RNA Virus Replication Depends on Enrichment of Phosphatidylethanolamine at Replication Sites in Subcellular Membranes

RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes

Kai Xu and Peter D. Nagy1

Department of Plant Pathology, University of Kentucky, Lexington, KY 40546

Edited by David C. Baulcombe, University of Cambridge, Cambridge, United Kingdom, and approved February 25, 2015 (received for review October 1, 2014) Intracellular membranes are critical for replication of positive- for PI4P facilitates (+)RNA virus replication. However, our strand RNA viruses. To dissect the roles of various lipids, we have knowledge on the roles of various phospholipids in RNA virus developed an artificial phosphatidylethanolamine (PE) vesicle- replication is currently incomplete. By using tombusviruses, small based Tomato bushy stunt virus (TBSV) replication assay. We dem- model RNA viruses of plants that can replicate in a yeast surro- onstrate that the in vitro assembled viral replicase complexes gate host (15), which has the advantage of tolerating large changes (VRCs) in artificial PE vesicles can support a complete cycle of re- in different phospholipid composition, a major role for global plication and asymmetrical RNA synthesis, which is a hallmark of phospholipid and sterol biosynthesis, have been revealed (16– (+)-strand RNA viruses. Vesicles containing ∼85% PE and ∼15% 18). In this paper, a viral replicase reconstitution assay based on additional phospholipids are the most efficient, suggesting that artificial phospholipid vesicles identified the essential role of TBSV replicates within membrane microdomains enriched for phosphatidylethanolamine (PE) in replication of Tomato bushy PE. Accordingly, lipidomics analyses show increased PE levels stunt virus (TBSV). It has also been shown that TBSV could recruit in yeast surrogate host and plant leaves replicating TBSV. In ad- and enrich PE to the sites of viral replication in yeast and plant cells. dition, efficient redistribution of PE leads to enrichment of PE at Moreover, genetic changes that either increase or decrease PE viral replication sites. Expression of the tombusvirus p33 replica- levels in yeast greatly stimulated or inhibited TBSV replication, tion protein in the absence of other viral compounds is sufficient confirming the key role of PE in the formation of TBSV replicase. to promote intracellular redistribution of PE. Increased PE level due to deletion of PE methyltransferase in yeast enhances replication of Results TBSV and other viruses, suggesting that abundant PE in subcellular Efficient Replication of TBSV RNA in Artificial PE Vesicles. To test membranes has a proviral function. In summary, various (+)RNA what type of phospholipids are required for tombusvirus repli- viruses might subvert PE to build membrane-bound VRCs for robust cation, we developed an artificial vesicle (liposome)-based rep- replication in PE-enriched membrane microdomains. lication assay involving purified recombinant tombusvirus p33 and p92pol replication proteins, TBSV (+) replicon RNA (repRNA), plant virus | virus–host interaction | phospholipid | viral replicase complex | and cellular cytosolic proteins present in yeast cell-free extract host factor (CFE; Fig. 1A). Interestingly, artificial vesicles prepared from PE supported TBSV repRNA replication, reaching about half of the any steps in the infection cycles of positive-strand RNA level that takes place in the standard total membrane fraction of Mviruses, including entry into the cell, replication, virion CFE obtained from WT yeast (Fig. 1A, lanes 3 and 4 versus 1 assembly, and egress, are associated with subcellular membranes and 2) (19). On the contrary, vesicles consisting of only phos- (1–4). Therefore, viruses have to interact with different lipids, phatidylcholine (PC; Fig. 1A, lanes 5 and 6) or lysophosphati- such as phospholipids and sterols, which affect the biophysical dylethanolamine (lysoPE) showed 5% viral RNA replication features of membranes, including the fluidity and curvature (5, 6). The subverted cellular membranes could protect the viral Significance RNA from recognition by the host nucleic acid sensors or from destruction by the cellular innate immune system. In addition, Positive-strand RNA viruses are major pathogens of plants, membranes facilitate the sequestration of viral and coopted host animals, and humans. These viruses subvert intracellular mem- proteins to increase their local concentrations and promote mac- branes for virus replication, and lipids are critical due to interaction romolecular assembly, including formation of the viral replicase with viral and coopted host proteins. To dissect the roles of vari- complex (VRC) or virion assembly. To optimize viral processes, ous lipids in Tomato bushy stunt virus (TBSV) replication, we have RNA viruses frequently manipulate lipid composition of various – developed artificial vesicle-based replication assay. Vesicles con- intracellular membranes (6 13). Overall, the interaction between sisting of a major phospholipid, namely phosphatidylethanolamine cellular lipids and viral components is emerging as one of the (PE), can support TBSV replication by assembling viral replicase possible targets for antiviral methods against a great number of complexes and performing a complete replication cycle. Monitor- viruses. Understanding the roles of various lipids in RNA virus ing PE distribution reveals that PE is enriched at the sites of TBSV infections is important to ultimately control harmful RNA viruses. replication in plant and yeast cells. Increasing PE level in cells leads Among the various lipids, the highly abundant phospholipids to enhanced replication of TBSV and other viruses, suggesting are especially targeted by RNA viruses (2). In general, phos- that abundant PE in subcellular membranes has proviral function. pholipids likely affect the replication of most RNA viruses, which takes place within membranous structures (1, 3, 4). Accordingly, Author contributions: K.X. and P.D.N. designed research; K.X. performed research; K.X. lipidomics analyses of cells infected with Dengue virus and contributed new reagents/analytic tools; K.X. and P.D.N. analyzed data; and K.X. and hepatitis C virus (HCV) (8, 9) revealed enhanced virus-induced P.D.N. wrote the paper. lipid biosynthesis, resulting in changes in the global lipid profile The authors declare no conflict of interest. of host cells. Also, the less abundant regulatory phosphatidyli- This article is a PNAS Direct Submission. nositol-4-phosphate (PI4P) was shown to be enriched at sites of 1To whom correspondence should be addressed. Email: [email protected]. enterovirus and HCV replication due to recruitment of cellular This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lipid kinases (7, 14), suggesting that a microenvironment enriched 1073/pnas.1418971112/-/DCSupplemental.

