Yeast Lipin Acts As A Host Limiting Factor That Controls Viral Replication Levels And Host Growth Rates By Regulating Phospholipid Synthesis

Brome mosaic virus(BMV),a positive-stranded RNA((+)RNA)virus,is the type member of Bromovirus,in Bromoviridae.It was first isolated from the Bromus inermis in the Kansas State of the U.S.and it was reported to be infectious to other cereals and grasses around the world.In recent decades,BMV has been used as a model to study the replication of(+)RNA virus and its interaction with host.BMV replicates in the nER membrane in Saccharomyces cerevisiae.Phospholipids constitute the matrix of the membrane and they are synthesized from phosphatidate(PA)de novo.On the other hand,PA can also be converted to diacylglycerol(DAG)by PAH1 encoded phosphatidate phosphatase(PAP)and further to triacylglycerol(TAG)as storage lipids.Pahlp is the only lipin ortholog in yeast,and it decides using PA for phospholipid synthesis or for storage lipids synthesis.This study focuses on how BMV replication is reguated by Pahlp-regulated lipid metabolism in yeast.It is reported that deleting Nemlp or Spo7p,which are required for Pahlp activity,enhanced the expression level of Renilla luciferase mediated by BMV replication.The Nemlp and Spo7p constituted complex dephosphorylates the hyperphosphorylated Pah1p to make it active.Here we found that BMV replication was promoted?2-fold when deleting NEM1 or SPO7 and deleting both genes did not enhance viral replication further.Deleting PAH1 directly promoted BMV replication?2-fold and deleting both PAH1 and NEM1 performed best.The above results indicated that Pahlp deficiency could facilitate BMV replication.What's more,BMV replication was inhibited?50%in the presence of over-expressed PAH1,NEM1 or SPO7.To verify whether this contribution by PAP deficiency is specific to BMV,we also tested replication of spring beauty latent virus(SBLV),which is in the same genus with BMV,in PAH1 deletion mutants and we found that SBLV replication was enhanced?3-fold,which indicates that PAP deficiency promotes a group of(+)RNA viruses replication.BMV 1a targets to the round nER membrane and induces formation of VRC in wt yeast.It was reported that deleting PAH1,NEM1 or SPO7 induced extension and proliferation of ER membranes and we found the same phenotype in pah1?nem1?,also both 1a and double-stranded RNA(dsRNA),a hallmark of the(+)RNA virus replication,co-localized to the extended nER in the double mutant.Besides,we found spherules,the dominant form of BMV VRC,were?2.4-fold greater in number but smaller in size than that in wt yeast,which indicates that smaller spherules can support effective viral replication.In Pah1p-disrupted yeast,two major consequences that are induced by PAH1 deletion might contribute to the enhanced BMV replication:the extended nER membrane that may provide more room for viral replication and the increased total phospholipids.To determine which is the major one,we took advantage of a double mutant pah1?dgk1?,in which nER membrane is recovered to wt but total phospholipids remain high and even higher than that in pah1?.We first confirmed the nucleus was back to normal by transmission electron microscopy(TEM)method,we next found that BMV 1a and dsRNA colocalized to the normal nER.We next confirmed that the total phospholipids in pah1?nem1? and pah1?dgk1? are higher than that in wt cells.Surprisingly,the BMV replication was promoted about?2-fold and spherules were smaller in size but greater in number,which is very similar with what we found in pah1?nem1?.Thus the increased total phospholipids,not the extended nER membrane,is the major contributor for the enhanced viral replication in Pahlp deficient yeast.The increased RNA accumulation in pah1?sct1?,which partially recovers the nER to the wt,also suggested that the extended nER is not the major contributor.To further verify the important role of phospholipids play in BMV replication,we deleted SCS2 or CHO2 in the pah1? cells.Scs2p and Cho2p are responsible for regulating synthesis of phospholipids and phosphatidylcholine(PC),respectively.In addition,PC makes up?50%of total phospholipids.We found that BMV replication were inhibited in pah1?scs2? and pah1?cho2? mutants compared to that of pah1? cells.However,over-expression of CHO2 promoted viral replication?70%.The aforementioned results indicate that increased total phospholipids contribute most to the promoted viral replication in Pahlp disrupted yeast.We also found that highly expressed Arabidopsis LIPIN inhibited BMV replication in both yeast cells and Nicotiana benthamiana leaves,which indicates that our finding is also applicable in plant host.We checked the relationship of BMV replication with the formation of lipid droplets,which were stained by Nile red.We found that wt yeast have 8.4 lipid droplets per cell in average.In pah1?,pah1?nem1? and pah1?sct1?,which support robust viral replication,the number of lipid droplet per cell dropped to less than 3.However,11.3 lipid droplets per cell were found in pah1?dgk1?,which also support enhanced viral replication,but pah1?scs2?,which only slightly increased BMV replication,had 3.4 lipid droplet per cell in average.Thus these data indicate that there is no clear correlation between viral replication and lipid droplet formation.Besides contributing to the viral replication,we found that increased total phospholipids also facilitate yeast cell growth.In the absence of BMV,wt and mutants grow similarly,with doubling time are all between 3-4h.In the presence of BMV replication,the doubling time of wt yeast increased to 10.7h.But for the above-mentioned mutants,which have high levels of phospholipids due to the disrupted Pahlp,their doubling time range from 5h to 7h,which is much shorter than wt.Over-expression of DGK1 and CHO2 promoted yeast cell growth while over-expression of PAH1 slowed down cell growth by 30%.More directly,supplementation of choline(Chol)or methymonoethanolamine(MME),which activate the Kennedy pathway to produce phospholipids,promoted yeast cell growth by?40%compared to that without any supplementation.Thus,viruses outcompete host for limited phospholipid resources for its own use and increased levels of total phospholipids facilitated both viral replication and host cell growth...