Positive-Strand RNA Viruses Stimulate Host Phosphatidylcholine Synthesis at Viral Replication Sites

Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

Jiantao Zhanga,1, Zhenlu Zhanga,b, Vineela Chukkapallic, Jules A. Nchoutmboubed, Jianhui Lia, Glenn Randallc, George A. Belovd, and Xiaofeng Wanga,2

aDepartment of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061; bDepartment of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, People’s Republic of China; cDepartment of Microbiology, The University of Chicago, Chicago, IL 60637; and dVirginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved January 12, 2016 (received for review October 6, 2015) All positive-strand RNA viruses reorganize host intracellular mem- expression of 1a alone in yeast induces spherule formation (6), branes to assemble their viral replication complexes (VRCs); how- which requires 1a’s amphipathic α-helix (1a amino acids 392–407) ever, how these viruses modulate host lipid metabolism to (8), helix A, and 1a–1a interactions (9, 10). In addition, several accommodate such membrane proliferation and rearrangements is host proteins, including membrane-shaping reticulons (RTNs) not well defined. We show that a significantly increased phospha- (11) and an ESCRT (endosomal sorting complex required for tidylcholine (PC) content is associated with brome mosaic virus transport) component, Snf7p (sucrose nonfermenting7) (12), are (BMV) replication in both natural host barley and alternate host recruited by 1a to form spherules. yeast based on a lipidomic analysis. Enhanced PC levels are primarily Similar to other (+)RNA viruses, BMV promotes host lipid associated with the perinuclear ER membrane, where BMV replica- synthesis and requires balanced lipids for the formation and action takes place. More specifically, BMV replication protein 1a interacts tivity of VRCs. Expression of 1a in yeast induces a ∼30% increase with and recruits Cho2p (choline requiring 2), a host enzyme in- in total fatty acids (FAs) per cell (13). BMV also requires a high volved in PC synthesis, to the site of viral replication. These results level of unsaturated FA (UFA) because a ∼12% decrease of suggest that PC synthesized at the site of VRC assembly, not the UFAs in the yeast ole1w mutant blocks its replication more than transport of existing PC, is responsible for the enhanced accumula- 20-fold (13). In addition, deleting host ACB1 (Acyl-CoA-binding 1) tion. Blocking PC synthesis by deleting the CHO2 gene resulted in gene results in formation of spherules that are smaller in size VRCs with wider diameters than those in wild-type cells; however, but are in greater number than in wild-type (WT) cells (14). BMV replication was significantly inhibited, highlighting the critical ACB1 encodes acyl-CoA binding protein, which binds long-chain role of PC in VRC formation and viral replication. We further show fatty acyl-CoAs and is involved in maintaining lipid homeostasis. that enhanced PC levels also accumulate at the replication sites of Supplemented long-chain UFAs largely complement the BMV hepatitis C virus and poliovirus, revealing a conserved feature among replication defects in cells lacking ACB1, indicating that the a group of positive-strand RNA viruses. Our work also highlights a altered lipid composition is primarily responsible for BMV repli- potential broad-spectrum antiviral strategy that would disrupt PC cation defects (14). synthesis at the sites of viral replication but would not alter Cellular membranes are mainly composed of phospholipids, and cellular processes. in particular, phosphatidylcholine (PC) constitutes ∼50% of total phospholipids (15). PC is synthesized via the CDP–DAG (cytidine positive-strand RNA viruses | viral replication complexes | diphosphate–diacylglycerol) and Kennedy pathways in eukaryotes virus–host interactions | virus control | phospholipids (16). PC synthesis is significantly enhanced during infection of

ll positive-strand RNA viruses [(+)RNA viruses], which in- Significance Aclude numerous important human, animal, and plant pathogens, share similar strategies for genomic replication. A highly Positive-strand RNA viruses [(+)RNA viruses] include many im- conserved and indispensable feature of their replication is the portant human, animal, and plant pathogens. A highly con- proliferation and reorganization of host cellular membranes to as- served and indispensable feature of (+)RNA virus infection is semble viral replication complexes (VRCs). Despite this cen- that these viruses proliferate and reorganize host membranes tral importance, it is largely unknown how cellular membranes to assemble viral replication complexes (VRCs). We show that are rearranged by the viral replication proteins and how cellular brome mosaic virus (BMV) stimulates phosphatidylcholine (PC) lipid metabolism is modulated to accommodate membrane pro- synthesis at the viral replication sites. BMV recruits a host en- liferation and remodeling. zyme involved in PC synthesis to support proper VRC formation Brome mosaic virus (BMV) serves as a model for understanding and genomic replication. We further show that hepatitis C virus VRC formation of (+)RNA viruses (1). BMV is the type member and poliovirus also promote accumulation of PC at the viral of the plant virus family Bromoviridae and a representative member replication sites, revealing a feature common to a group of of the alphavirus-like superfamily, which includes many human, (+)RNA viruses. This virus-specific step can be targeted to de- animal, and plant-infecting viruses (2). BMV encodes two repli- velop a broad-spectrum antiviral strategy with the least side cation proteins, 1a and 2apol.2apol serves as the replicase, whereas effects on host growth. 1a has an N-terminal methyltransferase domain (3, 4) and a C-terminal ATPase/helicase-like domain (5). Together, 1a and Author contributions: J.Z., G.R., G.A.B., and X.W. designed research; J.Z., Z.Z., V.C., J.A.N., and pol J.L. performed research; X.W. coordinated the research; J.Z., Z.Z., V.C., J.A.N., J.L., G.R., G.A.B., 2a are necessary and sufficient for BMV replication. BMV and X.W. analyzed data; and J.Z., G.R., G.A.B., and X.W. wrote the paper. Sac- induces vesicular structures in its surrogate host, the yeast The authors declare no conflict of interest. charomyces cerevisiae ,anditsnaturalhost,barley(6,7).These This article is a PNAS Direct Submission. structures, termed spherules, have been shown to be the VRCs in 1 pol Present address: Department of Pharmacology and Toxicology, The University of Ari- yeast as 1a, 2a , and nascent viral RNAs reside in the interior zona, Tucson, AZ 85721. of these compartments. Spherules are invaginations of the outer 2To whom correspondence should be addressed. Email: [email protected]. perinuclear endoplasmic reticulum (ER) membrane into the ER This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lumen and are about 60–80 nm in diameter (6). Remarkably, 1073/pnas.1519730113/-/DCSupplemental.