E1782–E1791 | PNAS | Published online March 25, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1418971112 Downloaded by guest on October 2, 2021 PNAS PLUS

A recombinant vesicles + S40 TBSV p33/p92 + S40 (CFE) +S40 P40 PE PC - template RNA

Artificial RNA repRNA Vesicles synthesis +ATP/GTP CTP/32P-UTP 1 2 3 4 5 6 7 8 100±23 51±2 3±1 4±1 % repRNA

B P40 PE C PE vesicles + S40

DI-72 (+)RNA + triton X100 0 1.0 0.1 0.01 0.001 0.0001

repRNA DI-72 (-)RNA 1 2 3 4 5 6 100 <1 <1 10 48 52 % repRNA 11:1 10:1 (+):(-) RNA ratio ±2 ±6 ±7 D

100 98 96 94 92 90 88 86 84 82 80 70 60 40 20 0 % PE repRNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 100 130 125 120 123 185 211 225 190 152 107 14 5 4 13 20% repRNA ±25 ±7 ±5 ±4 ±2 ±11 ±26 ±13 ±5 ±5 ±1 ±1 ±1 ±1 ±3 % 250 MICROBIOLOGY

200

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0 100 90 80 70 60 40 20 0% PE

Fig. 1. In vitro reconstitution of the TBSV replicase in artificial PE vesicles. (A) Scheme of the replicase assembly assay. Purified recombinant p33 and p92pol replication proteins of TBSV in combination with the TBSV-derived (+)repRNA were added to PE or PC vesicles or the P40 membrane fraction of yeast CFE. The S40 fraction of CFE was also added to each sample to provide soluble host factors required for TBSV VRC assembly. The denaturing PAGE analysis of the 32P-labeled repRNA products obtained is shown. The full-length repRNA is denoted by an arrow. The CFE-based replication assay was chosen as 100% (lanes 1 and 2). (B) Asymmetrical RNA synthesis by TBSV VRCs assembled in PE vesicles. The amounts of TBSV (+)- and (–)-stranded RNA products produced by the reconstituted TBSV VRCs are measured by using the 32P-labeled repRNA probes generated in the in vitro assays. The blot contains the same amount of cold (+)- and (–)-stranded DI-72 RNA. (C) TBSV RNA synthesis by the reconstituted VRCs in PE vesicles requires vesicles/membrane bilayers. The PE vesicles were disrupted by Triton-X100 treatment as shown. The denaturing PAGE analysis of the replicase products is as in panel A.(D) Increased VRC activity in PE vesicles containing a fraction of other phospholipids. The PE vesicles contained the shown percentage of PE plus a mixture of other phospholipids (the ratio in the phospholipid mix was 54.5 of PC, 6.1 of PI, 1.2 of PS, 8.3 of PG, 0.9 of LysoPE, and 0.6 of LysoPC), which was based on their ratios in TBSV-infected N. benthamiana leaves. The denaturing PAGE analysis of the replicase products is as in panel A.

activity compared with PE vesicles (Fig. S1A, lanes 3 and 4 and synthesis by adding various concentrations of Triton X-100, which 13 and 14), whereas phosphatidylglycerol (PG), phosphatidylserine could disrupt lipid bilayers in the vesicles. Viral RNA synthesis was (PS), phosphatidylinositol (PI), cardiolipin (CA), and lysophospha- inhibited up to 90% in the presence of 0.01%, whereas it was com- tidylcholine (lysoPC) vesicles did not support TBSV RNA replica- pletely blocked by the presence of 0.1 or 1.0% Triton X-100 (Fig. 1C), tion at detectable levels (Fig. S1A). These data support a model that suggesting that the membranous environment is needed for TBSV PE is the only phospholipid required for TBSV replication in vitro, replicationinvitro.TotestifTBSVreplicasecouldformanuclease- whereas the other phospholipids are not sufficient by themselves to resistant compartment, as is the case in cells and in the yeast CFE (19, support robust TBSV replication. 20), we performed the in vitro assays in the presence of micrococcal To examine the nature of TBSV replication in PE vesicles, we nuclease, which can destroy the unprotected viral RNAs. These measured viral (–)- versus (+)-strand RNA synthesis. These ex- studies revealed that TBSV replicase formed in the presence of PE periments revealed that TBSV replication led to the production vesicles was much less protective of the viral RNA against the nu- of ∼10-fold more (+)- than (–)-stranded RNAs, similar to the clease than the replicase assembled in yeast CFE (Fig. S1B). This ratio seen in yeast CFE preparation containing yeast membranes finding suggests that not only PE but also additional phospholipids, (Fig. 1B) (19). Thus, the in vitro assembled TBSV replicase in arti- other types of lipids, or membrane proteins in the yeast CFE might ficial PE vesicles can support a complete cycle of replication and contribute to the assembly of the authentic TBSV replicase. asymmetrical RNA synthesis, which is a hallmark of (+)-strand RNA To identify the optimal PE concentration in the lipid bilayer viruses. We also tested if the PE vesicles are required for RNA for supporting the most efficient TBSV replication, we made

