Inactivation of the host lipin gene accelerates RNA virus replication through viral exploitation of the expanded endoplasmic reticulum membrane.

RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.

(+)RNA viruses either target existing subcellular membranes or they extensively remodel membranes to support the VRC assembly. Interestingly, tombusviruses can utilize pre-existing membranes, but also remodel subcellular membranes by forming multivesicular body-like structures in infected cells [5,6,38,39]. Tombusviruses are useful for membrane remodeling studies, because they can utilize peroxisomal membranes [e.g., TBSV and the closely related cucumber necrosis virus (CNV)] [5,39], or they can switch to endoplasmic reticulum (ER) membranes in the absence of peroxisomes [40,41], or replicate in ER or mitochondrial membranes in vitro [42].
Genetic diseases can alter critical cellular processes, which might affect pathogens that have to take advantage of cellular resources. The host lipins are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage [43][44][45]. Spontaneous mutations in the LIPIN1 gene in mammals, which cause impaired lipin-1 function, contribute to common metabolic dysregulation and several major diseases, such as obesity, hyperinsulinemia, type 2 diabetes, fatty liver distrophy and hypertension [44,46,47].
The yeast PAH1 (phosphatidic acid phosphohydrolase) gene is the homolog of the mammalian fat-regulating protein Lipin-1 [43,45,48]. Like the three mammalian lipin genes, the single copy yeast PAH1 codes for a phosphatidate phosphatase (PAP), which dephosphorylates phosphatidic acid (PA), yielding diacylglycerol (DAG) (Fig. 1A). Pah1p is involved in synthesis of DAG and triacylglycerol (TAG) storage lipids, and in the absence of PAH1, the ER/nuclear membrane expands considerably and the total phospholipid content of the cell increases by ,2-fold [49,50]. Thus, Pah1p sits at the crossroads between membrane biogenesis and lipid storage (i.e., the decision to store fat or build membranes) [45]. The mammalian or plant lipins can complement Pah1p function in yeast, demonstrating the functional similarity among these enzymes [46,51]. Pah1p is the only yeast PAP protein involved in the synthesis of TAG and the regulation of phospholipid biosynthesis [52].
Since (+)RNA viruses likely depend on membrane biogenesis, we examined the relationship between the host lipins and tombusviruses, based on yeast model host. In this paper, we document that deletion of the yeast lipin gene, PAH1, whose inactivation is responsible for proliferation and enlargement of the ER membrane, facilitates robust RNA virus replication in yeast. Thus, surprisingly, a host gene whose homologs are involved in genetic diseases in humans, greatly affects virus replication.

Deletion of yeast PAH1 gene, a lipin ortholog, increases tombusvirus replication in yeast
Since TBSV and the closely related CNV (Table S1) induce membrane proliferation and they replicate by utilizing peroxisomal membranes for VRC assembly in vivo [5,[40][41][42] and ER membranes in vitro [42], we tested if deletion of PAH1 gene, which leads to ER membrane enlargement and proliferation [43,45,46], could alter TBSV and CNV replication in yeast cells. We found that the TBSV replicon (rep)RNA accumulated to ,7-fold higher in the presence of TBSV replication proteins (Fig. 1B, lanes 4-6 versus 1-3) and ,2.5-fold higher levels in case of CNV (lanes 10-12 versus 7-9) in pah1D yeast. These data suggest that the enlarged ER might provide favorable microenvironment for TBSV and CNV replication or tombusviruses might be able to take advantage of the increased phospholipid content of the cell. Interestingly, the levels of p33 and p92 pol replication proteins were increased in pah1D yeast (Fig. 1B).
Finally, we also tested the effect of over-expression of Dgk1p diacylglycerol kinase, which catalyzes the production of PA from DAG, the opposite reaction with Pah1p (Fig. 1A), on TBSV replication. Overproduction of the ER-localized Dgk1p induces the enlargement of ER-like membranes in yeast [54,55]. We found that overproduction of Dgk1p in yeast led to increased TBSV replication (Fig. S1, lanes 4-6). Altogether, the above data support that the enlarged ER or the increased phospholipid content of the cell is a major advantage for TBSV and CNV replication.
To test if the high level of TBSV accumulation is due to increased TBSV repRNA replication, we measured the level of TBSV repRNA accumulation at various time points after induction of replication in pah1Dnem1D or pah1D yeast. These experiments revealed that TBSV repRNA accumulated to 2.5-to-4-fold higher level even at the early time points ( Fig. 2A-B, 5 and 8 hour time points). Similarly, TBSV repRNA accumulation was ,5-fold higher at an early time point in pah1D yeast (Fig. S2). These data suggest that more robust TBSV replication occurs earlier in pah1Dnem1D and pah1D yeasts than in the wt yeast, indicating that VRCs might be able to assemble faster in the mutant yeast cells.
