Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication
© Castorena et al; licensee BioMed Central Ltd. 2010
Received: 30 July 2009
Accepted: 17 March 2010
Published: 17 March 2010
Cellular membranes are crucial host components utilized by positive-strand RNA viruses for replication of their genomes. Published studies have suggested that the synthesis and distribution of membrane lipids are particularly important for the assembly and function of positive-strand RNA virus replication complexes. However, the impact of specific lipid metabolism pathways in this process have not been well defined, nor have potential changes in lipid expression associated with positive-strand RNA virus replication been examined in detail.
In this study we used parallel and complementary global and targeted approaches to examine the impact of lipid metabolism on the replication of the well-studied model alphanodavirus Flock House virus (FHV). We found that FHV RNA replication in cultured Drosophila S2 cells stimulated the transcriptional upregulation of several lipid metabolism genes, and was also associated with increased phosphatidylcholine accumulation with preferential increases in lipid molecules with longer and unsaturated acyl chains. Furthermore, targeted RNA interference-mediated downregulation of candidate glycerophospholipid metabolism genes revealed a functional role of several genes in virus replication. In particular, we found that downregulation of Cct1 or Cct2, which encode essential enzymes for phosphatidylcholine biosynthesis, suppressed FHV RNA replication.
These results indicate that glycerophospholipid metabolism, and in particular phosphatidylcholine biosynthesis, plays an important role in FHV RNA replication. Furthermore, they provide a framework in which to further explore the impact of specific steps in lipid metabolism on FHV replication, and potentially identify novel cellular targets for the development of drugs to inhibit positive-strand RNA viruses.
The relatively small genome of most positive-strand RNA viruses compels these pathogens to use cellular machinery to complete their replication cycles. The search for these "host factors" utilized by positive-strand RNA viruses is at the forefront of virology research, due in part to the possibility that cellular proteins or processes may represent more stable drug targets or provide broader antiviral activity when disrupted . One diverse host factor that has been identified as crucial for positive-strand RNA virus replication are intracellular membranes [2–5]. Although viruses that contain a lipid envelope as a structural component clearly utilize cellular membranes to form infectious virions, all positive-strand RNA viruses, both enveloped and non-enveloped, also depend on host intracellular membranes for the assembly and function of the viral RNA replication complexes essential for genome amplification. The precise functions of cellular membranes in this process have not been fully defined, but may include: (i) serving as structural scaffolds for replication complex targeting and assembly; (ii) protecting viral RNA or replication intermediates from cellular antiviral defense responses; or (iii) providing essential protein or lipid cofactors for optimal viral enzymatic activities. These proposed functions are not mutually exclusive, and it is likely that cellular membranes and their constituent components play multiple roles in viral RNA replication.
Apart from the membrane localization of its viral RNA replication complexes, additional lines of evidence point to the importance of membranes, and in particular lipids, in FHV replication. Protein A is a lipid-binding protein with particular affinity for specific anionic phospholipids , which may mediate in part the protein A-membrane interactions required for replication complex assembly. FHV RNA replication induces dramatic mitochondrial membrane rearrangements [7, 11, 15, 16], reminiscent of structures seen with other positive-strand RNA viruses . The fatty acid synthetase inhibitor cerulenin disrupts FHV RNA replication , consistent with the activity of this inhibitor on other positive-strand RNA viruses [18, 19]. Finally, FHV RNA replication complex activity in isolated membrane fractions analyzed in vitro is disrupted by certain detergents , and can be augmented by the addition of exogenous phospholipids . These results all suggest a central role of cellular lipid metabolism in FHV RNA replication complex assembly and function. However, none of them provide direct evidence for a functional impact of specific lipid metabolism pathways on FHV RNA replication within intact cells.
In this report, we use complementary transcriptomic, lipidomic, and targeted functional genetic analyses to specifically examine the role of lipids in FHV RNA replication in cultured Drosophila cells. We demonstrate that FHV infection or replicon expression upregulates the transcription of a distinct set of cellular genes, several of which are involved in lipid metabolism. Furthermore, FHV RNA replication induces global changes in cellular phospholipid content, and in particular phosphatidylcholine (PC), and pharmacologic or genetic disruption of PC synthesis within cells inhibits FHV RNA replication.