E1064–E1073 | PNAS | Published online February 8, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1519730113 Downloaded by guest on September 28, 2021 Dengue virus (DENV) (17), Flock House virus (FHV) (18), and the 1a signal (∼60% of the population), PC colocalized with 1a in PNAS PLUS poliovirus (19, 20). A 70% and 35% increase of total PC levels >92% of cells. was recorded in DENV-infected mosquito cells (17) and FHV- PE is the substrate of PC in the CDP–DAG pathway, and in infected Drosophila cells (18), respectively. At the peak time of addition, PE is enriched at the viral replication sites of several poliovirus replication in HeLa cells, a ∼37% increase of PC (+)RNA viruses (24). To determine whether PE distribution is content was observed after a 30-min chase (19). It was further affected by BMV, we used a PE biosensor, duramycin peptide found that poliovirus promotes the import of FAs, which were (25). In the absence of BMV, PE is predominantly present in the subsequently channeled to the viral replication sites. In addition, cytoplasm. We found that a portion of PE signal, albeit very FAs were mainly incorporated into PCs (20). weak, colocalized with that of 1a in the presence of BMV rep- One critical question based on the aforementioned research is lication (Fig. S2). whether the enhanced PC is synthesized in association with the VRCs or elsewhere in cells and subsequently transported into BMV Promotes PC Accumulation at the Site of Viral Replication in the VRCs. If PC is produced in association with the VRCs, what Plants. To verify that BMV promotes an increase in PC levels key enzymes are recruited? We report here that several (+)RNA during natural infection, we analyzed phospholipid compositions viruses, including BMV, hepatitis C virus (HCV), and poliovirus, in mock- and BMV-inoculated barley systemic leaves at 7 d post- promote significantly enhanced accumulation of PC content at inoculation (dpi), a time when the systemic leaves started to show the viral replication sites, revealing a common feature of viral symptoms. Similar to BMV-replicating yeast cells, the amount replication among a group of (+)RNA viruses. We further dem- of total phospholipid and PC increased by ∼24% and ∼29%, re- onstrate that BMV 1a interacts with and redistributes the host spectively, in BMV-infected plants relative to mock-inoculated enzyme, Cho2p (choline requiring 2), to the viral replication sites. plants (Fig. 1E). In cell wall-free protoplasts prepared from mock- As Cho2p converts phosphatidylethanolamine (PE) to PC in the inoculated leaves, the PC signal was evenly distributed throughout CDP–DAG pathway, the relocalization of Cho2p suggests the the cytoplasm but was excluded from the nucleus (Fig. 1F). A more VRC-localized PC synthesis. Deleting CHO2 inhibits BMV rep- intense PC signal was occasionally detected surrounding the nu- lication up to 30-fold and results in formation of spherules that are cleus (Fig. 1F, Top Left). Agreeing with previous reports showing larger than those of WT cells. This work highlights the importance that BMV 1a, 2apol, and nascent viral RNAs reside in the perinuclear of PC in VRC formation and the possibility of developing a novel region in barley protoplasts (26), the 1a signal surrounded the nu- and broad-spectrum antiviral strategy by specifically disrupting PC cleus in the majority of BMV-infected cells (Fig. 1F). Similar to yeast synthesis at the viral replication sites but not general PC synthesis. cells, the majority of the PC signal in BMV-infected barley cells colocalized with that of 1a in the perinuclear ER (Fig. 1F). Thus, Results enhanced PC accumulation at the sites of BMV viral replication BMV Specifically Promotes PC Accumulation at the Site of Viral occurred both in yeast and plant host cells. Replication in Yeast. To determine whether phospholipid synthesis is modulated during BMV replication, total lipids were extracted BMV 1a Stimulates Perinuclear Accumulation of the Host PC Synthesis from yeast cells in the absence or presence of BMV replication Enzyme Cho2p. We next tested whether BMV has the ability to and quantified by electrospray ionization tandem mass spec- modulate the distribution of enzymes involved in the production trometry. In cells with BMV replication, the molar amount of of PC. PC is synthesized via two pathways in eukaryotes: the total phospholipids and PC increased 19% and 28%, respectively, CDP–DAG and Kennedy pathways (Fig. 2A) (27). In routine ex- compared with cells without BMV replication (Fig. 1 A and B). perimental settings with no choline present in the growth medium, The 28% increase in PC content (P < 0.05) was comparable to the PC is primarily synthesized via the CDP–DAG pathway in yeast via 35% and 37% increase in PC content seen during FHV (18) and three sequential methylations of the amino head group of PE (Fig. poliovirus (19) infections, respectively, but lower than the 70% 2A). The two enzymes catalyzing the conversion of PE to PC are increase detected during DENV infection in mosquito cells (17). Cho2p, the PE methyltransferase, and Opi3p, the phospholipid In addition, the amount of phosphatidylserine (PS) increased N-methyltransferase (Fig. 2A). Cho2p converts PE to Phosphati- by 40%, whereas phophatidylglycerol (PG) increased by 56% dylmonomethylethanolamine (PMME) by transferring a single (Fig. 1A). Conversely, a nearly twofold decrease in phospha- methyl moiety to PE, and Opi3p adds two extra methyl groups tidic acid (PA) levels was identified (Fig. 1A). The compositional to PMME to produce PC (Fig. 2A). In the presence of 1a or BMV changes of phospholipid species are shown in Fig. S1. Based on replication, we observed a significant increase in Cho2p protein the molar percentage of individual phospholipids (the percentage levels (Fig. 2B), suggesting that 1a is able to directly or indirectly of molar amount of each lipid against total phospholipids), the promote the expression of certain lipid synthesis enzymes. increase in PC levels due to BMV RNA replication was ∼8% (Fig. Cho2p localizes to ER membranes, both to the peripheral ER 1C), which is subtle but statistically highly significant (P < 0.01). (the larger outer ring in Fig. 2C) and perinuclear ER (the smaller However, because the components necessary for BMV RNA inner ring). This localization pattern of Cho2p is similar to that replication were expressed from plasmids, the changes in PC of Dpm1p, another ER protein, but differs from that of Rtn1p, content are likely underestimated, as plasmids are lost at a 20% which localizes mainly to the peripheral ER (Fig. 2C). In the rate due to segregation inefficiency during mitosis (21). presence of 1a, Cho2p was depleted from the peripheral ER and We next wanted to determine if the increase in PC levels was enriched in the perinuclear ER (Fig. 2C). The ratio of Cho2p global or specifically associated with the sites of viral replication. signal in the perinuclear ER against that of the whole cell (Fp/Ft) To do this, we used a PC-specific monoclonal antibody (mAb), increased from 30% in the absence of 1a to 54% in the presence JE-1, which does not recognize other phospholipids, such as PA, of 1a (Fig. 2D). The Fp/Ft ratio of Rtn1p, a protein known to be PE, PS, or phosphatidylinositol (PI) (22, 23). In the absence of redistributed from the peripheral to the perinuclear ER by 1a BMV components, using JE-1 as a primary antibody, PC showed (11), increased from ∼15% to about 50% in the presence of 1a a faint and diffused pattern throughout the cell by immunoflu- (Fig. 2 C and D). Conversely, the distribution of Dpm1p only orescence (IF) confocal microscopy (Fig. 1D). In contrast, a strong changed slightly (Fig. 2 C and D). Thus, BMV 1a specifically re- PC signal was detected in cells expressing BMV components (Fig. distributes Cho2p from the peripheral ER to the perinuclear ER. 1D), consistent with enhanced PC levels as observed in lipid Opi3p localized primarily to the perinuclear ER, and its distri- profiles (Fig. 1 A–C). In particular, the great majority of signal bution was not affected by the expression of 1a. Nevertheless,

colocalized with BMV 1a in the perinuclear ER membrane, the most of 1a colocalized with Opi3p, but not all Opi3p colocalized MICROBIOLOGY site of viral replication (Fig. 1D). Moreover, among cells showing with 1a (Fig. 2C).