Xu and Nagy PNAS | Published online March 25, 2015 | E1783 Downloaded by guest on October 2, 2021 pol A viral RNA recruitment the highest efficiency when p33/p92 replication proteins were present (Fig. 2B). The association of the viral (+)repRNA to top artificial PG, PS, PI, CA, and lysoPC vesicles was low (Fig. 2B). pol low vesicle- Therefore, we suggest that binding of p33/p92 replication CF bound proteins to PE, PC, and lysoPE phospholipids facilitates the recruitment of the viral (+)repRNA to membranes, a required step high sucrose conc. not bound repRNA* for VRC assembly and RNA replication. vesicles bottom p33/p92 PE Is Enriched at the Sites of TBSV Replication. Because TBSV B Top fraction requires membranes with high PE content to assemble the functional VRCs in vitro, we wondered if PE, which is among the PE PC PG PS PI CA lysoPE lysoPC most abundant phospholipids in yeast, is enriched at the sites of + - + - + + + + + - + - p33/p92 replication. We used biotinylated duromycin, which specifically binds to PE (23), to monitor the distribution of PE during TBSV repRNA* replication. Interestingly, PE was highly enriched in the subcellular locations containing GFP-p33 in yeast cells replicating 1 2 3 4 5 6 7 8 9 10 11 12 the TBSV repRNA (Fig. 3A). Expression of the p33 replication 100 33 288 9 <1 <1 <1 <1 69 11 4 8 % repRNA protein in the absence of other viral compounds was sufficient to ±2 ±21 ±2 ±2 ±4 ±3 ±3 promote intracellular redistribution of PE (Fig. 3A). Of all of the p33-positive cells examined, >90% showed redistribution of PE. Fig. 2. TBSV p33/p92-mediated binding of TBSV RNA to artificial vesicles These PE-enriched sites were colocalized with both the Pex13p- containing different phospholipids. (A) Scheme of the in vitro binding assay GFP peroxisomal marker and red fluorescent protein (RFP)-p33 32 and membrane-flotation experiments. The P-labeled TBSV (+)repRNA (Fig. 3B), indicating that PE is redistributed to the sites of TBSV (DI-72) was incubated with artificial vesicles in the presence of purified re- replication in the peroxisomal membrane. On the contrary, the combinant TBSV p33 and p92 (plus the S40 fraction of yeast CFE to provide soluble cellular factors, such as heat shock protein 70), followed by centri- peroxisomal membrane was not enriched with PE in the absence fugation in 10–70% sucrose density gradient. The top fraction of the su- of TBSV replication proteins and PE was dispersed in many crose gradient was tested for the presence of 32P-labeled TBSV (+)repRNA. parts of the yeast cell (Fig. 3B, Bottom). We confirmed the above (B) Denaturing RNA gel analysis of the presence of 32P-labeled TBSV (+) findings using PE molecules with fluorescently labeled fatty-acid repRNA in the top fraction. The amount of 32P-labeled TBSV (+)repRNA with chain (named NBD-PE) added to the culture media. Accord- the PE vesicles was chosen as 100%. ingly, NBD-PE was enriched in subcellular locations also containing RFP-p33 and Pex13p-BFP (blue fluorescent protein) (Fig. 4A). On the contrary, NBD-PE was not enriched in the artificial vesicles containing PE and increasing amounts of a peroxisomal membranes in the absence of TBSV replication mixture of other phospholipids (including PC, PI, PS, PG, proteins (Fig. 4B). Unlike NBD-PE, NBD-PC was not highly D lysoPE, and lysoPC) (Fig. 1 ). The highest level of TBSV rep- enriched at the sites of TBSV replication (Fig. 4C). Based on – lication was observed with the vesicles containing 82 90% PE these data, we propose that PE molecules are efficiently D (Fig. 1 ). On the other hand, vesicles containing less than 70% relocalized to and enriched at the sites of viral replication in PE supported inefficient TBSV replication in vitro. To test the the peroxisomal membranes with the help of a p33 replica- effects of various phospholipids on TBSV replication, we also tion protein. tested PE and other phospholipids in pair-wise combinations. To test if similar phenomena also occur in plant cells during These in vitro assays revealed that the presence of only 10% of TBSV replication, we stained TBSV-infected Nicotiana ben- PC or lysoPE in artificial PE vesicles enhanced TBSV replication thamiana protoplasts (single cells lacking cell walls) with bio- by more than 50%, whereas these phospholipids were inhibitory tinylated duromycin and also treated them with an anti-p33 A when applied in higher than 20% concentrations (Fig. S2 and antibody. Importantly, confocal imaging showed high enrichment B ). In contrast, the presence of other phospholipids (PS, PI, CA, of PE in subcellular locations containing the p33/p92 replication and lysoPC) was inhibitory to TBSV replication, except for 10% proteins (Fig. 3C). On the contrary, the subcellular distribution of PG (Fig. S2B). Thus, various phospholipids (other than PE) of PE was dramatically different in uninfected plant cells (Fig. have inhibitory effects on TBSV replication when present in 3C). Based on all these data, the emerging picture is that PE, higher than 20% amounts. These results indicate that TBSV unlike PC, is efficiently redistributed to and enriched in the replication is greatly affected by different kinds of phospholipids. peroxisomal membranes in yeast and plant cells to facilitate ro- To test whether phospholipids affect the membrane associa- bust TBSV replication. tion of the viral replication proteins, we performed membrane 35 flotation experiments with artificial vesicles and S-labeled p33 Increased PE Level in Yeast and Plant Cells Supporting TBSV replication protein. As expected, in the absence of membranes/ Replication. To test if TBSV replication alters phospholipid me- vesicles, p33 stays at the bottom of the sucrose gradient (Fig. tabolism in the host cells to facilitate viral replication, we per- S3B), whereas ∼30% of p33 are present in the top fraction in the formed lipidomics of yeast cells replicating TBSV repRNA or presence of either PE or PC vesicles (Fig. S3 C and D). In ad- lacking all TBSV components. These experiments revealed that dition, p33 can strongly associate with PG, PS, and CA vesicles, the relative level of PE increased from 17.6% to 29.3% of total whereas binding to PI vesicles is poor (Fig. S3). These data phospholipids in yeast replicating TBSV (Fig. 5A). On the con- suggest that the viral p33 replication protein can bind to different trary, PC and PI levels, which are two of the most abundant phospholipids. Thus, the inhibitory effects of high PC, PG, PS, phospholipids, are decreased by ∼6% and 8%, respectively, and CA concentrations on TBSV replication are not due to the when yeast supported TBSV replication (Fig. 5A). These data interference of these phospholipids with membrane association suggest that TBSV replication leads to an increased PE level in of p33 to block TBSV replication in vitro. yeast. The overall phospholipid content of yeast cells increased Similar studies with the viral (+)repRNA (Fig. 2A), which has by 38%, suggesting that yeast cells are induced by TBSV to to be recruited with the help of the viral replication proteins to produce new phospholipids (Fig. S4A). Overall, the total PE the sites of replication in the membranes (19, 21, 22), revealed content of yeast cells replicating TBSV increased by ∼2.3-fold. that the (+)repRNA bound to PE, PC, and lysoPE vesicles with This increased level of PE in yeast cells likely serves the virus’s

E1784 | www.pnas.org/cgi/doi/10.1073/pnas.1418971112 Xu and Nagy Downloaded by guest on October 2, 2021 A B C PNAS PLUS duramycin GFP-p33 merged DIC Pex13- duramycin 33p BA merged DIC dCnicymaru 33p-PFG mRFP degrem ID +p33/ p92 +p33/ repRNA p92 repRNA -TBSV +TBSV

+p33 Pex13- nicymarud GFP RFP-p33 degrem - GFP-p33 merged DIC

+p33 +p33/ p92 dBnicymaru 33p A degrem DIC duramycin - merged DIC repRNA

-TBSV Pex13- - nicymarud GFP degrem -TBSV -TBSV

Fig. 3. Enrichment of PE at TBSV replication sites in yeast and plant cells. (A) Confocal laser microscopy images show the enrichment of PE and its colocalization with the GFP-tagged TBSV p33 expressed from the GAL1 promoter during TBSV replication (top two images) or when only GFP-p33 was expressed. Differential interference contrast (DIC) images are shown on the right. Localization of PE is detected by using biotinylated duramycin peptide and streptavidin conjugated with Alexa Fluor 405. The bottom image shows the more even cellular distribution of PE in the absence of viral components. Note that the GFP- p33 is functional and fully supports TBSV replication in yeast. (B) Peroxisomal enrichment of PE in the presence of TBSV replication proteins. Peroxisomal MICROBIOLOGY membranes are visualized with the help of mRFP-tagged (top images) or GFP-tagged (middle images) yeast Pex13 protein. The bottom image shows the lack of PE enrichment in peroxisomes in the absence of viral components. See further details in A.(C) Enrichment and colocalization of PE with p33/p92 replication proteins in N. benthamiana protoplasts replicating TBSV genomic RNA. The TBSV p33/p92 replication proteins were detected with p33-specific primary antibody and secondary antibody conjugated with Alexa Fluor 488. The images at the bottom show the more even distribution of PE in the absence of viral components. Note that the infectious WT TBSV genomic RNA produced the natural (untagged) p33 and p92 in these experiments. See further details in A.