FHV RNA replication induces transcriptional upregulation of lipid metabolism genes in Drosophila S2 cells
We examined the transcriptional responses of Drosophila S2 cells to FHV RNA replication using genome-wide microarray analyses. To focus on cellular responses related to RNA replication rather than those associated with other steps in the viral life cycle, such as virion attachment, entry, uncoating, encapsidation, and virion release, we conducted parallel analyses of cells either infected with FHV or transfected with an RNA1 replicon-expression plasmid. The RNA1 segment of the FHV genome encodes the viral RNA-dependent RNA polymerase (Fig. 1A) and is the only genome segment required for RNA replication (Fig. 1C). Thus, FHV RNA replication can be initiated in S2 cells by the introduction of a self-replicating RNA1 via the inducible plasmid pS2F1 (Figs. 1B and 1C) . To maximize the number of S2 cells expressing the replicon, we first generated stable cell lines containing pS2F1 or a control plasmid (pS2F1fs, see Fig. 1B legend) containing a translation-defective RNA1 segment. Although there was a low level of baseline FHV RNA replication in pS2F1-expressing cells due to leaky metallothionein promoter activity, induction with copper sulfate resulted in a dramatic increase in subgenomic RNA3 and protein B2 accumulation, indicative of highly active viral RNA replication complexes (data not shown).
Drosophila genes co-upregulated in S2 cells either infected with FHV or expressing an RNA1 replicon.
Lethal (3) 02649
CTP:phosphocholine cytidylyltransferase 1
CTP:phosphocholine cytidylyltransferase 2
Poly U binding factor 68 kDa
Daughter of sevenless
Ecdysone-induced protein 75B
Peptidoglycan recognition protein LA
Serine protease 7
Protein tyrosine phosphatase 61F
Suppressor of cytokine signalling at 36E
Ets at 21C
Heat shock protein 23
FHV RNA replication alters phospholipid levels in Drosophila S2 cells
To evaluate potential lipid changes associated with FHV replication in S2 cells in more detail we used electrospray ionization tandem mass spectrometry (ESI-MS/MS) to quantitate polar phospholipids in extracts from FHV-infected cells. This analysis allowed the determination of relative individual phospholipid species content as a molar percentage of total recovered polar phospholipids but also provided details on total acyl chain length and saturation levels. As controls for these experiments we used cells treated with miltefosine to decrease PC levels or cells cultured with 1 mM oleic acid (cis-9-octadecenoic acid), which increases the production of phospholipids with individual C18:1 acyl chains and hence total sum acyl chain compositions of C36:2. Complete detailed results from the ESI-MS/MS analyses are provided in Additional File 4. Control membrane extracts from S2 cells contained predominantly PC and phosphatidylethanolamine (PE) as the primary phospholipids, with the latter representing the majority (~40%) of all phospholipids (Fig. 3B). Although PC is typically the most prominent phospholipid in most eukaryotic cell membranes, Diptera species such as Drosophila contain relatively high levels of PE . As expected, miltefosine dramatically reduced PC content with compensatory increases in the percentages of PE and phosphatidylinositol (PI). In contrast, total cellular membranes from FHV-infected S2 cells showed a trend towards an increase in the molar percentage of PC and phosphatidylserine (PS) with an associated decrease in PE content, but these differences were not statistically significant (Fig. 3B). However, when we analyzed PC species based on total acyl chain length or saturation level there were modest but statistically significant changes in the percentage of PC molecules with total acyl chain lengths of 32 or 34 carbons (Fig. 3C) and 0, 1, or 2 total double bonds (Fig. 3D), where FHV infection increased the fraction of PC molecules with longer unsaturated acyl chains. Furthermore, the selective increase in longer unsaturated acyl chains in FHV-infected cells was seen specifically with PC and not PE, PI, or PS (Fig. 3E and data not shown). These quantitative lipid analysis results suggested that FHV infection induces selective changes in PC metabolism, and are consistent with the observation that FHV RNA replication complex activity in isolated membrane fractions analyzed in vitro is preferentially stimulated by phospholipids with increased acyl chain length and decreased saturation .