Zhang et al. PNAS | Published online February 8, 2016 | E1065 Downloaded by guest on September 28, 2021 A C 8  70 wt wt+BMV 60 wt  6 50 wt+BMV celles)  40

600 4 30

2 (mol%) 20  10    Phospholipids  Phospholipids 0 0 (nmol/OD Total PA PS PE PC PI PG PA PS PE PC PI PG B 160 E 50  wt wt+BMV  Mock BMV 40 140  30  120 20 % of wt 100 10  (nmol/mg DW) 80 Phospholipids 0 Total PE PC Total PA PS PE PC PI PG D - BMV + BMV

Anti-PC DAPI Merge BF Anti-1a Anti-PC DAPI Merge BF (Alexa 488) (Alexa 594) (Alexa 488)

F - BMV + BMV

Anti-PC DAPI Merge BF Anti-1a DAPI Merge BF (Alexa 488) (Alexa 546)

Anti-PC Anti-1a Merge BF Anti-PC Anti-1a Merge BF (Alexa 488) (Alexa 405) (Alexa 488) (Alexa 405)

Fig. 1. BMV promotes PC accumulation at the sites of viral replication. (A) Molar amount of phospholipids in yeast cells in the absence (WT) or presence of BMV replication (WT+BMV). (B) Percentage change in total phospholipids, PE, and PC content based on the molar amount. (C) The relative level of each phospholipid class (molarity percentage, mol%) without or with BMV replication. *P < 0.05; **P < 0.01. (D) IF confocal microscopic images of PC distribution. PC was detected by using an anti-PC mAb (JE-1) and a secondary antibody conjugated to Alexa Fluor 488. BMV 1a was detected with an anti-1a antiserum and a secondary antibody conjugated to Alexa Fluor 594. The yellow color in the merged images indicates the colocalization of PC and 1a signals. The nucleus was stained with DAPI. (Scale bar, 1 μm.) (E) Molar amount of phospholipids in mock- or BMV-inoculated barley plants. (F) PC distribution in barley protoplasts prepared from mock- and BMV-infected systemic leaves. 1a was detected using an anti-1a antiserum. The secondary antibodies for detecting 1a were conjugated to Alexa Fluor 546 or 405 in the Top or Bottom panel, respectively. (Scale bar, 100 μm.)

We further verified that Cho2p was redistributed by and colo- four fractions (Fig. 2E). In addition, Cho2p-HA localized in the calized with 1a biochemically. Lipid rafts or detergent-resistant same fractions as that of 1a, indicating that 1a enriched Cho2p at membrane (DRM) fractions are membrane microdomains that the DRMs. This result was also consistent with the fact that 1a are enriched with sterol and sphingolipids (28). Although the redistributed Cho2p to the perinuclear ER membrane and colo- majority of DRMs are localized at the plasma membrane, they are calized with 1a (Fig. 2C). also present in ER membranes in yeast cells (29). Treatment of cellular membranes with 1% Triton X-100 (TX100) at 4 °C dis- BMV 1a Interacts with Cho2p. Redistribution of Cho2p to the peri- solves most of the membrane structures but not DRMs. After ul- nuclear ER and its enrichment at DRMs in the presence of 1a may tracentrifugation through a density gradient, DRMs and associated be due to an interaction between 1a and Cho2p or due to an in- proteins, such as Gas1p (29), float to the top fractions and the direct and homeostatic redistribution because of ER reorganization dissolved membranes as well as associated proteins (e.g., Dpm1p) induced by 1a. To verify which of the above hypotheses is correct, sediment to the bottom fractions (Fig. 2E). Under such conditions, the potential Cho2p–1a interaction was first tested by a coimmu- the majority of 1a fractionated to the top 3 fractions, suggesting that noprecipitation (co-IP) assay. In yeast cells coexpressing 1a-His6 1a is associated with DRMs (Fig. 2E). Although a large fraction of and Cho2p-HA, the anti-HA antibody pulled down both Cho2p- Cho2p-HA was found in DRMs in the absence of 1a, a portion of HA and 1a-His6, and the anti-His antibody pulled down both 1a- Cho2p-HA also localized in the bottom four fractions (Fig. 2E). In His6 and Cho2p-HA (Fig. 3A). Conversely, Dpm1p-HA was not the presence of 1a, however, the vast majority of Cho2p-HA floated pulled down by the anti-His antibody and the anti-HA antibody did to the top three fractions, and no signal was detected in the bottom not precipitate 1a-His6 (Fig. 3A) in cells expressing both 1a-His6

E1066 | www.pnas.org/cgi/doi/10.1073/pnas.1519730113 Zhang et al. Downloaded by guest on September 28, 2021 PNAS PLUS A PI CDP-DAG PA G3P B -BMV +BMV Cho2p-HA PS Psd1p 1a Psd2p Pgk1p PE Eth D Cho2p Rtn1p * PMME MME Cho2p Opi3p *

CDP-DAG pathway PDME Opi3p Opi3p -1a Cpt1p Pct1p Cki1p Dpm1p PC Cho +1a 0 20 40 60 80 Kennedy pathway Fp/Ft (%)

C - 1a-His6 + 1a-His6 Anti-HA Anti-HA Anti-His (Alexa 594) DAPI Merge BF (Alexa 594) (Alexa 488) DAPI Merge BF Rtn1p -HA

Cho2p -HA

Opi3p -HA

Dpm1p -HA

E - 1a + 1a 1 234 56789 1 23456789 Cho2p-HA 1a FLAG-Gas1p

Dpm1p

Fig. 2. BMV redistributes Cho2p to the viral replication sites. (A) Diagram of the CDP–DAG and Kennedy pathways involved in PC synthesis. Key enzymes are listed. (B) Accumulated Cho2p, 1a, and pgk1p in the absence or presence of BMV replication. Cho2p was tagged with HA and expressed from its endogenous promoter in cho2Δ cells, and Pgk1p served as a loading control. (C) IF images showing the distributions of host Rtn1p, Cho2p, Opi3p, and Dpm1p in the absence or presence of 1a. (Scale bar, 1 μm.) (D) Percentage of the fluorescent signals [Fp/Ft (%)] of each host protein localized to the perinuclear ER (Fp) versus total fluorescence in the cell (Ft) in the absence or presence of 1a. **P < 0.01. (E) Cho2p is enriched in the DRM fractions in the presence of 1a. Gas1p and Dpm1p serve as positive and negative controls, respectively.

