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Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication.

ABSTRACT: BACKGROUND: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. RESULTS: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. CONCLUSION: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.

SUBMITTER: Castorena KM

PROVIDER: S-EPMC2847973 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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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 Kathryn M KM Stapleford Kenneth A KA Miller David J DJ

BMC genomics 20100317

<h4>Background</h4>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 wit ...[more]

PMID: 20236518

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Project description:The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account for the subtle phenotype of these mutants are discussed.

Dataset Information

Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication.

Publications

Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication.

Similar Datasets

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