need to build new membrane-bound replicase complexes. Lip- vitro TBSV replicase assembly assay, the increased TBSV rep- idomics analysis revealed no significant changes in the fatty acid licase activity in the membrane fraction of cho2Δ CFE suggests length or saturation status of PE in yeast replicating TBSV that the high accumulation level of TBSV repRNA is due to versus the control yeast (Fig. S4B). enhanced replicase activity in cho2Δ yeast (Fig. 6A). The CFE- The relative PE level was also increased in TBSV-infected based assay with comparable amounts of purified TBSV repli- plant leaves from 21.2% to 28.8% (Fig. 5B). In contrast to yeast, cation proteins suggests that TBSV could build more VRCs the PC level also increased in plants from 37.8% to 55.4%. The when PE is abundant and it excludes that the increased PE level total phospholipid content was also increased in TBSV-infected in cho2Δ yeast enhances TBSV replication due solely to the pres- plant leaves by 20.6%, suggesting active phospholipid synthesis ence of higher amounts of replication proteins (Fig. 6A). Alto- occurring in infected cells. Overall, the lipidomics data support gether, these findings strongly support the stimulatory function of the increased synthesis of PE in TBSV-infected plant cells, high PE level on tombusvirus replicase assembly. similar to its yeast counterpart. Confocal microscopy analysis of cho2Δ yeast showed robust redistribution of PE to the sites of TBSV replication containing Increased PE Level in cho2Δ Yeast Promotes TBSV Replication. To the viral replication proteins and peroxisome membranes (Fig. examine if PE level can directly affect TBSV replication in 6D). Thus, similar to the WT yeast, PE becomes highly enriched cells, we deleted CHO2, which codes for PE methyltransferase at the viral replication sites in cho2Δ yeast. To test if the high (PEMT), in yeast. Cho2p catalyzes the first step in the conver- accumulation of PE in the peroxisome membranes is critical for sion of PE to PC, and in its absence, PE level is increased up to TBSV replication, we deleted the PEX3 peroxisome biogenesis 40–45%, whereas PC level is reduced down to 15–20% (24, 25). gene in cho2Δ yeast. In the absence of PEX3, there is no per- We find that TBSV replication is increased by ∼10-fold in cho2Δ oxisome or peroxisome membrane remnants in yeast cells (27, yeast in comparison with WT yeast (Fig. 6A, lanes 5–8 versus 28), and TBSV switches to the ER membranes to perform its 1–4). In addition, the amounts of p33 and p92pol replication pro- replication (29). ER membranes can support as robust a TBSV teins were also increased (Fig. 6A). Lipidomics analysis of cho2Δ replication as the peroxisomes in yeast (29–31). We find that yeast supporting TBSV replication showed that PE becomes the TBSV replication is increased by ∼13-fold in cho2Δpex3Δ yeast most abundant phospholipid by reaching up to the ∼42% level (Fig. S5), suggesting that TBSV can take advantage of increased (from ∼18%inWTyeastlackingTBSV)anda2.5-foldhigher PE level in the ER membrane in the absence of peroxisomes. We amount than in WT BY4741 yeast replicating TBSV (Fig. 6B). observed enrichment of PE (Fig. S5B) or NBD-PE (Fig. S5C)at We also performed in vitro TBSV replicase assembly assay in sites containing p33 in the ER membranes. These data highlight isolated membrane fractions from CFEs obtained from WT or the emerging scheme that PE enrichment at the site of replication cho2Δ yeasts (19, 26). The in vitro assembled viral replicase on is critical regardless of peroxisomal or ER localization of the membranes derived from cho2Δ yeast showed ∼threefold higher tombusvirus replicase. activity than comparable viral replicase from WT yeast (Fig. 6C). Because we used the same amounts of the purified recombinant Depletion of PE in Yeast Blocks TBSV Replication. To study if de- viral proteins and the soluble fraction from WT yeast in the in pletion of PE has a negative effect on TBSV replication, we

Xu and Nagy PNAS | Published online March 25, 2015 | E1785 Downloaded by guest on October 2, 2021 Pex13- NBD-PE RFP-p33 merged DIC Pex13- NBD-PC RFP-p33 merged DIC A BFP C BFP

+p33

90 %

30 D Pex13- BFP DMSO RFP-p33 merged DIC 10 B Pex13- - BFP NBD-PE merged DIC +p33

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Fig. 4. Redistribution and enrichment of exogenous PE in peroxisomes containing the p33 replication protein in yeast cells. (A and B) NBD-PE was added to yeast cultures expressing the TBSV RFP-p33 replication protein or lacking p33. The peroxisomes were visualized by expressing Pex13p-BFP. ImageJ software was used to show the enrichment of PE (green line) in peroxisomes (blue line) containing TBSV p33 (red line). (C) NBD-PC was added to yeast cultures expressing the TBSV RFP-p33 replication protein. See panel A for details. (D) Control panel to show colocalization of Pex13p and p33 in the absence of exogenous PE. DMSO solvent was added to yeast cultures.

generated a yeast strain with deletion of three genes involved in replication in yeast, whereas the PSD1, PSD2,andDPL1 genes are PE production—PSD1, PSD2, and DPL1 (32). Briefly, the de not directly involved in the replication process. novo synthesis of PE from PS was eliminated via deletion of the To further test the role of PE in TBSV replication, we pre- two phosphatidylserine decarboxylase genes, psd1Δ and psd2Δ, pared the membrane fraction from yeast with depleted PE whereas the Kennedy pathway was inhibited through deletion of content, followed by reconstituting the functional TBSV repli- dihydrosphingosine-1-phosphate, dpl1Δ, and exclusion of etha- case using purified recombinant p33 and p92 replication proteins, the viral (+)repRNA, and the soluble fraction from CFE of nolamine from the culture media (33). We found that TBSV ∼ psd1Δ/ WT yeast. Interestingly, the CFE membrane fraction with de- repRNA accumulation was inhibited by 7.5-fold in yeast ( pleted PE supported ∼eightfold less TBSV replication than the psd2Δ/dpl1Δ E ) with depleted PE content (Fig. 6 ,lanes3and4 CFE membrane fraction from WT yeast (Fig. 6F, lanes 3 and 4 psd1Δ/psd2Δ/ versus 1 and 2). Restoring PE production in yeast ( versus 1 and 2). Because only the membrane fractions were dpl1Δ) via the Kennedy pathway due to inclusion of ethanolamine different in these CFE-based experiments, it is likely that the in the culture media led to full recovery of TBSV replication (Fig. depleted PE level in psd1Δ/psd2Δ/dpl1Δ yeast membrane is re- 6E, lanes 7 and 8). Thus, PE seems to be required for TBSV sponsible for the poor TBSV replicase assembly/activity in vitro.