and Dpm1p-HA. A previously reported interaction between Rtn1p expressed well and localized to the ER membranes in yeast cells and 1a was also confirmed in the co-IP assay (Fig. S3) (11). (Fig. 2C). In such a setting, fluorescent dots were present in To verify the 1a–Cho2p interaction and determine the lo- perinuclear ER membranes in cells expressing 1a-His6 and cation within the cell where the interaction occurs, we used a Cho2p-HA, suggesting that 1a interacts with Cho2p. Based on proximity ligation assay (PLA). If two proteins interact with the positive results from both co-IP and PLA, we concluded that each other, a fluorescent dye will be deposited at the sites of 1a recruited Cho2p to the viral replication sites most likely via interactionafterperforminganinsituPLAprocedure(30). the 1a–Cho2p interaction. Using PLA, we detected perinuclear ER-localized dot signals (Fig. 3B) from two positive interactions, one between 1a-His6 Deletion of CHO2 Inhibited BMV Replication. Because BMV 1a re- and 1a-FLAG based on a 1a self-interaction (9, 10) and the cruits Cho2p for viral replication, deletion of the CHO2 gene other between 1a-His6 and Rtn1p-HA (11) (Fig. 2C and Fig. should significantly affect BMV replication. For the experiments cho2Δ S3). No such signal was detected in cells coexpressing 1a-His6 described below, WT and cells expressing BMV compo- MICROBIOLOGY and Dpm1p-HA (Fig. 3B) even though both proteins were nents were grown in the absence or presence of choline. Because

Zhang et al. PNAS | Published online February 8, 2016 | E1067 Downloaded by guest on September 28, 2021 A and cho2Δ cells as the PE signals were primarily detected in the Total IP His IP HA cytoplasm without BMV but weakly associated with perinuclear +++ +++ +++1a-His ER with BMV replication (Fig. S2). It has been reported that supersized lipid droplets (SLDs) form in cho2Δ cells due primarily + -- + -- + -- Cho2p-HA to an enhanced PE/phospholipids ratio (33). Although no SLDs -- + -- + -- + Dpm1p-HA were observed in WT cells, SLDs were formed in cho2Δ cells in IB 1a 1a-His the absence or presence of BMV replication (Fig. S5). Never- theless, the relationship between LD formation and BMV repli- Cho2p-HA cation is currently unclear and awaits further investigation. IB HA Dpm1p-HA BMV-Induced Membrane Rearrangements Are Affected in cho2Δ Cells. Pgk1p To identify the specific step(s) of BMV RNA replication that were affected by deleting CHO2, we tested several steps that occur be- B PLA DAPI Merge BF fore negative-stand RNA3 synthesis. As shown in Fig. 4 A and B,we only observed a minor decrease in 2apol accumulation associated 1a-FLAG cho2Δ 1a-His6 with layer replication complexes in cells, which could be complemented by the addition of choline. The accumulation and membrane association of 1a were not affected, based on Western blotting and membrane flotation assay, respectively Rtn1p-HA (Fig. 4). It should be noted that in the engineered BMV-yeast 1a-His6 system, a stop codon was incorporated into the 5′ end of the coat protein (CP) coding sequence, and thus, no BMV CPs are produced and no viral particles are assembled (Fig. S4). Cho2p-HA We also tested the assembly of spherule and layer replication 1a-His6 complexes in cho2Δ cells. Among more than 100 spherules measured, 1a-induced spherules were ∼72 ± 8 nm in diameter in WT cells (Fig. 6 A and F). However, in the absence of choline, Dpm1p-HA larger spherules with a wider range in diameter formed upon 1a-His6 deletion of CHO2 (Fig. 6 B–E, pointed to by the white arrow- head). In cho2Δ cells, the average spherule diameter was about 90 ± 34 nm, an increase of ∼25% compared with that of spherules Fig. 3. BMV 1a interacts with Cho2p. (A) Co-IP of 1a-His6 and Cho2p-HA. Cell P < F lysates were subjected to IP using anti-His mAb (IP His) or anti-HA pAb (IP HA), formed in WT cells ( 0.001; Fig. 6 ). followed by Western blotting using anti-HA (IB HA) or anti-His (IB His) antibodies, respectively. Dpm1p served as a negative control. (B)Insitudetection of the 1a–Cho2p interaction via PLA. In cells expressing various protein pairs, PLA signals (red dots) were detected by incubating fixed yeast spheroplasts A -choline +choline with anti-His mAb and anti-FLAG pAb or anti-HA pAb following the protocol layers spherules layers spherules detailed in Experimental Procedures. The interactions between 1a-FLAG and 1a-His6 as well as Rtn1p-HA and 1a-His6 served as positive controls. The wt cho2 wt cho2 wt cho2 wt cho2 Dpm1p-HA and 1a-His6 pair served as a negative control. (Scale bar, 1 μm.) +RNA3 % of wt +RNA4 cho2Δ cells grew slowly after 5–6 generations in liquid culture +RNA4 100±16 6±1 100±11 25±3 100±14 30±2 100±8 37±2 without supplemented choline due to depleted PC content (31), -RNA3 – – % of wt we harvested cells after 20 24 h (2 3 generations) for analyzing -RNA3 100±18 3±1 100±6 18±6 100±16 27±9 100±2 52±1 viral replication in the absence of choline. 18S BMV replication was tested under conditions that normally rRNA lead to spherule or double-membrane layer replication com- 1a plexes. Coexpression of 1a with low levels of 2apol (e.g., expressed pol under the control of weak promoters) (Fig. S4) induces spherules. 2a When 1a is coexpressed with high levels of 2apol, it leads to the Pgk1p formation of double-membrane layer replication complexes (32). B wt cho2 Although morphologically different, spherule and layer replication complexes support similar levels of BMV RNA replication (32). 1 2 3 4 5 6 1 2 3 4 5 6 When cultured in the absence of choline, negative-strand RNA3 1a levels in cho2Δ cells were only 3% and 18% of WT levels in layer Pgk1p or spherule conditions, respectively, indicating that BMV replication was substantially affected as early as negative-strand RNA Dpm1p cho2Δ synthesis. Positive-strand RNA4 accumulation in the cells Fig. 4. BMV replication is blocked in cho2Δ cells. (A) BMV RNA replication similarly decreased by 16- or fourfold in layers or spherules, re- wasmeasuredinWTorcho2Δ cells under conditions that led to the formation spectively (Fig. 4A). The addition of choline, which activates PC of double-membrane layers or spherules in the absence or presence of choline. production via the Kennedy pathway, partially complemented the Positive- and negative-strand viral RNAs were detected using BMV strand- BMV replication defect in cho2Δ cells (Fig. 4A). Consistent with specific probes. 18S rRNA served as a loading control. Total proteins were extracted from the same numbers of cells and subjected to Western blotting viral replication data, we observed a decrease in PC levels and an pol cho2Δ with anti-1a or -2a antibodies. Pgk1p served as a loading control. (B)1a increase of PE in cells compared with WT cells even in the associated similarly with membranes in both WT and cho2Δ cells. 1a was de- presence of choline (Fig. 5B). Nevertheless, we did not find an tected in the membrane fractions along with positive control Dpm1p after an obvious difference in the localization of PE signals between WT iodixanol density gradient. Pgk1p served as a control for soluble proteins.