Testing the replicase activity in the isolated membrane fraction containing the viral replicase/viral RNA complex from pah1D nem1D (Fig. 2D-F) or pah1D yeast (Fig. S3A-B) also showed ,4-to-5-fold increase over the replicase activity observed with the isolated membrane fraction from wt yeast at both early and late time points. The isolated membrane fractions were adjusted to contain comparable amount of the p92 pol replication protein ( Fig. 2E-F, S3B), thus the increased in vitro replicase activity in the samples from pah1Dnem1D or pah1D yeasts indicates that the tombusvirus replicase in pah1Dnem1D or pah1D yeasts is more active than in wt yeast. Interestingly, unlike p92 pol , the amounts of the tombusvirus p33 replication protein, the Sec61p ER resident protein and the Ssa1p Hsp70 chaperone, which is co-opted for TBSV replication, all increased in the isolated membrane fraction from pah1Dnem1D (Fig. 2D-E) or pah1D yeast (Fig. S3B). The presence of elevated amounts of p33 [56] and Ssa1p [37,57,58] has been shown to increase TBSV replication, which is in agreement with the increased in vitro replicase activity in the isolated membrane fraction from pah1Dnem1D or pah1D yeasts.

Author Summary
Genetic diseases alter cellular pathways and they likely influence pathogen-host interactions as well. To test the relationship between a key cellular gene, whose mutation causes genetic diseases, and a pathogen, the authors have chosen the cellular lipins. Lipins are involved in a key cellular decision on using lipids for membrane biogenesis or for storage. Spontaneous mutations in the LIPIN1 gene in mammals, which cause impaired lipin-1 function, contribute to common metabolic dysregulation and several major diseases, such as obesity, hyperinsulinemia, type 2 diabetes, fatty liver distrophy and hypertension. In this work, the authors tested if tomato bushy stunt virus (TBSV), which, similar to many (+)RNA viruses, depends on host membrane biogenesis, is affected by deletion of the single lipin gene (PAH1) in yeast model host. They show that pah1D yeast supports increased replication of TBSV. They demonstrate that TBSV takes advantage of the expanded ER membranes in lipin-deficient yeast to efficiently assemble viral replicase complexes. Their findings suggest possible positive effect of a genetic disease caused by mutation on the replication of an infectious agent.
Since p33 replication protein is an integral membrane protein [39,58], the observation of increased p33 level in the isolated membrane fraction from pah1Dnem1D yeast suggests that p33 might be more stable in the mutant yeast than in the wt yeast. Indeed, estimation of the half-life of p33 revealed ,4-fold increased stability of p33 in pah1Dnem1D yeast in comparison with wt yeast (Fig. 2G).
Enhanced in vitro assembly of the tombusvirus replicase complex in cell-free extract from yeast lacking PAH1 Based on the above data, it is possible that TBSV VRCs could assemble more efficiently in pah1Dnem1D yeast due to the presence of extended ER membranes and abundant amounts of phospholipids. To test this possibility, we utilized an in vitro tombusvirus VRC assembly assay based on purified recombinant replication proteins and cell-free extracts (CFE) obtained from pah1Dnem1D or wt yeast (Fig. 3A). In this assay, the tombusvirus (+)repRNA performs one cycle of asymmetrical replication supported by the tombusvirus VRCs assembled in vitro [37,59]. Since we use recombinant viral proteins and repRNA in this assay, we can make sure that only the CFEs, involving the cellular membranes and possibly host factors, are different. The yeast CFEs were adjusted to contain comparable amounts of Pgk1p (a cytosolic protein) (Fig. 3B) and total proteins. Interestingly, the in vitro RNA replication supported by CFE was ,7-fold higher when assembled in CFE obtained from pah1D-nem1D yeast than from wt yeast (Fig. 3B, lanes 5-8 versus 1-4). These data strongly suggest that the tombusvirus replicase assembly in the CFE derived from pah1Dnem1D is more efficient than in the CFE from wt yeast.
To test if TBSV replication indeed includes a full cycle in the CFE from pah1Dnem1D yeast, we analyzed the (2) and (+)-strand RNAs in the CFEs (Fig. 3C). The membrane-fraction of the CFE at the end of the replication assay contains both single-stranded (ss)RNA [representing the newly made (+)-stranded progeny RNA] and dsRNA [representing the annealed (2) and (+)RNAs].
In addition, the soluble fraction contains the newly released (+)RNAs from the membrane-bound VRCs. We found that the amounts of both ssRNA and dsRNA were ,2-fold higher in the membrane fraction and the ssRNA was ,2-fold higher in the soluble fraction of the CFE prepared from pah1Dnem1D yeast than from the wt yeast (Fig. 3D). These results suggest that the CFE obtained from pah1Dnem1D yeast performs all the replication steps more efficiently than the CFE prepared from wt yeast cells.