A TBSV-replication in yeast B TBSV-replication in N. benthamiana

% % 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 PPC PE PI SGP PPC PE PI SGP lyso- lyso- PC PE

Fig. 5. Increased level of PE in yeast and plant cells replicating TBSV. Relative levels of phospholipids in yeast (24 h time point, in A) and plants (from systemic leaves showing symptoms, 6 d after infection, in B) replicating TBSV RNA (black columns) or the TBSV-free control (gray columns) were determined using mass spectrometry analysis.

E1786 | www.pnas.org/cgi/doi/10.1073/pnas.1418971112 Xu and Nagy Downloaded by guest on October 2, 2021 PNAS PLUS A TBSV-replication in peroxisomes B phospholipid content (relative) C recombinant 50 p33/p92 + WT S40 WT cho2Δ template 45 RNA 40 35 membrane fraction RNA repRNA 30 WT or cho2Δ synthesis 25 +ATP/GTP CTP/32P-UTP 20 WT cho2Δ 1 2 3 4 5 6 7 8 15 100±8 1014±41 % repRNA 10 5 repRNA 0 rRNA % PC PE PSPI PG 1 2 3 4 phospholipid content (absolute) 100±16 278±14 % p92 total membrane 100 protein p33 90 80 D cho2Δ total 70 duromycin GFP-p33 merged 60 50 E TBSV-replication 40 30 +p33/ - + ethanolamine 20 p92 psd1Δ psd1Δ 10 repRNA psd2Δ psd2Δ WT dpl1Δ WT dpl1Δ 0 % PC PE PSPI PG Pex13- repRNA F in vitro replication YFP-p33 merged - + ethanolamine CFP 1 2 3 4 5 6 7 8 psd1Δ psd1Δ psd2Δ psd2Δ +p33/ 100±12 15±8 154±35 162±38 % repRNA WT dpl1Δ WT dpl1Δ p92 repRNA

rRNA MICROBIOLOGY repRNA p92 1 2 3 4 5 6 7 8 p33 100±1 13±1 95±12 111±4 % repRNA total membrane total protein

Fig. 6. Deletion of the CHO2 PEMT gene enhances TBSV repRNA accumulation in yeast. (A, Top) Replication of the TBSV repRNA in WT and cho2Δ yeast was measured by Northern blotting 24 h after initiation of TBSV replication. Yeast coexpressed the TBSV p33 and p92 replication proteins with the DI-72 (+)repRNA. The accumulation level of repRNA was normalized based on the ribosomal RNA (rRNA). Each sample is obtained from different yeast colonies.

(A, Middle and Bottom) The accumulation levels of His6-p92 and His6-p33 were tested by Western blotting. Note that in the absence of Cho2p, which catalyzes the first step in the conversion of PE to PC, the PE level is increased. Each experiment was repeated at least three times. (B) Relative and absolute levels of phospholipids in cho2Δ (red columns) versus WT yeasts (blue columns, measured at 24 h time point) replicating TBSV RNA were determined using mass spectrometry analysis. (C) Enhanced TBSV repRNA replication in CFE prepared from cho2Δ yeast. Shown is the scheme of the CFE-based TBSV replication assay. Purified recombinant TBSV p33 and p92pol replication proteins, DI-72 (+)repRNA in combination with the soluble fraction (S40 fraction from WT yeast), were added to the membranous fraction (P40) of cho2Δ or WT CFEs. Denaturing PAGE analysis of the 32P-labeled repRNA products obtained is shown. The full- length single-stranded repRNA is denoted by an arrow. (D) Confocal laser microscopy images show the enrichment of PE at peroxisomal sites of TBSV p33 accumulation in cho2Δ yeast. See further details in Fig. 3. (E) Depletion of PE in yeast inhibits TBSV replication. PE production was inhibited in a yeast strain (psd1Δ/psd2Δ/dpl1Δ) due to the exclusion of ethanolamine in the culture media (“– ethanolamine” samples). Restoring PE production in yeast (psd1Δ/psd2Δ/ dpl1Δ) via the Kennedy pathway due to inclusion of ethanolamine in the culture media led to full recovery of TBSV replication. See further details in panel A. (F) Poor TBSV repRNA replication in CFE prepared from yeast with a depleted PE level. Purified recombinant TBSV p33 and p92pol replication proteins, DI-72 (+)repRNA in combination with the soluble fraction (S40 fraction from WT yeast), were added to the membranous fraction (P40) from a triple mutant (psd1Δ/ psd2Δ/dpl1Δ) or WT yeasts. Note that omission of ethanolamine in the culture media (– ethanolamine samples) leads to PE depletion in yeast (psd1Δ/psd2Δ/ dpl1Δ), whereas inclusion of ethanolamine in the culture media (“+ ethanolamine” samples) leads to PE production. Denaturing PAGE analysis of the 32P- labeled repRNA products obtained is shown. See further details in panel C.