E1068 | www.pnas.org/cgi/doi/10.1073/pnas.1519730113 Zhang et al. Downloaded by guest on September 28, 2021 WT cells expressing 1a or supporting BMV replication grew PNAS PLUS A 2.5 A 2 twiceasslowlyasBMV-freecells(Fig.5 ). Supplementing sub- strates that stimulate the Kennedy pathway, like Eth, MME, or 1.5 choline (Fig. 2A), reduced the doubling time of BMV-replicating 1 cells by 25–40% (Fig. 5A), suggesting that the rate of PC synthesis is (folds) 0.5 one of the limiting factors for cell growth during BMV replication. Growth Rate 0 In the presence of choline and free of BMV, PC accounted for None Cho MME Eth None 30% of phospholipids in cho2Δ cells (Fig. 5B), a significant in- wt + BMV wt Doubling crease from the reported 10–20% of total phospholipids in the time (h) 7.8±0.3 5.5±0.2 5.4±0.6 6.0±0.3 3.8±0.1 absence of choline (31). Despite this discrepancy, PC levels were B still ∼9% lower in cho2Δ cells compared with WT. A corre- 50 sponding ∼8% increase in PE levels was seen in cho2Δ cells (Fig. wt 40 5B), suggesting that the Kennedy pathway can complement but (mol%) wt-S – 30 not completely replace the CDP DAG pathway. Nevertheless, wt-L BMV RNA replication under both spherule and layer conditions 20 promoted a 28% increase in relative PC levels in cho2Δ cells 10 -S supplemented with choline (Fig. 5 B and C). Thus, BMV appears -L 0 to have the ability to modulate PC synthesis through both the Phosphalipids PA PS PE PC PI PG CDP–DAG (Fig. 1) and Kennedy pathways (Fig. 5C). In addition, this increase was much higher than the 8% increase in WT C 160 cells without choline during BMV replication (Fig. 1C). This is 150 No BMV cho2Δ Spherules consistent with significant increases in BMV replication in 140 Layers   cells supplemented with choline (Fig. 4A). This also agrees well 130   with the results that BMV infection significantly stimulated the 120 E F 110 amount of PC in barley cells (Fig. 1 and ), where the Kennedy 100 pathway is dominant in PC production (34). PC Content

(% of No BMV) 90 80 Virus-Stimulated and Viral Replication Site-Localized PC Accumulation wt cho2 Is a Conserved Feature Among Some (+)RNA Viruses. Although DENV and polioviruses stimulate PC synthesis (17, 20), it is not Fig. 5. BMV induces PC synthesis via the Kennedy pathway. (A) Growth rate clear whether HCV infection increases total PC levels. Never- of WT cells in the media supplemented with choline, MME, or Eth in the theless, certain PC species, such as PC 30:0, increased significantly, presence of BMV replication. (B) Phospholipid compositions (mol%) in WT or cho2Δ cells with supplemented choline in the absence and presence of BMV whereas some decreased (e.g., PC 35:1) upon HCV infection (35). replication. S and L, BMV replication as spherule- or layer-replication com- To determine if the enhanced synthesis/accumulation of PC at the plexes. (C) Relative PC levels (mol%) in WT and cho2Δ cells without or with site of viral replication is a common feature associated with BMV replication in the presence of choline to activate the Kennedy pathway; (+)RNA virus infections, we checked distribution and signal intensity **P < 0.01. of PC and PE in cells infected with DENV, HCV, or poliovirus. PE is abundantly present in the cytoplasm primarily associated with ER membranes but excluded from the nucleus in the ab- pol Although coexpression of 1a and a low level of 2a induces sence of viral infection (Fig. S6). Upon infection with HCV or pol pol spherule formation, high levels of 2a (compare 2a signals in DENV, there is no significant redistribution of the PE signal. For spherule and layer complexes in Fig. 4A) along with 1a support the instance, in some infected cells, HCV NS5A and DENV NS3, formation of layer replication complexes, which are stacks of 1–4 which are the replication proteins and components of the VRCs double-membrane layers surrounding the nucleus (32). As shown of each virus, colocalized with PE signal (Fig. S6). However, in in Fig. 7A, three stacks of membranes were formed in WT cells. many cells, we observed strong viral protein signal, but no cor- Consistent with decreased accumulation of PC (Fig. 5B)and2apol responding increase in PE signal was present (boxed areas in Fig. (Fig. 4A), the number of stacked membranes surrounding the S6), suggesting neither virus modulates PE distribution. It should nucleus was reduced to one (79% of the cells with layer structures) be noted that PC is primarily synthesized by the Kennedy path- or two (21%) in cho2Δ cells (Fig. 7 B–E). way in mammalian cells, and thus, choline, but not PE, is the primary substrate for PC. BMV Replication Promotes PC Synthesis Through the Kennedy Pathway As seen in yeast and barley cells probed with the JE-1 mAb, as Well. In the Kennedy pathway, choline is first converted to the PC signal was weak and evenly distributed throughout mock- A B cholinephosphate by Cki1p (choline kinase) and, subsequently, to infected Huh-7.5 cells (Fig. 8 and ). Upon HCV infection, the PC signal was not only more intense, but it colocalized with NS5A CDP-choline by Pct1p (cholinephosphate cytidyltransferase), and A B then to PC by Cpt1p (choline phosphotransferase) (Fig. 2A). In in the perinuclear ER membrane (Fig. 8 ). As shown in Fig. 8 , the increase in the intensity of the PC signal in HCV-infected over addition, ethanolamine (Eth) and monomethylethanolamine (MME) mock-infected cells was statistically highly significant (P < 0.001; can also be converted to PE and PMME, respectively, via the Ken- Fig. 8B). Moreover, the colocalization of PC with NS5A at the nedy pathway. PE and PMME are subsequently converted to PC via – A perinuclear ER membrane had a correlation coefficient of 0.7 as the CDP DAG pathway (Fig. 2 ). analyzed by both Manders’ and Peasons’ approaches, indicating Because yeast cells were grown in synthetic-defined medium that HCV promoted PC accumulation at the sites of viral repli- lacking choline, the Kennedy pathway does not contribute cation. Conversely, no enhanced PC signal based on IF micros- significantly to PC production. To determine possible contri- copy was detected during DENV infection (Fig. 8C). bution of the Kennedy pathway to BMV replication, WT and To monitor the synthesis of PC in poliovirus-infected HeLa cho2Δ cells were grown in media supplemented with choline to cells, we used a choline analog, propargyl-choline, which is ef- stimulate PC production (Fig. 2A). In the latter case, PC pro- ficiently incorporated into PC without affecting the overall PC

duced from the Kennedy pathway accounted for most of the PC composition (36). In uninfected cells, propargyl-choline was MICROBIOLOGY in cho2Δ cells. evenly incorporated into cellular organelles but excluded from