To test if the membrane-fraction of the CFE from pah1Dnem1D yeast is important for the enhanced TBSV RNA replication, we separated the soluble and membrane fractions of the CFEs prepared from pah1Dnem1D and wt yeasts and then used various  The scheme of the CFE-based TBSV replication assay. Purified recombinant TBSV p33 (7 pmol) and p92 pol (4 pmol) replication proteins in combination with DI-72 (+)repRNA (0.5 mg) were added to the CFEs. After the VRC assembly in the presence of rATP and rGTP, the membrane fraction of the CFE was collected by centrifugation and the replication assay was performed in the presence of the shown ribonucleotides. (B) TBSV replication assay based on CFEs prepared from wt (lanes 1-4) or pah1D nem1D yeasts (lanes 5-8). Denaturing PAGE analysis of the 32 P-labeled repRNA products obtained is shown. The full-length single-stranded repRNA is pointed at by an arrow. Note that, prior to the TBSV replication assay, the CFEs were adjusted to contain comparable amounts of the cellular Pgk1p, a cytosolic protein marker. Bottom image shows coomassie-stainde SDS PAGE of the total proteins in the CFEs. (C) The scheme of the modified CFE-based TBSV replication assay to show full in vitro replication. Note that the membrane fraction of the CFEs contain the viral VRCs and the bound TBSV RNAs, while the soluble fraction contains the released (+)RNA after replication. (D) Left panel: The denaturing PAGE analysis of the 32 P-labeled repRNA products in the soluble fraction of the CFEs prepared from wt (lanes 1-2) or pah1D nem1D yeasts (lanes 3-4) is shown. Right panel: Non-denaturing PAGE analysis of the 32 P-labeled repRNA products in the membrane fraction of the CFEs prepared from wt (lanes 5-6) or pah1D nem1D yeasts (lanes 7-8) is shown. The full-length single-stranded repRNA and the double-stranded repRNA are pointed at by an arrow. The even numbered lanes represent replicase products, which were not heat-treated (thus both ssRNA and dsRNA products are present), while the odd numbered lanes show the heat-treated replicase products (ssRNA is present). The amount of ssRNA and the ratio of ssRNA/dsRNA in the samples are shown. Note that, in the nondenatured samples, the dsRNA product represents the annealed (2)RNA and the (+)RNA, while the ssRNA products combinations of these fractions for in vitro TBSV replication (Fig.  S4A). Interestingly, the CFE containing the mixture of the membrane fraction from pah1Dnem1D yeast and the soluble fraction from the wt yeast supported almost as efficient in vitro TBSV replication as the CFE consisting of both fractions from pah1Dnem1D yeast (Fig. S4B, compare lanes 3-4 and 7-8 with 1-2). Altogether, it seems that the membrane fraction when derived from pah1Dnem1D yeast was able to support ,5-fold higher TBSV replication than the membrane fraction from wt yeast in the CFEbased replication assay. This is not surprising since PAH1 deletion is expected to dramatically change the ER membranes that could be utilized by TBSV for assembly of the VRCs.
Tombusvirus replicase utilizes the ER membrane derived from yeast lacking PAH1 more efficiently than from wt yeast To obtain direct evidence that the expanded ER structures and membranes in pah1Dnem1D yeast are utilized efficiently for TBSV replication, we took advantage of an isolated ER-based tombusvirus replication assay [42]. In this assay, the tombusvirus (+)repRNA can also perform full replication supported by the tombusvirus VRCs assembled in the ER membrane (Fig. 4A) [42]. As expected, the isolated ER preparations contained the Sec61p ER-resident protein, while lacked the cytosolic Pgk1p and the peroxisomal (Fox3p) proteins (Fig. 4B, lanes 1-3 versus 4-6, representing the total CFE). Larger amounts of the p33 replication protein were associated with the ER fraction obtained from pah1Dnem1D or pah1D yeasts than from the wt yeast (Fig. 4B, lanes 2 and 3 versus 1), suggesting that p33 utilized the ER membranes more efficiently in yeast lacking the PAH1 gene.
When we used similar amounts of isolated ER membranes (based on adjusted cellular Sec61p level) for TBSV replication, we observed that the replication of TBSV RNA was 2-to-4-fold higher in ER preparations obtained from pah1Dnem1D or pah1D yeasts than from wt yeast (compare lanes 5-6 with 4, Fig. 4C). These data suggest that the ER membranes derived from yeast lacking PAH1 are more efficiently utilized by the tombusvirus replicase than the ER membrane from wt yeast. On the contrary, when we adjusted the p33 levels in the isolated ER preparations, then we observed comparable levels of in vitro tombusvirus replication in ER membranes from all three yeast strains (Fig. 4C, lanes 1-3). This indicates that the relative activity of the tombusvirus replication protein expressed in these yeast strains is similar. Therefore, the higher activity of tombusvirus replicase in pah1Dnem1D or pah1D yeasts are likely due to the more efficient assembly of the tombusvirus VRCs, resulting in larger number of replicationally active VRCs than in wt yeast.