As discussed above, the CFE-based assay with comparable in yeast cells (Fig. S6B). In contrast, depletion of the PE level in amounts of purified TBSV replication proteins suggests that psd1Δ/psd2Δ/dpl1Δ yeast resulted in less than 10% CNV replication TBSV could build less VRCs when PE is depleted in subcellular (Fig. S6C, lanes 3 and 4 versus 1 and 2). Thus, the proviral role of membranes. The CFE-based assay also excludes that the low PE PE seems to be similar for TBSV and CNV. level in psd1Δ/psd2Δ/dpl1Δ yeast inhibits TBSV replication due To study if viruses replicating in other subcellular compart- solely to the presence of reduced amounts of replication proteins ments could take advantage of the increased PE level in cho2Δ (Fig. 6E). Altogether, these findings strongly support that the PE yeast, we used Carnation Italian ringspot virus (CIRV, a tom- level plays a direct role in tombusvirus replicase assembly. busvirus), which replicates in the outer mitochondrial membranes (26, 34). CIRV accumulation is increased by ∼fivefold in PE Level Also Affects Replication of Other Tombusviruses and the cho2Δ yeast (Fig. 7A). Moreover, the p36 replication protein of Insect Nodamura Virus in Yeast. To test if the proviral role of PE CIRV induced the efficient enrichment of PE in the same sub- also extends to other viruses, we analyzed replication of the closely cellular locations that harbor p36 (Fig. 7B). Depletion of the PE related Cucumber necrosis tombusvirus (CNV), which also repli- level in psd1Δ/psd2Δ/dpl1Δ yeast reduced CIRV replication to cates on peroxisomal membranes. Similar to TBSV, CNV replica- ∼10% of that found in WT yeast (Fig. 7C, lanes 3 and 4 versus 1 tion was increased in cho2Δ yeast (Fig. S6A)andtheexpressionof and 2), confirming the essential role of PE in CIRV replication. CNV p33 replication protein alone induced the relocalization of PE Replication of another mitochondrial RNA virus, the unrelated

Xu and Nagy PNAS | Published online March 25, 2015 | E1787 Downloaded by guest on October 2, 2021 Nodamura virus (NoV) insect RNA virus (35), also benefitted replicase assembly based on artificial vesicles that facilitate from the increased PE level in cho2Δ yeast (Fig. 7D). Interestingly, TBSV replication only when PE is present above 70%, (ii) the protein A replication protein of NoV is localized at highly viral replication protein induces a relative increase in PE level at PE-enriched subcellular locations (Fig. 7E). Depletion of the PE level the expense of other phospholipids in yeast and plant cells rep- reduced NoV replication by ∼sixfold in psd1Δ/psd2Δ/dpl1Δ yeast licating TBSV, (iii) highly localized enrichment of PE in the (Fig. 7F, lanes 3 and 4 versus 1 and 2), further supporting a pro- peroxisomal membranes (the sites of tombusvirus replication) to viral role of PE in NoV replication. Therefore, we suggest that promote tombusvirus replication, (iv) cell-based results showing replication of different tombusviruses and NoV in peroxisomal or increased TBSV replication in cho2Δ yeast that contains a high level mitochondrial subcompartments depends on the PE level in yeast. of PE due to a defect in conversion of PE into PC, (v) a yeast strain Similar to TBSV, the replication proteins of these viruses can with depleted PE level supports low-level viral replication, and likely induce the efficient enrichment of PE at the sites of virus (vi) in vitro CFE-based assay to reconstitute TBSV replicase shows replication, suggesting that different RNA viruses use active, albeit enhanced replicase activity in a highly PE-enriched membrane yet unraveled, mechanisms to create a PE-enriched microenvi- fraction from cho2Δ yeast and poor replicase activity when a ronment in infected cells. PE-depleted yeast membrane fraction is used.

Discussion Essential Role of PE in TBSV VRC Assembly. Based on in vitro PE Is an Essential Host Factor Subverted for TBSV Replication. It is approaches, PE plays a major function during the assembly of universally accepted that plant and animal positive-strand RNA the VRCs that synthesize the viral RNAs. We show that active viruses require cellular membranes for their propagation in VRCs are only formed and functional in the presence of artificial infected cells (1–4). These viruses replicate in various subcellular PE vesicles, whereas other phospholipids are insufficient to compartments that contain a unique composition of lipids. support efficient VRC assembly. Moreover, other phospholipids However, it is currently poorly understood how different lipids are inhibitory to active VRC assembly when present in 20% could affect the viral replication process. By using the highly or higher concentration in artificial vesicles. In addition, a CFE tractable tombusviruses, we show that PE is a coopted host factor membrane fraction prepared from cho2Δ yeast, which contains that plays an essential role in viral replication. The supporting a high PE level, supports greatly enhanced TBSV replication evidence includes (i) the requirement of PE for in vitro viral in vitro, suggesting more efficient VRC assembly when PE is

A CIRV-replication in mitochondria B C CIRV-replication WT cho2Δ duramycin GFP-p36 merged DIC - + ethanolamine psd1Δ psd1Δ psd2Δ psd2Δ WT WT repRNA dpl1Δ dpl1Δ +p36/ p95 1 2 3 4 5 6 7 8 repRNA repRNA 100±23 513±30% repRNA 1 2 3 4 5 6 7 8 rRNA 100±7 9±4 321±68 351±66 % repRNA

p95 rRNA +p36 p95 p36 p36 total total

D NoV-replication in mitochondria E F NoV-replication WT cho2Δ Anti-FLAG - + ethanolamine duramycin Alexa488 merged DIC psd1Δ psd1Δ psd2Δ psd2Δ RNA1 WT dpl1Δ WT dpl1Δ

RNA1

+FLAG -protA +RNA1 RNA3 RNA3 1 2 3 4 5 6 1 2 3 4 5 6 7 8 ± ± 100±46 16±4 501±141 425±86 % RNA1 100 37 604 119% RNA1 ± ± ± ± 100±14 388±9 % RNA3 100 15 46 22 329 56 275 26 % RNA3 rRNA rRNA

Fig. 7. Increased PE level facilitates CIRV and NoV RNA accumulation in cho2Δ yeast. (A, Top) Replication of CIRV repRNA in WT and cho2Δ yeast was measured by Northern blotting 24 h after initiation of CIRV replication. Yeast coexpressed the CIRV p36 and p95 replication proteins with the (+)repRNA.

(A, Middle and Bottom) The accumulation levels of His6-p95 and His6-p36 were tested by Western blotting. Each experiment was repeated. (B) Confocal laser microscopy images show the enrichment of PE at mitochondrial sites of CIRV p36 accumulation in WT yeast. See further details in Fig. 3. (C) Depletion of PE in yeast (psd1Δ/psd2Δ/dpl1Δ) inhibits CIRV replication. See further details in Fig. 6E.(D) Replication of NoV RNA1 and RNA3 in WT and cho2Δ yeast was measured by Northern blotting 24 h after initiation of NoV replication. (E) Confocal laser microscopy images show the enrichment of PE at mitochondrial sites of NoV Flag-tagged protA replication protein accumulation in WT yeast. See further details in Fig. 3. (F) Depletion of PE in yeast (psd1Δ/psd2Δ/dpl1Δ) inhibits NoV replication. See further details in Fig. 6E.