Manipulation of glycerophospholipid metabolism gene expression modulates FHV RNA replication in Drosophila S2 cells
Global approaches such as transcriptomic, proteomic, and functional genomic analyses have provided important clues to critical host-pathogen interactions that influence virus replication and pathogenesis [40–47]. However, these approaches when used in isolation often provide an overwhelming amount of information that requires careful selection and validation. We have used an alternative approach that incorporates more targeted analyses including lipidomics to specifically examine the role of glycerophospholipid metabolism in FHV RNA replication. The results presented in this report further support the well described crucial role that intracellular membranes play in positive-strand RNA virus replication [3–5], but emphasize that cellular lipids are key membrane constituents for this particular host-pathogen interaction. Furthermore, this report provides new details on the impact of specific lipid metabolism pathways on viral RNA replication, and in particular PC biosynthesis. The identification of specific lipid metabolism pathways is an essential first step in the rationale design of antiviral strategies that target cellular rather than viral components. Indeed, the recognition that cholesterol metabolism is important for hepatitis C virus replication in cultured cells [48, 49] has led to direct clinical trials using cholesterol synthesis inhibitors .
The observation that PC is important for FHV RNA replication in cells is consistent with results published almost twenty years ago, which demonstrated that phospholipids enhance FHV RNA replication complex activity in isolated membrane fractions analyzed in vitro . It also supports the hypothesis that one potential role cellular membranes play in viral RNA replication is to provide functional co-factors such as phospholipids for optimal RNA polymerase activity, and is consistent with published observations on the functional impact that phospholipids have on Semliki Forest virus nsP1 methyltransferase activity . The precise mechanism(s) whereby phospholipids enhance FHV RNA replication complex activity is unknown, and there are multiple steps during process of viral RNA replication that could be influenced by these cellular components (see Fig. 1C). Interestingly, the observation that individual knockdown of Cct1 or Cct2 expression had an apparent preferential effect on subgenomic RNA3 production (Fig. 5) suggests that this particular step may be especially sensitive to cellular phospholipids. In addition to their potential roles as functional cofactors, membrane-resident lipids may also play other roles during RNA replication, such as providing a scaffold for replication complex targeting and assembly. Indeed, we recently demonstrated that FHV protein A is a lipid-binding protein with particular affinity for anionic phospholipids, including the mitochondrial-specific phospholipid cardiolipin . Interestingly, we could not detect a significant physical interaction between FHV protein A and PC using in vitro assays , suggesting that PC may influence protein A activity indirectly or interact via some as yet unidentified intermediate protein or lipid.
Transcriptional array results suggested that FHV RNA replication stimulated PC synthesis in part via Cct1 or Cct2 upregulation, but the molecular mechanisms whereby FHV modulates lipid biosynthesis are unknown. One hypothesis is that FHV simply takes advantage of the cellular stress response to virus infection that may induce changes in phospholipid metabolism. However, published microarray results with cultured S2 cells infected with various pathogens, including viruses, bacteria, parasites, or fungi do not demonstrate a consistent upregulation of glycerophospholipid metabolism-related genes [44, 47, 52]. An alternative hypothesis is that FHV directly and specifically modulates glycerophospholipid metabolism. Interestingly, the enzyme encoded by Drosophila Cct1 is activated by cellular lipids, and in particular cardiolipin , suggesting a potential link with the observed protein A-cardiolipin interaction . Phosphatidic acid, which represents the essential precursor in the synthesis of all glycerophospholipids  (see also Fig. 4B), also physically interacts with FHV protein A  and could serve as a potential conduit for regulation. Although these hypotheses are speculative and cannot directly explain transcriptional upregulation, they are readily testable with FHV using established in vitro and in vivo systems.