To test how efficiently the p33 and p92 replication proteins can utilize the expanded ER membranes in pah1Dnem1D yeast versus the wt yeast, we performed confocal laser microscopy with fluorescently tagged tombusvirus p33 replication protein. When we looked at the localization of YFP-p33 at an early time point (4 hours), we observed that a large portion of YFP-p33 was still cytosolic and a small number of punctate structures were forming in wt yeast (Fig. 5A). At the same time point, the YFP-p33 localized efficiently in the ER membranes in pah1Dnem1D yeast and only a smaller fraction of YFP-p33 showed cytosolic localization pattern (Fig. 5B). At the 6 hr time point, the YFP-p33 also showed mostly punctate pattern in wt yeast (Fig. 5C). A fraction of YFP-p33 was present in the ER membrane or was diffused in the cytosol, while other YFP-p33 molecules were likely localized in the peroxisomal membranes in wt yeast. In contrast, most YFP-p33 localized in the ER membranes in pah1Dnem1D represents the newly made (+)RNA products. Bottom panels show Western blot analysis of the CFEs for Pgk1p, Sec61p and Ssa1p cellular proteins. Note that, prior to the assay, the CFEs were adjusted to contain comparable amounts of the cellular Pgk1p. doi:10.1371/journal.ppat.1003944.g003 yeast at the 6 h time point (Fig. 5D). At the late 24 h time point, as expected, most of the p33 was localized in punctate structures separate from the ER membranes [likely representing the peroxisomal membranes as shown previously [39][40][41]], although a small fraction of p33 did co-localize with the ER in wt yeast cells (Fig. 5E). In contrast, most p33 is localized in the ER membranes, forming large elongated structures in pah1Dnem1D yeast (Fig. 5F). Altogether, these data showed that p33 is rapidly localized to the expanded ER membranes in pah1Dnem1D yeast, while the localization of p33 from the cytosol to membranes is slower in wt yeast, and likely involves the peroxisomal membranes as shown before. It seems that a small fraction of p33 does target the ER membrane even in the wt yeast cells. Therefore, we conclude that the tombusvirus replication proteins efficiently exploit the expanded ER membranes in pah1Dnem1D yeast cells.
To test if peroxisomes are available for TBSV replication in pah1Dnem1D yeast cells, we used CFP-tagged Pex13p peroxisome membrane marker protein [41]. The distribution of Pex13p showed the characteristic punctate structures in pah1Dnem1D yeast cells (Fig. 6). However, the co-localization of Pex13p and the tombusvirus p33 differed in wt and in pah1Dnem1D yeast cells (Fig. 6A-B). While Pex13p showed remarkably good co-localization with the tombusvirus p33 in wt yeast (67% co-localization and 28% partial co-localization of Pex13p and p33 puncta and only 15% of p33 puncta were not co-localized with Pex13p puncta; Fig. 6A), the tombusvirus p33 was largely present in different compartment than Pex13p in pah1Dnem1D yeast cells (Fig. 6B). However, a small fraction of p33 (less than 10%) was completely or partially co-localized with Pex13p in pah1Dnem1D yeast cells, suggesting that peroxisome membranes are still utilized by tombusviruses in the mutant yeast. In addition, both Pex13p and the tombusvirus p33 were present in several large foci in wt yeast, indicative of membrane proliferation and peroxisome aggregation induced by the tombusvirus p33 [40,41,60]. In contrast, Pex13p was mostly present in several smaller foci in pah1Dnem1D yeast cells, suggesting lack of membrane proliferation and peroxisome aggregation (Fig. 6B). Altogether, these data is compatible with the model that the expanded ER membranes are more efficiently utilized by the tombusvirus replication protein than the peroxisomal membranes in pah1Dnem1D yeast cells, although peroxisomes are also available in these cells.
To test if the VRC assembly could indeed be more efficient in the pah1Dnem1D yeast cells, we used the CFE-based assay to assemble the membrane-bound VRCs [37]. After the assembly and activation of the VRCs in the CFEs, we solubilized and affinity-purified the tombusvirus replicase and tested the replicase activity on added RNA template (Fig. 7A). This assay depends on the efficiency of VRC assembly and the activation of the tombusvirus p92 pol replication protein, which is originally inactive when expressed in E. coli, yeast or plants [31]. The activation of p92 pol replication protein occurs in the membrane-bound VRCs and depends on many factors, including p33, cis-acting elements in the viral (+)RNA, host factors and cellular membranes [30,32,37,61,62]. We found that the CFE prepared from pah1Dnem1D yeast cells resulted in ,3-fold higher replicase activity in vitro (Fig. 7B), suggesting that the VRC assembly and activation of p92 pol replication protein is more efficient than that in the similar CFE from wt yeast.
CIRV and NoV replicating in the mitochondrial membranes are not benefitted from the expanded ER membranes in yeast lacking PAH1 To study if other RNA viruses that replicate in the mitochondrial membranes could take advantage of the expanded ER membranes or increased phospholipid synthesis in pah1D yeast, first we used Carnation Italian ringspot virus (CIRV) (Table S1), a tombusvirus closely related to TBSV [29]. CIRV replicates on the outside surface of the mitochondrial membranes in vivo and in vitro [42,63,64]. Interestingly, the level of CIRV replication was not changed in pah1D yeast (Fig. 8A, lanes 6-10). Also, the membrane-fraction obtained from pah1D yeast resulted in similar level of RNA replication supported by the CIRV p36 and p95 pol replication proteins than the membrane-fraction from wt yeast (Fig. 8B, lane 2 versus 1). The accumulation levels of p36 and p95 pol were comparable in pah1D yeast, while the accumulation of cellular Sec61p and Ssa1p was increased as expected (Fig. 8B). Based on these data, we suggest that CIRV replication is not affected in yeast lacking the PAH1 gene and the expanded ER membranes do not seem to be utilized by CIRV in yeast.