E1788 | www.pnas.org/cgi/doi/10.1073/pnas.1418971112 Xu and Nagy Downloaded by guest on October 2, 2021 abundant in membranes. In contrast, the CFE-based assay using to increase the PE level via new PE/phospholipid synthesis, as PNAS PLUS yeast membranes with depleted PE is remarkably inefficient in shown by lipidomics data from yeast and plant cells. Then, the supporting TBSV replication in vitro. newly synthesized PE molecules are likely subverted for TBSV Interestingly, PE does not seem to be essential at the very replication in a currently unknown manner. Another way to in- early steps of replication (before the VRC assembly), because crease the local concentration of PE is to redistribute PE from the TBSV p33 replication protein can associate not only with PE various subcellular membranes to the site of replication. Indeed, but other phospholipids too. Also, PC is even more efficient than confocal microscopy images show the robust accumulation of PE PE for the p33/p92-driven recruitment of the viral (+)repRNA to at peroxisomal sites where TBSV p33 replication protein accu- artificial membranes in vitro (Table 1). After the initial binding mulates (to form VRCs). Interestingly, PE molecules (presented to the membranes, p33 will likely induce the local enrichment of as NBD-PE) provided in the yeast culture media found their ways PE that is required for VRC assembly. In addition, binding of to the p33-containing peroxisomal membrane sites, suggesting that p33/p92 to PE might stabilize the replication proteins, because PE is efficiently redistributed to the sites of TBSV replication we observed elevated levels of p33/p92 in cho2Δ yeast and de- from the preexisting cellular PE pool. Overall, it seems that TBSV creased levels of replication proteins in psd1Δ/psd2Δ/dpl1Δ yeast induces cellular PE synthesis as well as subcellular redistribution in comparison with WT yeast. We have shown previously that of PE, resulting in a PE-enriched microenvironment, which serves phospholipids are important for p33/p92 stability in yeast (17). the virus’s need during VRC assembly. In contrast to the preassembly of p33/p92 and the viral + (+)RNA in membranes, for which PE might not be essential, we A Wide-Spread Role of PE in ( )RNA Virus Replication? Because + find that PE is absolutely critical for the final assembly of the many ( )RNA viruses build vesicle-like structures for replication TBSV VRC. Accordingly, the development of artificial vesicle- that requires membrane deformation and negative membrane based TBSV replication unambiguously demonstrates that TBSV curvature (1, 3, 4), it is possible that these viruses also depend on requires PE for active VRC assembly and viral RNA synthesis. local enrichment of PE at the replication sites. We directly tested the role of PE in replication of several (+)RNA viruses by using TBSV replicase assembled on the PE vesicles could support a cho2Δ complete cycle of RNA replication, including (–)- and (+)-RNA yeast, which lacks one of the PEMTs to convert PE to PC synthesis in an asymmetrical manner, producing ∼10 times more (25), with an especially high cellular PE level. Interestingly, the (+)-strands than (–)-strands. Asymmetrical replication of the TBSV-related CNV (peroxisomal replication) and CIRV (mitochondrial replication) and the unrelated NoV (mitochondrial RNA genome is one of the hallmarks of (+)-strand RNA viruses.

replication) all supported enhanced replication when PE is MICROBIOLOGY However, optimal TBSV replication also requires additional abundant in membranes of cho2Δ yeast. Moreover, PE was phospholipids, because the highest level of TBSV RNA synthesis shown to become highly enriched at the sites of viral replication was observed with vesicles containing ∼15% additional phos- protein accumulation, which represent VRCs for these viruses. pholipids and ∼85% PE. Also, “forcing” TBSV to switch to ER membranes in the absence In comparison with yeast CFE, which supports twofold more of peroxisomes in cho2Δ yeast (due to the pex3Δ background) efficient TBSV replication than PE vesicles, the artificial PE still resulted in efficient TBSV replication, suggesting that tom- vesicles cannot fully protect the viral RNA from nucleases during busviruses could take advantage of abundant PE in various in vitro RNA synthesis, suggesting that PE is not sufficient to subcellular membranes. In summary, the emerging picture from allow the formation of complete authentic TBSV VRCs. It is our work is that various (+)RNA viruses could subvert cellular possible that sterols and coopted cellular proteins in combina- PE to build VRCs in the PE-enriched microenvironment, leading tion with additional phospholipids are also needed for the for- to efficient viral replication in infected cells. mation of spherule structures (vesicles with narrow neck-like In addition to the above presented critical role for PE in openings), which are the characteristic replication structures for tombusvirus and NoV VRC assembly and replication, other – – TBSV (36 38) and many other RNA viruses (1 4). We suggest phospholipids could also be exploited by plus-strand RNA that the subverted PE molecules, due to their conical molecular viruses. For example, PC biosynthesis is increased in poliovirus- structures, facilitate the formation of spherules by introducing infected cells, leading to formation of replication organelles from a negative curvature into lipid bilayers. Indeed, these TBSV- newly made lipids (39, 40). Inhibition of PC biosynthesis in induced negatively deformed membranes are visualized by elec- Drosophila cells reduced the replication of Flock house virus + tron microscopy in yeast cells missing the ESCRT Vps4p AAA (FHV) (41). However, the absence of direct binding between the ATPase protein (37), which is required for spherule formation. FHV replication protein and PC complicates the interpretation of the PC role in the replication process (41). Interestingly, TBSV Builds a PE-Enriched Microenvironment for Replication. Based Drosophila cells contain an unusually high level of PE (41), which on TBSV replication studies with artificial vesicles, the emerging might naturally facilitate FHV replication. A template-depen- picture is that TBSV requires a high local concentration of PE at dent RNA-dependent RNA polymerase extracted from FHV- the sites of replication (above 70%; Fig. 1). However, PE is infected cells was stimulated by phospholipids, including PC and below that level in subcellular membranes, such as peroxisomes, PE (42), suggesting that phospholipids are important for FHV in the virus-free stage. Therefore, TBSV likely stimulates the cell replication in vitro. However, the precise function of PC in replication or VRC assembly in the case of poliovirus or FHV is currently unknown. Table 1. Role of various phospholipids in TBSV replication Vesicles Replication p33 membrane association RNA recruitment Experimental Procedures In Vitro TBSV Replication Assay Using Artificial Phospholipid Vesicles. The ++ + PE procedure for in vitro replication assay using phospholipid vesicles was PC – ++adapted from the previously published procedure using purified yeast PG – + – organelles (26), except that the 100,000 × g supernatant (S100) was replaced PS – + – with the S40 fraction of CFE. Briefly, 2 μL of phospholipid vesicles and 1 μL PI –– –of the S40 fraction were incubated at 25 °C for 1 h in 8 μL buffer containing CA – + – 30 mM Hepes-KOH (pH 7.4); 150 mM potassium acetate; 5 mM magnesium lysoPE – ++acetate; 0.6 M sorbitol; 15 mM creatine phosphate; 1 mM ATP, CTP, and GTP; 0.025 mM UTP; 0.1 μL[32P]UTP; 0.1 mg/mL creatine kinase; 0.1 μL RNase lysoPC – + – inhibitor (Thermo Scientific); 10 mM DTT; 0.5 μg DI-72 RNA transcript; and