We focused on PC for this report, but our results do not exclude the potential important role of other cellular phospholipids in FHV RNA replication. Indeed, both transcriptomic (Fig. 2A) and functional genetic (Fig. 4A) analyses identified numerous additional candidate lipid metabolism-associated genes linked to FHV RNA replication. Furthermore, the lipidomics analysis examined total cellular membrane lipid content, whereas FHV RNA replication complexes localize to outer mitochondrial membranes and induce dramatic morphological and structural changes [11, 15]. Although most glycerophospholipids, and in particular the highly abundant PC and PE, are widely distributed in membranes throughout the cell [29, 53], detailed analyses of mitochondrial outer membrane lipids may reveal interesting and potentially more dramatic changes than we observed with the total cellular lipid analyses. Experiments are currently in progress to isolate and examine mitochondrial and submitochondrial fractions from cells with active FHV RNA replication complexes using well established techniques . In addition, the observation that specific acyl chain modifications that result in longer unsaturated PC species were seen in FHV-infected cells suggests that particular lipid changes apart from the phospholipid head group also may play important roles in viral RNA replication. This is consistent with the demonstration that brome mosaic virus RNA replication is suppressed in yeast with a deletion of the enzyme Δ9 fatty acid desaturase . FHV replicates robustly in Saccharomyces cerevisiae [12, 56, 57], and preliminary results suggest that FHV RNA replication in yeast is also influenced by alterations in PC metabolism (K. Stapleford and D. Miller, unpublished results). Phospholipid metabolism pathways and their regulation have been well studied in yeast , and therefore this genetically tractable host provides an excellent companion for parallel studies with Drosophila cells to further examine the impact of lipid metabolism on FHV RNA replication.
In this study we demonstrate through a combination of complementary targeted and global analyses that glycerophospholipids, and in particular PC, play an important role in FHV RNA replication. These results enhance our understanding of the essential function that cellular membranes have in facilitating positive-strand RNA virus replication, and suggest that modulating cellular phospholipid synthesis may represent a novel approach for targeted antiviral development.
Cells and virus
Drosophila S2 cells were cultured in Schneider's Drosophila media (SDM) and transfected with inducible expression plasmids as previously described . Sucrose gradient-purified FHV  was used for all infection experiments.
Plasmids, antibodies, and chemicals
Standard molecular biology procedures were used for all cloning steps. The FHV RNA1 replicon expression plasmid pS2F1 (Fig. 1B), the protein A expression plasmid pS2FA-HA, and the control plasmid pS2LacZ have been previously described . The control plasmid pS2F1fs, which contains an early frameshifting mutation in the protein A coding region (Fig. 1B) and hence will not produce a functional cis replicon transcript [6, 12], was generated by cloning the ScaI/BsrGI fragment from pF1fs  into the MscI/Acc65I sites of pMT-V5/HisA (Invitrogen, Carlsbad, CA). Rabbit polyclonal antibodies against FHV protein A have been previously described , and antibodies against FHV protein B2 were generously provided by Paul Ahlquist (University of Wisconsin-Madison). Rabbit polyclonal antibodies against the hemagglutinin (HA) epitope tag or the voltage-dependent anion channel porin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) or Affinity Bioreagents (Golden, CO), respectively, while monoclonal antibodies against tubulin were purchased from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). All secondary reagents for ELISA and immunoblot analyses were purchased from Jackson Immunoresearch (West Grove, PA). The CTP:phosphocholine cytidyltransferase inhibitor miltefosine (1-hexadecylphosphorylcholine) was purchased from Calbiochem (San Diego, CA) and stored as a 100 mM stock solution in dimethylsulfoxide at -20°C. Oleic acid (cis-9-octadecenoic acid) was purchased from Sigma (St. Louis, MO) and stored as an undiluted stock solution at -20°C.