The second RNA virus tested was Nodamura virus (NoV) (Table S1), an insect RNA virus not related to tombusviruses. NoV RNA replicates on the outer mitochondrial membranes in yeast cells by expressing a single replication protein termed protein A [65][66][67]. We found similar level of NoV RNA accumulation in pah1D and wt yeasts (Fig. 8C), suggesting that NoV does not take advantage of the cellular changes caused by the deletion of PAH1.

Over-expression of Arabidopsis Pah2p interferes with tombusvirus replication in Nicotiana bethamiana
To obtain evidence if the conserved lipin-like PAP phosphatidate phosphatase also plays a role in tombusvirus replication in plants, we over-expressed the Arabidopsis Pah2p protein in N. benthamiana leaves using an Agrobacterium-based expression system. Plants have two phosphatidate phosphatase-coding genes, PAH1 and PAH2, which are highly homologous with the yeast PAH1 gene and they can complement the phenotypes in pah1D yeast [68][69][70]. Also, deletion of PAH1 and PAH2 in Arabidopsis thaliana causes ER expansion and increased phospholipid synthesis, similar to the phenotypes observed in pah1D yeast [69].
We found that the over-expression of AtPah2p caused a 6-fold drop in the genomic RNA accumulation of CNV, a TBSV-like tombusvirus, which replicates in the peroxisomal membrane [39], in N. benthamiana leaves (Fig. 9A). The lethal necrotic effect of the CNV tombusvirus was also attenuated in N. benthamiana expressing the AtPah2p (Fig. 9B). The accumulation of TBSV RNA also decreased by ,3-fold in N. benthamiana leaves over-expressing AtPah2p (Fig. 9C). On the contrary, the replication of another tombusvirus, CIRV (Table S1), which uses the mitochondrial membrane [63], was not changed in N. benthamiana leaves overexpressing AtPah2p (Fig. 9D). Based on these observations, we suggest that the plant phosphatidate phosphatase also plays a role in TBSV and CNV tombusvirus replication that can use peroxisomes and ER membranes, but does not affect the replication of the mitochondrial CIRV in plants.
To test if the inhibitory effect of AtPah2p over-expression is specific to peroxisomal tombusviruses, we studied the accumulation of red clover necrotic mosaic virus (RCNMV) (Table S1), which is a distantly related RNA virus replicating in the ER membranes of the host cells [71]. We observed that the accumulation of RCNMV RNA decreased by ,3-fold in N. benthamiana leaves over-expressing AtPah2p (Fig. 9E). Thus, another plant RNA virus, RCNMV, is also affected by the plant lipin-like gene, suggesting that the effect of this host gene on ER membranes is critical for replication of RNA viruses taking advantage of the ER membrane to build replication complexes.

Discussion
Tombusviruses, like many (+)RNA viruses, depend on host membrane biogenesis during replication. Thus, conditions that induce membrane biogenesis in cells might affect replication of some RNA viruses. Accordingly, the key finding in this paper is that tombusviruses (TBSV and CNV) can take advantage of expanded ER membrane surface, which is due to deletion of the cellular Pah1p PAP enzyme leading to massive enlargement of ER membranes and increased phospholipid biosynthesis in yeast [43,49,55,72], to build VRCs efficiently. The evidence supporting this model is extensive and includes: (i) increased TBSV repRNA accumulation in pah1D (or in the functionally similar pah1Dnem1D lipin deficient) yeasts or Dgk1p diacylglycerol kinase overproducing yeast; (ii) increased accumulation and stability of tombusvirus p33 replication protein in pah1D yeast; (iii) the enhanced in vitro assembly of the tombusvirus VRCs in a CFEbased assay derived from yeast lacking Pah1p; (iv) detection of highly abundant p33 replication proteins in isolated ER fraction or using confocal microscopy in yeast lacking Pah1p; (v) the higher in vitro activity of the tombusvirus replicase purified from CFE obtained from yeast lacking Pah1p than from wt yeast; and (vi) the stimulatory role of the membrane fraction of pah1D yeast in the CFE-based TBSV replication assay. The efficient utilization of the expanded ER membrane is reflected by the rapid localization of the p33 replication protein to the ER membrane at an early time point in yeast lacking Pah1p. In addition, it appears that the tombusvirus replicase assembles faster in yeast lacking Pah1p than in wt yeast. Thus, our model proposes that TBSV and CNV efficiently subvert the expanded ER membranes and utilizes the abundant phospholipids generated in pah1D or pah1Dnem1D lipindeficient yeast, leading to robust viral replication. These tombusviruses replicate in the expanded ER membranes in lipin deficient yeast although the peroxisomal membranes are also present (and some of these membranes are still utilized by TBSV and CNV), suggesting that the expanded ER likely contains favorable microenvironment (membrane microdomains) for these tombusviruses. Therefore, it seems that TBSV and CNV are flexible in utilizing peroxisomal and ER membranes depending on cellular conditions. Accordingly, TBSV is capable to assemble the VRCs on ER and mitochondrial preparations in vitro [42], indicating that TBSV could exploit a range of subcellular membranes in cells. Altogether, we document that deletion of the lipin gene results in strong stimulatory effect on TBSV replication. We also show that TBSV could readily switch to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support robust viral replication instead of utilizing the peroxisomal membranes as observed in wt yeast and plants [5,39]. Thus, the increased phospholipid synthesis and the expanded ER membranes in lipin-deficient cells provide highly suitable environment for TBSV and CNV for efficient viral replication.