Zhang et al. PNAS | Published online February 8, 2016 | E1069 Downloaded by guest on September 28, 2021 A B F wt cho2 Cyto 40  100nm wt 30 cho2 Nuc 20

10 Nuc Distribution (%) 0 40 60 80 100 120 140 200nm Cyto 200nm 100nm Diameter of spherules (nm)

C D E cho2 cho2 cho2 Nuc Cyto Cyto Nuc

Cyto Nuc 200nm 100nm 200nm 100nm 200nm

Fig. 6. Enlarged spherules are formed in cho2Δ cells. Electron microscopic images of WT (A)orcho2Δ cells (B–E) expressing BMV 1a are shown. Arrows in A point to spherules, and arrowheads in B–E point out spherules with a wider diameter. Images on the Right are higher magnification micrographs of the boxed areas from the images on the Left.(F) Distribution of spherule diameters in WT and cho2Δ cells. ***P < 0.001. Cyto, cytoplasm; Nuc, nucleus.

the nucleus (Fig. 8D). Upon poliovirus infection, a substantially different viruses replicate in different organelle membranes and enhanced propargyl-choline signal was incorporated into cell may modulate and require different cellular lipids for the as- structures surrounding the nucleus (Fig. 8D). We also tested sembly and function of their VRCs. Host phosphatidylinositol- the distribution of the poliovirus 2B protein, which serves as a 4-kinase III α (PI4KIIIα)andPI4KIIIβ, which convert PI to maker of poliovirus VRCs. Detergent treatment, which per- PI-4-phosphate (PI4P), are recruited by HCV (39–41) and Cox- meabilizes cells and enables antibodies to access the antigens, sackievirus B3 (CVB3, a picornavirus) (42), respectively. Although also affects PC detection. To preserve the integrity of the mem- HCV stimulates the activity of PI4KIIIα to promote PI4P pro- branes and PC, we used 0.5% saponin in simultaneous detection duction for VRC formation (39, 40), PI4P produced by PI4KIIIβ is of poliovirus 2B and PC. Due to the suboptimal permeabilization required for activating or recruiting CVB3’s replicase protein (42). conditions used, the 2B signals were mainly detected in the pe- Other (+)RNA viruses, like tomato bushy stunt virus (TBSV), en- riphery of the PC-positive membranes (Fig. 8D, Middle). Never- rich and require high levels of PE at the viral replication sites (24). theless, in the majority of cells where a 2B signal was detected, the Although the expression of TBSV p33 replication protein alone is propargyl-choline signal colocalized with that of 2B (Fig. 8D), sufficient to induce the enrichment of PE at the places colocalizing suggesting that poliovirus promotes PC accumulation at the viral with that of p33 (24), it is not clear whether p33 directly or indirectly replication sites. induces the site-specific PE synthesis or transports PE to the site of viral replication. Conversely, it has been well-documented that PC Discussion accumulation is significantly enhanced during DENV infection in In this report, we examined how certain (+)RNA viruses mod- mosquito cells (17), in addition to the infection by FHV (18) and ulate cellular phospholipid synthesis during infection, including poliovirus (19, 20). We did not observe obvious increases in PC BMV, DENV, HCV, and poliovirus. Among them, we found that accumulation in DENV-infected Huh7 cells (Fig. S6)basedonthe BMV, HCV, and poliovirus enhanced PC synthesis and/or accu- IF confocal microscopy, which may reflect differences in cell type or mulation at the viral replication sites. These three viruses infect other experimental conditions compared with ref. 17. Nevertheless, plants or humans and belong to the alphavirus-, flavivirius-, or it is not clear where the virus-stimulated PC synthesis occurs picornavirus-like superfamily, respectively. They also induce very during infections of DENV, FHV, and poliovirus. different membrane-bound VRCs: BMV forms single-membrane Our data indicated a localized PC synthesis and/or accumu- spherules that remain connected to the ER membrane (6), lation at the viral replication sites of a group of diverse (+)RNA whereas HCV forms convoluted membrane webs and DMVs viruses (Figs. 1 and 8). In particular, our data support a working (double-membrane vesicles) (37) and poliovirus induces DMVs model that BMV 1a redistributes host Cho2p to the viral repli- and tubular structures (38). Despite the topological differences in cation sites to promote a localized PC synthesis, and this extra VRC structures, these viruses specifically promoted PC synthesis/ pool of PC is likely designated for the VRC formation and re- accumulation at the viral replication sites, suggesting a common quired for BMV replication. feature shared by them and possibly by other (+)RNA viruses. We have previously shown that poliovirus 2A protein stimulates activity of host long chain acyl-CoA synthetase3 for the Virus-Induced Local Synthesis and/or Accumulation of PC Is a Strategy enhanced import of FAs, which are retargeted from lipid drop- Shared by a Group of (+)RNA Viruses. Although (+)RNA virus RNA lets to the viral replication sites. We have further shown that replication invariably takes place in membrane-bound VRCs, these FAs are incorporated into PCs (20). Our data here