FHV infection, replicon induction, and total RNA isolation
Cells were either infected with FHV at a multiplicity of infection of 10 as previously described  and harvested at 12 to 24 h after infection, or transiently transfected with pS2F1 and pS2LacZ as previously described  and harvested at 18 h after induction with 0.5 mM copper sulfate. For microarray and phospholipid analyses, S2 cells stably transfected with pS2F1 or pS2F1fs were induced with 1 mM copper sulfate and harvested at 18 h after induction. Total RNA was isolated using TRIzol reagent (Invitrogen) per the manufacturer's instruction. For microarray and RT-PCR analyses, RNA was further processed by digestion with RQ1 DNAse and subsequent purification with RNAsy columns (Qiagen, Valencia, CA). Total RNA purity, integrity, and concentration were checked by spectrophotometry and agarose gel electrophoresis, and samples were stored at -80°C until analysis.
Affymetrix microarray data acquisition and analysis
Affymetrix Drosophila Genome 1.0 or 2.0 microarrays were used for FHV infection or replicon experiments, respectively, and biotinylated amplified cRNAs were produced using Affymetrix GeneChip target labelling kits per the manufacturer's instructions. Microarrays were scanned with an Affymetrix Scanner 3000, and complete original data files for all microarray experiments have been deposited in the Gene Expression Omnibus (GEO) database http://www.ncbi.nlm.nih.gov/geo/ under the accession number GSE15469.
Genomatix ChipInspector software package was used for primary microarray data analyses. This program uses a single probe method with an enhanced statistics package based on a SAM algorithm  that incorporates a t-test with a permuted artificial background to reduce false-positives. The following parameters were chosen to identify sets of up- or downregulated transcripts: (i) false-discovery rate of 1%; (ii) three probe minimum coverage; and (iii) 1.5-fold change from control. Similar results were obtained when microarray data were analyzed with the Affymetrix package of Bioconductor . The FlyBase database (FB2008_09; http://flybase.org/) was used to identify Gene Ontology (GO) terms, genetic interaction partners, and yeast-human orthologs for Drosophila genes identified in the microarray analyses.
Semiquantitative RT-PCR validation
Sequence (5' → 3')
Total PC content was determined using a modified phospholipase D-based enzymatic method initially developed for measurement of plasma PC levels . Briefly, S2 cells were pelleted at 1,000 × g for 5 min at 4°C, washed twice with Tris-buffered saline (TBS) (100 mM sodium chloride, 50 mM Tris, pH 7.2), lysed with 1% Triton X-100, and incubated with reaction buffer containing 50 mM Tris (pH 7.2), 0.64 M calcium chloride, 0.73 M N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyanaline, 0.73 M 4-aminoantipyrine, 120 U/ml PC-specific phospholipase D, 0.5 U/ml choline oxidase, and 20 U/ml peroxidase. Reactions were incubated at 37°C for 2 h and the absorbance at 595 nm was measured using a FLUOstar Omega microplate reader (BMG Labtech, Durham, NC). Quantitative PC concentrations were calculated using a standard curve generated with purified PC. Parallel total protein concentrations were determined with cell lysates prepared using TBS and 1% n-octyl-β-D-glucopuranoside and BioRad total protein reagent with bovine serum albumin as the standard. For analysis of individual cellular phospholipids, S2 cells were pelleted, washed twice with TBS, and lipids were extracted with chloroform:methanol using the method of Bligh and Dyer . Final extract samples were dried under vacuum and analyzed for polar lipid content by ESI-MS/MS at the Kansas Lipidomics Research Center http://www.k-state.edu/lipid/lipidomics/index.htm.
