes (VRCs), which contain multiple viral and host components, on intracellular membranes. Phospholipids are major constituents of cellular membranes; however, the function(s) of phospholipids in genome replication of (+)RNA viruses remains largely unknown. Here, we show that Red clover necrotic mosaic virus (RCNMV), a plant (+)RNA virus, induces a high accumulation of phosphatidic acid (PA) in infected plant leaves. PA-producing enzymes, phospholipase Dα (PLDα) and PLDβ, are associated with RCNMV VRCs. PA interacts with the viral replication protein and enhances the viral replication by upregulating the activity/assembly of the VRCs in vitro. In summary, RCNMV alters cellular lipid metabolism via PLD to establish a suitable environment for viral replication.