E1070 | www.pnas.org/cgi/doi/10.1073/pnas.1519730113 Zhang et al. Downloaded by guest on September 28, 2021 A B (Fig. 4) suggested that the changes in membrane phospholipid PNAS PLUS composition might affect the 1a–2apol interaction. wt cho2  Besides its role in maintaining membrane structure and composition, PC serves as a reservoir for producing secondary messengers: Nuc Nuc PA, DAG, and lysoPC. Enhanced PC levels may lead to the increased production of such signaling molecules that may stimulate viral replication. To support this notion, red clover necrotic mosaic virus promotes PA production by recruiting phospholipase D-α and 500nm Cyto 500nm Cyto -β, which convert PC to PA by removing choline from PC (44). Interfering phospholipase-mediated PA production inhibited repli- C D cation of both red clover necrotic mosaic virus and BMV (44). cho2  Cyto cho2  Cyto The size of the VRCs is regulated by several factors. For Semliki Forest virus, the length of the viral templates influences the size of VRCs (45). It has been previously shown that the size of Nuc BMV spherules can be affected by several manipulations. Smaller spherules are formed in cells lacking ACBP and thus with the Nuc altered lipid composition (14). Moreover, substitutions within the 500nm 500nm membrane-anchoring helix A of 1a also lead to the formation of smaller spherules (8), as does depleting the pool of host RTNs (11). In contrast, cho2Δ cells displayed spherules whose diameter E was about 25% greater than those in WT cells but were not fully # of stacks 1 23 functional (Fig. 4A). These results provide further evidence that strain the composition of the membrane at the site of viral replication is wt 19% 38% 43% crucial for both proper assembly and function of VRCs. cho2 79% 21% In conclusion, we revealed a common feature shared by a group of (+)RNA viruses in that they promote significantly enhanced Fig. 7. Decreased stacks of double-membrane layers are assembled in cho2Δ synthesis and/or accumulation of PC content at the viral rep- cells. Electron micrographs of WT (A)orcho2Δ (B–D) cells expressing BMV 1a lication sites. This pool of virus-stimulated PC likely contributes and high levels of 2apol are shown. (E) Percentage of cells with visible layer to the formation of VRCs. Although blocking general PC syn- replication complexes that contained a different number of stacked mem- thesis evidently affects host cell growth, looking for ways to block branes. Cyto, cytoplasm; Nuc, nucleus. the viral replication site-localized PC accumulation could be a new way to specifically control viral replication with the least possible side effects on host growth. provided direct evidence that the enhanced PC content during poliovirus infection was produced at the viral replication sites Experimental Procedures (Fig. 8D). Hosts, Viruses, Plasmids, and Antibodies. Yeast strain YPH500 and routine Our data also suggested that BMV stimulates PC production culture conditions have been described previously (14). BMV components via the Kennedy pathway (Fig. 5), the dominant pathway in higher were expressed from various constructs described in ref. 14 and illustrated in eukaryotes. Among the three enzymes in the Kennedy pathway Fig. S4. A 4-bp insertion in CP coding sequence results in an early stop codon (Fig. 2A), Pct1p (or CCT in other organisms) is the rate-limiting so no CPs are produced. Choline chloride, Eth, or MME was added to a final concentration of 1 mM. enzyme. Pct1p/CCT is inactive in its soluble form but is activated Barley plants (cv Rust) were grown in a growth chamber under 14 h light/ upon binding to membranes (16, 43). FHV infection increases 10 h dark at 24 °C. The first expanded leaves of 6-d-old barley seedlings Cct1 Cct2 transcription of the CCT-encoding and genes by were inoculated with BMV or buffer, and second expanded leaves were ∼twofold (18). It is possible that BMV, HCV, or poliovirus may harvested for lipid analysis and IF 7 DPI. target CCT to stimulate the Kennedy pathway-mediated PC pro- Rabbit anti-1a and mouse anti-2apol (provided by Paul Ahlquist, University duction for their replications. of Wisconsin, Madison, WI), mouse anti-His (Genescript), anti-Dpm1p (Life Technologies), and anti-Pgk1p antibodies (Life Technologies) as well as PC Synthesis Is Required for Proper Assembly of BMV VRCs. Blocked rabbit anti-FLAG (Sigma) and anti-HA (Life Technologies) were used at 1:100 PC synthesis by the deletion of CHO2 inhibited BMV replication dilution for IF and 1:10,000 or 1:3,000 dilution for Western blotting. Mouse anti-PC antibody (JE-1, a gift from Masato Umeda, Kyoto University, Kyoto) up to 30-fold, showing the importance of PC synthesis in BMV was used at 1:40 dilution for IF. replication (Fig. 4A). However, this could be an overestimate of ’ CHO2 PC s contribution to BMV replication as deleting pre- Lipid Analysis. Total lipids were extracted following the protocol provided by

vented PC synthesis not just at the site of viral replication but also the Kansas Lipodomics Research Center. Ten OD600 units of yeast cells were throughout the cell, which may affect BMV replication indirectly. processed. For barley, second expanded leaves were harvested at 7 DPI. All Because BMV 1a induces spherule formation and is also in- lipid samples were analyzed on a triple quadruple MS/MS equipped for ESI at volved in recruiting Cho2p, the PC synthesized in the perinuclear the Kansas Lipodomics Research Center. ER is most likely incorporated into the membranes that are used IF Assay and PLA. Yeast cells were fixed with 4% (vol/vol) formaldehyde and to assemble VRCs. The fact that formation of both spherule and cho2Δ the cell wall removed by lyticase and permeabilized with 0.1% TX100. layer replication complexes was affected in cells supports Spheroplasts were incubated with specified primary antibodies overnight at this notion (Figs. 6 and 7). For instance, only one double-mem- 4 °C and followed by a secondary antibody at 1:100 dilution for 1 h at 37 °C: brane layer was formed in the majority of cho2Δ cells (Fig. 7). It is anti-rabbit antibodies conjugated to Alexa Fluor 405 or 488 or anti-mouse possible that not enough PC is available to form such layers. Al- antibody conjugated to Alexa Fluor 594 (Life Technologies). The nucleus was ternatively or in addition, altering the membrane lipid composi- stained with DAPI for 10 min. Barley protoplasts were isolated following tion might affect BMV 1a’s ability to interact with the membrane Loesch-Fries and Hall (46) and then processed using the same procedure as that pol pol in yeast. Images were captured using a Zeiss LSM 510 confocal microscope. For and/or 2a . Stability and accumulation of 2a are significantly PE detection, spheroplasts were incubated with biotin-conjugated duramycin

enhanced in the presence of 1a due to 1a-mediated recruitment of (Molecular Targeting Technologies) and then Texas Red-conjugated strepta- MICROBIOLOGY pol pol 2a into spherules or layers (32). Decreased 2a accumulation vidin (GenTex Corporation). Lipid droplets were detected by incubating live

Zhang et al. PNAS | Published online February 8, 2016 | E1071 Downloaded by guest on September 28, 2021 A PC NS5A B DAPI Merge (Alexa 594) (Alexa 488) *** Uninfected HCV-infected

propargal- co- 2B C PC NS3 DAPI Merge D choline Merge localization (Alexa 594) (Alexa 488) (Alexa 594) (Alexa 488) Uninfected Uninfected DENV-infected poliovirus-infected