Similar to tombusviruses, other (+)RNA viruses subvert various intracellular membranes for construction of VRCs, and the ER membrane is often critical for these processes [4,[73][74][75]. Indeed, we find that the ER-based RCNMV replication in plant host cells is also affected by the lipin gene. Therefore, our findings with tombusviruses presented in this paper could be relevant for other RNA viruses of plants and animals. However, not all viruses could benefit from lipin mutations, since we find that CIRV tombusvirus and NoV insect RNA virus, both of which replicate on the mitochondrial membrane surfaces, could not take advantage of the expanded ER membranes generated in pah1D yeast. CIRV was also more restrictive in the CFE-based replication assay, utilizing the mitochondrial membranes more efficiently than the ER membranes [42].
The role of PAH1 in tombusvirus replication based on yeast model host was further supported by data obtained in natural plant host by over-expression of AtPah2p. The over-expression of the yeast Pah1p (especially the constitutively active Pah1-7A mutant) in yeast and the AtPah2p in N. benthamiana strongly inhibited tombusvirus replication. This inhibition is likely due to strong competition between cellular phospholipid pathways driven by the over-expressed PAP enzyme and the need for phospholipidcontaining cellular membranes in tombusvirus replication [76]. We suggest that tombusviruses could find and/or induce phospholipid-rich microdomains in peroxisomal or ER membranes less efficiently when the over-expressed cellular PAP enzyme decreases the phosholipids level (by producing DAG) in cells.
Our findings might also influence the current view on genetic mutations and diseases versus pathogen infections. Mutations in cellular genes frequently alter cellular pathways and they can cause genetic diseases. The same gene mutations/changes might also influence pathogen-host interactions as well. For example, the cellular lipin, which is involved in a key cellular decision on using lipids for membrane biogenesis or for storage [43][44][45][46][47], could be one of those factors. We suggest that mutations in lipin genes, which are known to induce many genetic diseases in humans and animals [44,46,47,[77][78][79], not only change the physiology of the given organism, but they might affect pathogens and their interactions with the altered host. Although we used yeast as a model host, since it only has a single lipin gene (PAH1), the role of the PAP enzyme in phospholipid pathways are conserved in yeast, plants and animals. Deletion/mutations of the lipin gene is known to facilitate membrane biogenesis in all these organisms. Therefore, the effects seen with tombusviruses and RCNMV might also manifest with animal viruses possibly leading to exploitation of the expanded ER membranes during infections. Thus, it is possible that one disease state might facilitate the development of robust viral infection at the cellular level.
We found that the pah1Dnem1D yeast was more consistent than pah1D yeast in supporting high level of CNV and TBSV repRNA replication for yet unknown reasons. Therefore, most of the experiments were performed with both strains or with only pah1Dnem1D yeast strain.

Pah1p complementation and over-expression assays in yeast
Yeast wt RS453 and pah1D strain was transformed with pESC-CUP1-His-p92 (URA3 selection), pESC-GAL1-His-p33/GAL10-DI-72 (HIS3 selection) and LEU2 based plasmids: pYEplac181 (vector control), pYEplac181-wt-Pah1p (for expression of wt Pah1p) or pYEplac181-Pah1p-7A (for expression of constitutively active phosphorylation-deficient Pah1p), respectively [53]. Yeast was pre-grown at 23uC overnight in 2 ml SC-ULH 2 medium containing 2% galactose and then the cultures were harvested after 24 h 50 mM CuSO 4 induction. Replication protein stability assay To study the stability of p33 replication protein in yeast, RS453 and pah1Dnem1D strains were transformed with plasmid UpYES-GAL-Hisp33 expressing His 6 -tagged CNV p33 from the galactoseinducible GAL1 promoter. Yeast transformants were cultured overnight in SC-U 2 medium containing 2% glucose at 23uC. Yeast cultures were transferred to SC-U 2 medium supplemented with 2% galactose for 3 h at 23uC. Then, the cultures were shifted back to the SC-U 2 medium supplemented with 2% glucose and cycloheximide (at a final concentration of 100 mg/ml). The amount of p33 was detected by Western blotting with anti-His 6 antibody at given time points after cycloheximide treatment. Each sample loading was adjusted based on total protein levels as determined by SDS-PAGE [39].