Xu and Nagy PNAS | Published online March 25, 2015 | E1789 Downloaded by guest on October 2, 2021 0.5 μg MBP-tagged recombinant TBSV p33 and p92 replication proteins. 6 min and acetone for 30 s, respectively, at –20 °C. Biotinylated duramycin Then, the reaction mix was incubated for 3 h in 16 μL cell-free replication was added into PBS (pH 7.4) containing 0.05% Nonidet P-40 and 1% BSA buffer B (30 mM Hepes-KOH pH 7.4, 150 mM potassium acetate, 5 mM (15 μg/mL) and incubated overnight with the fixed cells at –4 °C. Slides were magnesium acetate) with 15 mM creatine phosphate; 1 mM ATP, CTP, and washed and incubated with Streptavidin conjugated with Alexa Fluor 405 GTP; 0.025 mM UTP; 0.2 μL[32P]UTP; 0.1 mg/mL creatine kinase; 0.2 μL RNase (Life Technologies) for 1 h before imaging. In each experiment, ∼20 cells inhibitor; 10 mM DTT; and 0.05 mg/mL actinomycin D. After reaction, total were observed under the confocal laser microscope. RNA was extracted and analyzed in a denaturing gel. Determination of the The distribution of PE was also monitored using fatty acid-labeled NBD-PE + – viral ( )RNA/( )RNA ratio as well as micrococcal nuclease treatment was and NBD-PC internalization. M-C6–NBD-PE [1-myristoyl-2-(6-[(7-nitro-2–1,3- described previously (26). benzoxadiazol-4-yl)amino]hexanoyl)-sn-glycero-3-phosphoethanolamine] and

M-C6–NBD-PC [1-myristoyl-2-(6-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino] Membrane Flotation Assay. [35S]methionine-labeled TBSV p33 (43) was in- hexanoyl)-sn-glycero-3-phosphocholine] (Avanti Polar lipids, Inc.) were dis- cubated with different artificial phospholipid vesicles as previously described solved in DMSO in 8 mM concentration (45) and stored at –20 °C. WT yeast using purified yeast organelles (26). The membrane flotation assay was transformed with pESC-mRFP-T33 and pRS315-Pex13p-BFP was pregrown in SC performed as previously described (44) with minor modifications. Briefly, the medium, then cultured in SC medium containing 2% galactose with 80 μM reaction mixture (24 μL) was mixed with 126 μL 85% (wt/vol) sucrose in NBD-PE or NBD-PC at 0.5 OD600, and incubated at 23 °C for 16 h. Cells were Hepes buffer in a final concentration of 71.25%, then overlaid with 900 μL washed with SCNaN3 medium (45) (SC medium with 2% sorbitol and 20 mM 65% (wt/vol) sucrose and 150 μL 10% (wt/vol) sucrose in Hepes buffer (30 mM, sodium azide) and subjected to confocal laser microscope analysis. pH 7.4). The gradient was centrifuged at 134,000 × g for 16 h at 4 °C in a N. benthamiana protoplasts were prepared and eletroporated with in swinging bucket rotor (Beckman TLS-55). vitro-transcribed TBSV full-length genomic RNA as described previously (46). Protoplasts were fixed with 3.7% formaldehyde in protoplast culture me- TBSV RNA Recruitment Assay. For the viral RNA recruitment assay, artificial dium (46), applied to poly-L-lysine–coated slides, and processed using the phospholipid vesicles were mixed with MBP-tagged recombinant TBSV p33 above procedure for PE staining. For dual staining of p33 and PE, the anti- and p92 replication proteins (0.5 μg each) as in the in vitro replication assay, p33 primary antibody was diluted (1:400) and incubated with fixed proto- except that UTP was omitted from the reaction mixture, and 1 μg/μL yeast plasts in PBS containing 1% BSA/0.05% Nonidet P-40 overnight. After tRNA was added as a nonspecific competitor and 1 μL radioactive [32P]UTP- washing three times with PBS/1% BSA/0.05% Nonidet P-40, cells were in- labeled DI-72 RNA transcripts was added. After 1 h incubation at 25 °C, the cubated with anti-mouse secondary antibody conjugated to Alexa Fluor 488 reaction mixtures were subjected to membrane flotation assay in sucrose (Life Technologies) for 1 h before imaging. gradients as described above. Total RNA from the top fraction of each Confocal images were obtained with an Olympus FV1000 microscope gradient was extracted and analyzed in a denaturing RNA gel (5% poly- (Olympus America). BFP/Alexa 405, GFP/Alexa 488, and RFP were excited using acrylamide gel containing 8 M urea). 405 nm, 488 nm, or 543 nm lasers, respectively. Images were obtained se- quentially and merged using Olympus FLUOVIEW 1.5 software. Relative Imaging of PE Distribution and Viral Protein Localization in Yeasts and Plant fluorescence intensity was estimated by ImageJ software and analyzed fur- Protoplasts. Yeast cultures were grown in glucose-containing media over- ther using Microsoft EXCEL software.

night and switched to galactose-containing media with an initial 0.3 OD600. To prepare spheroplasts, overnight cultures were harvested and the yeast ACKNOWLEDGMENTS. The authors thank Dr. Herman B. Scholthof (Texas cells were fixed with 3.7% formaldehyde for 40 min at room temperature in A&M) for the the anti-p33 primary antibody. This work was supported by the dark, washed twice with 0.1 M potassium phosphate (pH 7.5), and then National Science Foundation Grant MCB-1122039 (to P.D.N.). The lipid analyses described in this work were performed at the Kansas Lipidomics Research resuspended in SPP (0.1 M potassium phosphate pH 7.5, 1.2 M sorbitol) with Center Analytical Laboratory. The Kansas Lipsidomics Research Center was sup- zymolase 20T (1 mg/mL). Cells were incubated at 30 °C for 1 h, and then ported by National Science Foundation Grants EPS 0236913, MCB 0920663, DBI incubated with SPP with 50 mM NH4Cl for 15 min to quench free aldehyde 0521587, and DBI 1228622; the Kansas Technology Enterprise Corporation; groups. Spheroplasts were collected after washing twice with SPP and then K-IDeA Networks of Biomedical Research Excellence of the National Insti- applied to poly-L-lysine–coated slides. Slides were immersed in methanol for tutes of Health Grant (P20GM103418); and Kansas State University.

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