dsRNA production and RNAi induction
The O'Farrell RNAi library was purchased from the Drosophila Genome Research Center (DGRC). This collection contains 300-600 bp double-stranded cDNAs corresponding to greater than 7,000 individual phylogenetically conserved Drosophila genes , which can all be amplified by PCR using a common oligonucleotide primer (S2-RNAi-T7) that contains a GC-rich linker and T7 RNA polymerase promoter (Table 2). PCR products were generated using Eppendorf Mastermix with 200 nM primers, annealing temperature of 62°C, and 40 cycles. If no product was obtained after the first amplification, a second PCR was done using an aliquot of the first reaction as a template. PCR products were analyzed by non-denaturing gel electrophoresis and ethidium bromide staining and purified using a PCR cleanup kit (Promega) per the manufacturer's instruction. To generate the cDNA templates for Cct1 and Cct2 validation studies, candidate RNAi targets that differed from the O'Farrell library constructs were identified using the E-RNAi program . To generate control FHV RNA1-specific dsRNA we targeted nucleotides 40-735, which include the initiator AUG codon. RT-PCR with gene-specific primer sets that incorporated a T7 RNA polymerase promoter (Table 2) were used to generate PCR templates for dsRNA generation. Control LacZ and Hsp83 cDNAs were produced as previously described .
RNAs were generated using a T7 Megascript kit (Ambion) per the manufacturer's instruction, purified by phenol-chloroform extraction and isopropanol precipitation, and resuspended in RNAse-free water. dsRNAs were generated by heating samples to 65°C for 30 min and cooling slowly to room temperature. Samples were analyzed by non-denaturing agarose gel electrophoresis and ethidium bromide staining to ensure the formation of properly sized dsRNA products, quantitated by spectrophotometry, and stored at -20°C until used for RNAi studies. RNAi was induced in cultured S2 cells as previously described . Briefly, 10 μg dsRNA was added to 106 cells in 0.5 ml serum-free SDM, incubated at 25°C for 1 h, 0.5 ml SDM with 20% fetal bovine serum was added and cells were incubated for an additional 48-72 h prior to FHV infection or replicon transfection.
FHV RNA replication analyses
To facilitate the analysis of FHV RNA replication in a medium throughput format, an FHV protein B2 capture ELISA was developed. FHV protein B2-specific polyclonal antibodies for detection were purified from rabbit antisera by Staphylococcus aureus protein A affinity chromatography and biotinylated with 6-(biotinamidocaproylamido)caproic acid N-hydroxysuccinimide ester (Sigma) per the manufacturer's instructions. For capture antibodies, total immunoglobulins from FHV protein B2 antisera were isolated by saturated ammonium sulfate precipitation. Flexible microassay plates were coated with 5 μg/ml capture antibody in phosphate-buffered saline (PBS) (100 mM sodium chloride, 50 mM sodium phosphate, pH 7.4) overnight at 4°C, blocked with 1% non-fat milk, and S2 cell lysates in PBS with 0.5% Triton X-100 were incubated in duplicate wells for 2 h at room temperature. Plates were washed extensively with TBS and 0.1% Tween 20, incubated with 1.25 μg/ml biotinylated detection antibody followed by streptavidin-alkaline phosphatase. Plates were developed with 1 mg/ml p-nitrophenyl phosphate in 50 mM sodium carbonate buffer (pH 10.0) with 1 mM magnesium chloride and the absorbance at 405 nm was measured with the microplate reader described above. Initial optimization experiments demonstrated a high sensitivity for this assay, where lysates from less than ten S2 cells expressing a pS2F1-based replicon produced a positive signal (data not shown). Northern blot analyses for FHV-specific RNAs, immunoblot analyses for protein A accumulation, saponin-mediated permeabilization and differential centrifugation, and β-galactosidase assays were done as previously described [17, 22].
Viability assays were performed using 3- [4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) as previously described .
Microarray statistical analyses are described above. For additional statistical analyses a two-tailed t test assuming equal variances was used and a p value of < 0.05 was considered significant. All results are representative or a composite of at least three independent experiments, where quantitative data represent the mean ± standard errors of the mean.
We thank Donna Gschwend and Allison Simms for assistance and all laboratory members for their helpful comments on the research and manuscript. We thank Paul Ahlquist for provided reagents and James McDonald and the University of Michigan Affymetrix Microarray Core for assistance with microarray data acquisition and analyses. We also thank Mary Roth, Ruth Welti, and the Kansas Lipidomics Research Center Analytical Laboratory for ESI-MS/MS phospholipid analyses. This work was supported by National Institutes of Health grants K08-AI01770, R01-AI062749, and T32-GM007315.
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