In vitro replication assay using yeast membrane fractions
The membrane fractions were prepared as described previously [85]. Briefly, yeasts were transformed and cultured as described in the main text for TBSV or CIRV repRNA replication in yeast. Cultures were collected by centrifugation to obtain membrane fractions containing the in vivo-assembled tombusvirus replicase complexes. Each membrane fraction preparation was adjusted by the relative amounts of His 6 -tagged p92 and comparable amounts of replicase from each preparation were used in the subsequent replicase assay. The replicase assay was performed as described [85]. The in vitro reaction (50 ml) contained 10 ml of the normalized MEFs preparations, 50 mM Tris-Cl pH 8.0, 10 mM MgCl 2 , 10 mM DTT, 0.1 U RNase inhibitor, 10 mM ATP, 10 mM CTP, 10 mM GTP and 0.1 ml of 32 P-UTP (3000 Ci/mmol). Reaction mixtures were incubated for 3 h at 25uC, followed by phenol/ chloroform extraction and isopropanol/ammonium acetate (10:1) precipitation. The 32 P-UTP-labeled RNA products were analyzed in 5% acrylamide/8 M urea gels.
In vitro replication assay using yeast cell free extract (CFE) CEFs from RS453 (wt) or pah1Dnem1D were prepared as described earlier [37] and adjusted to contain comparable amounts of cellular Pgk1p, a cytosolic protein marker. The in vitro reaction was performed in 20 ml total volume containing 2 ml of adjusted CFE, 0.5 mg DI-72 (+)repRNA transcript, 0.  by phenol/chloroform extraction and isopropanol/ammonium acetate (10:1) precipitation. 32 P-UTP-labeled RNA products were analyzed in 5% acrylamide/8 M urea gels [37]. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32 P-labeled RNA samples were directly loaded onto the gel without heat treatment. Membrane and soluble fractions of these CFEs or in vitro reaction were separated by centrifugation at 35,000 g for 30 min and then mixed them in various combinations (described in the figure legends).

In vitro TBSV replication assay using isolated yeast ER fractions
Yeasts were transformed and cultured as described in the main text for TBSV or CIRV repRNA replication in yeast. The ER fractions were prepared as described earlier [42,86]. Briefly, yeast cells were made into spheroplasts by incubating with 5 mg/g (wet weight) Zymolyase 20T (Seikagaku), and then the spheroplasts were homogenized and lysed with a glass Dounce homogenizer in ice-cold HEPES lysis buffer (20 mM HEPES/KOH [pH 6.8], 50 mM potassium acetate, 100 mM sorbitol, 2 mM EDTA, 1 mM DTT and 1% [V/V] yeast protease inhibitor cocktail [Ypic]). The homogenized spheroplasts were then centrifuged at 1,000 g for 10 min at 4uC, and the supernatant was subjected to additional centrifugation at 27,000 g for 10 min at 4uC to obtain the membrane preparation. To further purify the ER fraction, the membrane preparation was subjected to centrifugation at 100,000 g on a sucrose step gradients (1.5 M and 1.2 M sucrose/HEPES). The purified ER fraction was recovered between the sucrose gradient interfaces and each fraction was adjusted by the amounts of the protein mentioned in figure legend. The in vitro TBSV replication assay was performed as described for in vitro replication assay with MEF except that using the purified ER fraction.
Affinity-purification of the in vitro assembled TBSV replicase 200 ml of the CFE-based replication assay was performed as described above, except that only rATP and rGTP were used. Also, the CFE assay contained the MBP and His 6 -tagged recombinant p33. At the end of the in vitro replicase assembly assay, the reaction mixture was diluted with 800 ml solubilization buffer, and the replicase complex was purified followed the procedure described previously [61]. In vitro RdRp activity assay was performed using DI-72 region I/III (2)RNA or region IV (+)RNA as template transcribed in vitro by T7 transcription.

Imaging yeast cells with confocal laser microscopy
To visualize the ER, Pho86-CFP was used as a marker, while peroxisomes were monitored with the help of Pex13-CFP (a peroxisome membrane marker) [39]. The yeast cells were transformed with UpESC-YFP-p33 and LpGAD-Pho86-CFP or LpGAD-ADH::Pex13-CFP. For the 24 h time point, transformed yeast cells were grown in SC-UL 2 medium containing 2% galactose at 23uC for 24 h and then sample were collected and analyzed by confocal microscopy as described [41]. For short time points, the transformed yeasts were pre-grown overnight at 23uC in SC-UL 2 medium containing 2% glucose and then transferred to media containing 2% galactose, and then, samples were collected for microscopy analysis at given time points.
Confocal laser scanning micrographs of yeast cells were acquired on an Olympus FV1000 microscope (Olympus America Inc., Melville, New York) as described [41]. ECFP was excited using 440 nm laser light, attenuated to 4.5% of the maximum laser power, while EYFP was excited using 515 nm laser line (3.5% of the maximum laser power). The images were acquired using sequential line-by-line mode in order to reduce excitation and emission cross-talk. The primary objective used was water-immersion PLAPO60XWLSM (Olympus). Image acquisition was conducted at a resolution of 5126512 pixels and a scan-rate of 10 ms/pixel. Image acquisition and exportation of TIFF files were controlled by using Olympus Fluoview software version 1.5.