Fig. 8. Newly synthesized PC colocalizes with viral proteins associated with the VRCs of HCV and poliovirus. (A) Huh-7.5 cells were infected with HCV (JC1) at an MOI of 0.05 for 48 h, fixed with 4% (vol/vol) paraformaldehyde, and permeabilized with 0.1% TX100. Cells were then incubated with anti-HCV NS5A mAb (9E10, IgG) and followed by anti-mouse IgG conjugated with Alexa Fluor 488. Cells were then reblocked, incubated with JE1, and followed by anti-mouse IgM (u chain) conjugated to Alexa Fluor 594. The yellow color in the merged images indicates the colocalization of PC and NS5A. DAPI stains the nucleus. (Scale bar, 13.5 μm.) (B) Comparison of PC signal intensity per cell in mock- and HCV-infected Huh-7.5 cells. ***P < 0.001. (C) Huh-7.5 cells were infected with DENV-2 at an MOI of 1 for 48 h. Cells were processed as those of infected by HCV. PC was detected using JE-1, and DENV NS3 was detected by an anti-NS3 antiserum. (Scale bar, 13.5 μm.) (D) HeLa cells were infected with poliovirus type I Mahoney at an MOI of 10. Growth medium with 200 μM of propargal-choline was added at 4 h postinfection and incubated for 1 h. Propargal-choline–labeled PCs were detected by Alexa Fluor 594-conjugated azide. Poliovirus 2B was detected by an anti-2B mAb, and the nucleus was stained with Hoechst-33342. The colocalization panel shows the colocalized red and green pixels of 2B and PC identified with ImageJ software. (Scale bar, 10 μm.)

cells with Nile red for 10 min. Images for PE and LD signals (Fig. S2 and Fig. S5) 1 and incubated for 48 h. The cells were processed as those infected by HCV. were acquired with a Zeiss LSM 880 confocal microscope. To detect the PC was detected using JE-1, and NS3 was detected using anti-NS3 antiserum. colocalization of two protein signals, signals were sequentially acquired to For poliovirus, detection of PC was done by a “click chemistry” method avoid any possible bleed-through. described in Jao et al. (36). HeLa cells were infected with an MOI of 10 of Huh-7.5 cells were infected with HCV (Jc1) at an MOI (multiplicity of poliovirus type I Mahoney. At 4 h postinfection, the incubation medium was ’ infection) of 0.05 for 48 h. Cells were fixed with 4% (vol/vol) para- replaced with Earls balanced salt solution with amino acids supplemented with 200 μM of propargyl-choline and incubated for 1 h; the cells were then formaldehyde, permeabilized with 0.1% TX100, and quenched with 50 mM fixed with 4% (vol/vol) formaldehyde in PBS. For immunostaining of the viral ammonium chloride. Cells were then blocked with 10% (vol/vol) goat serum antigen, cells were incubated with mouse anti-poliovirus 2B antibody in PBS and stained with NS5A mAb (9E10; a gift from Charlie Rice, Rockefeller with 0.05% saponin and followed by incubation with the anti-mouse sec- University, New York) and then with anti-mouse IgG-Alexa Fluor 488. Cells ondary antibodies conjugated with Alexa Fluor 594. Confocal images were were then blocked again before staining for PC (JE-1) and detected with obtained with a Zeiss LSM 510 microscope. Colocalization analysis was per- anti-mouse IgM (u chain) conjugated with Alexa Fluor 594. Cells were formed using ImageJ software with colocalization plugin by Pierre Bour- mounted with ProLong Gold anti-fade with DAPI (Life Technology) and vi- doncle, Institut Jacques Monod, Service Imagerie, Paris, France. sualized using Olympus spinning disk microscopy. To detect PC distribution PLA was performed following the standard procedure (Duolink, Sigma). in DENV-infected cells, Huh-7.5 cells were infected with DENV-2 at an MOI of Briefly, yeast spheroplasts were incubated with mouse anti-His6 and rabbit

E1072 | www.pnas.org/cgi/doi/10.1073/pnas.1519730113 Zhang et al. Downloaded by guest on September 28, 2021 anti-HA or anti-FLAG at 1:100 dilutions overnight at 4 °C and then with sec- were loaded onto a 10% (vol/vol) SDS/PAGE gel and used to detect target PNAS PLUS ondary antibodies conjugated with oligonucleotides (PLA probe MINUS and proteins. PLUS) for 1 h at 37 °C. After a 30-min ligation reaction and 100-min amplifi- cation reaction at 37 °C, samples were mounted with Duolink in situ mounting Electron Microscopy. Fixation, dehydration, and embedding of yeast cells medium with DAPI and observed using a Zeiss LSM Scanning 510 microscope. were performed as previously described (14). Images were obtained using a JEOL JEM 1400 transmission electron microscope at the Virginia-Maryland Assay for DRMs. The yeast lysate in TNE buffer (50 mM Tris, pH7.4, 150 mM Regional College of Veterinary Medicine.

NaCl, 5 mM EDTA, pH 8.0) from 20 OD600 units of cells was subjected to a density gradient at 201,000 × g at 4 °C for 5 h. The top fraction from the ACKNOWLEDGMENTS. We thank Drs. Glenda Gillaspy and Boris Vinatzer for membrane flotation assay was incubated with TX100 at 1% for 30 min on discussions; Haijie Liu, Sarah Lau, and Alicia Campos for general assistance; ice, adjusted to 40% (vol/vol) iodixanol, followed by the density gradient for Guijuan He for setting up the Nile red protocol; Drs. Yuji Hara and Masato Umeda for providing JE-1 anti-PC antibody; Kansas Lipidomics Research a second time. The gradients were divided into nine fractions and analyzed Center for processing lipid samples; and Drs. Arturo Diaz and Janet by Western blotting for target proteins. Webster for critical reading of the manuscript. We appreciate the help from Drs. Zackary Adelman, Kevin Miles, and Glady Hazitha Samuel for Co-IP Assay. The co-IP assay was performed as previously described in ref. 11. providing virus-infected mosquito cells. Research at the G.R. laboratory is

Briefly, 10 OD600 units of yeast cells were lysed in RIPA buffer (50 mM Tris at pH supported by NIH Grant DK102883, and V.C. is supported by an American 8.0, 1% Nonidet P-40, 0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate, Heart Association postdoctoral fellowship. Research at the G.A.B. labora- 5 mM EDTA, 10 mM NaF, 10 mM NaPPi, and protease inhibitor mix). After tory is supported by funds from the Maryland Agricultural Experimental Station and NIH Grant AI115383. Work in the X.W. laboratory is supported centrifugation to remove debris, the supernatant was mixed with Protein A by a Virginia Tech Startup fund, National Science Foundation Grant Sepharose beads (GE Healthcare) plus anti-His or anti-HA antibody and in- 1265260, and the Virginia Agricultural Experiment Station and Hatch cubated overnight at 4 °C. Beads were washed three times with RIPA buffer, Program of National Institute of Food and Agriculture, United States resuspended in 1× SDS gel loading buffer, and boiled for 10 min. The samples Department of Agriculture.

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