Western blotting
To prepare the total protein sample for Western blotting, we followed a previous protocol [31,39]. Briefly, 1 ml of yeast culture was harvested by centrifugation. Then, the samples were resuspended in 200 ml of 0.1M NaOH and incubated at room temperature with shaking for 20 min. The supernatant was removed after a short centrifugation, and the pellet was resuspended in 50 ml, 1X SDS-polyacrylamide gel electrophoresis (PAGE) buffer containing 5% b-mercaptoethanol and incubated at 85uC for 15 min. The supernatant was used for SDS/PAGE and Western blot analysis as described [87]. To detect the CNV or TBSV viral proteins, anti-His 6 antibody was used as the primary antibody (Invitrogen) and the secondary antibody was alkalinephosphatase conjugated anti-mouse IgG (Sigma). For cellular protein markers, the following antibodies were used: anti-3phosphoglycerate kinase (anti-PGK), and anti-heat shock protein 70 (anti-Hsc70) (purchased from Invitrogen, CA). Sec61p antibody was provided by Tom Rapoport, Harvard Medical School. Fox3p antibody was provided by Daniel J. Klionsky, University of Michigan.

TBSV and CIRV replication in Nicotiana benthamiana
Transient expression of Arabidopsis thaliana Pah2p in N. benthamiana leaves was performed by agroinfiltration [88]. A. thaliana PAH2 (At5g42870) was amplified by PCR using primers #4630 (GCCGGATCCATGAATGCCGTCGGTAGGATC) / #4631 (CGGCTCGAGTCACATAAGCGATGGAGGAGGC-AG) and genomic DNA as template. The obtained PCR product was digested with BamHI and XhoI, purified and ligated into pGD-L [6] previously digested with BamHI and SalI. The resulting plasmid was transformed into Agrobacterium tumefaciens C58C1 [6]. N. benthamiana plants were infiltrated with A. tumefaciens (OD 600 = 0.8) carrying pGD-L-PAH2 or the control empty plasmid pGD. Two days later, the same leaves were infiltrated with A. tumefaciens (OD 600 = 0.2) carrying pGD-CNV to launch CNV replication, or pGD-CIRV to launch CIRV replication [6]. Leaf samples were collected 5 days later and total RNA was extracted. CNV and CIRV RNA accumulation was analyzed by agarose gel electrophoresis and Northern blotting using 32 Plabeled probes complementary to the 39 end of the viral RNAs [6].
For studies with TBSV and RCNMV, N. benthamiana plants were infiltrated with A. tumefaciens (OD 600 = 0.8) carrying pGD-L-PAH2 or the control empty plasmid pGD. Two days later, the same leaves were inoculated with infectious saps containing TBSV or RCNMV virions. Leaf samples were collected 3 days later and total RNA was extracted. RNA accumulation was analyzed by agarose gel electrophoresis and Northern blotting using 32 Plabeled probes complementary to the 39 end of the TBSV RNA or RCNMV RNA1 [6]. Figure S1 Over-expression of Dgk1p enhances TBSV repRNA accumulation in wt yeast as shown by Northern blotting. Note that overproduction of Dgk1p, similar to PAH1 deletion, leads to ER membrane expansion. Bottom panel: detection of the overproduced Dgk1p in yeast. The His 6 -tagged Dgk1p, His 6 -p33 and His 6 -p92 were detected by Western blotting. (EPS) Figure S2 Comparable effects of single PAH1 and double PAH1 and NEM1 deletions on TBSV repRNA accumulation in yeast. Top panel: Replication of the TBSV repRNA in wt, pah1Dnem1D and pah1D yeasts was measured by Northern blotting 8 h after initiation of TBSV replication. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Each sample is obtained from different yeast colonies. Bottom panel: The accumulation levels of His 6 -p92 and His 6 -p33 were tested by Western blotting. Each experiment was repeated. (EPS) Figure S3 Comparable effects of single PAH1 and double PAH1 and NEM1 deletions on in vitro TBSV repRNA replication. (A) The scheme of the in vitro TBSV replication assay based on the isolated membrane fraction carrying the tombusvirus replicase and the bound RNA template. (B) Top panel: Comparable in vitro replication of TBSV repRNA in the isolated membrane fraction from pah1D yeast when compared with that from pah1D nem1D yeast. Note that the levels of His 6 -p92 replication protein were normalized as shown in the second panel. The third, fourth and fifth panels show the accumulation levels of His 6 -p33, and the cellular Sec61p (an ER marker) and Ssa1p Hsp70. Samples were taken at 24 h time point. (EPS) Figure S4 Enhanced TBSV replication depends on the membrane fraction of CFE prepared from pah1Dnem1D yeast.

Supporting Information
(A) The scheme of the CFE-based TBSV replication assay. Purified recombinant TBSV p33 (7 pmol) and p92 pol (4 pmol) replication proteins in combination with DI-72 (+)repRNA (0.5 mg) were added to the CFEs made by mixing the soluble and membrane fractions as shown. (B) Denaturing PAGE analysis of the 32 P-labeled repRNA products obtained is shown. The TBSV replication assays were based on the mixed soluble and membrane fractions of CFEs prepared from wt or pah1Dnem1D yeasts, as shown. The full-length single-stranded repRNA is pointed at by an arrow. (EPS)