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The Role of Cholesterol Pathways in Norovirus Replication

ABSTRACT: Norwalk virus (NV) is a prototype strain of the noroviruses (family Caliciviridae) which have emerged as major causes of acute gastroenteritis worldwide. We have developed NV replicon systems using reporter proteins such as a neomycin resistant protein (NV replicon-bearing cells) and a green fluorescent protein (pNV-GFP), and demonstrated that these systems were excellent tools to study virus replication in cell culture. In this study, we first performed DNA microarray analysis of the replicon-bearing cells to identify cellular factors associated with NV replication. The analysis demonstrated that genes in lipid (cholesterol) or carbohydrate metabolic pathways were significantly (p< 0.001) changed by the gene ontology analysis. Among genes in the cholesterol pathways, we found that mRNA levels of hydroxymethylglutaryl-CoA (HMG-CoA) synthase, squalene epoxidase and acyl-CoA:cholesterol acyltransferase(ACAT) 1, ACAT 2, small heterodimer partner, and low density lipoprotein receptor (LDLR)-related proteins were significantly changed in the cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE15520 | GEO | 2009/11/13

SECONDARY ACCESSION(S): PRJNA115691

REPOSITORIES: GEO

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Project description:The anabolic androgen 17M-NM-2-trenbolone (TB) can cause masculinization and reduce fecundity of fish. However, the underlying mechanisms of various biological pathways including metabolism, biosynthesis etc. are largely unknown. Here, we evaluated the effects of TB using the medaka DNA microarray representing 36,398 genes. Larval medaka, Oryzias latipes, (within 24 hrs posthatch) were exposed to TB at various concentrations (2, 6, 20, 60, 100, 200 ng/L) for up to 7 days. Dose-response relationships in gene expression levels of the categorized genes were analyzed using the cumulative chisquared method. Microarray analyses of the TB-exposed larvae showed that 117 and 32 genes were determined as up and down-regulated genes, respectively. The most significant GO term for biological process identified within this gene list was lipid metabolic process, which contained 26 genes in up-regulated genes. In this category, M-bM-^@M-^\cholesterol biosynthetic processM-bM-^@M-^] was highlighted as an important subcategory with 15 genes, including hydroxymethylglutaryl-CoA synthase cytoplasmic, squalene monooxygenase, lanosterol synthase etc. RT-PCR measurements in these genes were consistent with the microarray results in the direction and magnitude of these changes in gene expression. On the other hands, in the category of M-bM-^@M-^\sexual differentiation and developmentM-bM-^@M-^], genes related to hypothalamic-pituitary-gonadal (HPG) axis were not affected by TB treatment except for one gene encoded to cytochrome P450 19A1. Genes related to oogenesis, such as choriogenins and vitellogenins were weakly down regulated at 2-200 ng/L of TB. Our findings demonstrate that genes encoding cholesterol synthesis pathway via the mevalonate pathway were controlled by TB in larval medaka. TB concentrations used were 0 (control), 2, 6, 20, 60, 100 and 200 ng/L for 7 days of exposure. Each TB treatment had 90 larval medaka for each chamber. At day 7 of the exposures, triplicate samples (30 larvae/sample) from each chamber respectively were collected, flash-frozen in liquid nitrogen, and stored in liquid nitrogen until RNA extraction.

Project description:Pooled CRISPR knock-out (KO) screens using live viruses are a proven and valuable approach to identify essential host factors acting across the viral life cycle. Here we describe the development of a pooled, genome-wide CRISPR KO screening approach using stable viral replicon cell lines to specifically target host genes essential for viral replication complex formation and function. We benchmarked this approach with dengue virus 2 (DENV2), a model virus with well-established replicon systems and knowledge of the host factors requirements for replication. We developed a stable fluorescent DENV2 replicon cell line to perform a pooled genome wide FACS-based CRISPR KO screen. Our benchmark DENV2 screen successfully identified previously known host factors involved in DENV replication such as components of the ER membrane complex (EMC) and oligosaccharyltransferase (OST) complex. In addition, 2 other genes identified in the screen (DOHH and ZFP36L2) were validated to impact DENV2 replication. We extended the replicon screening approach to two different classes of viruses - Chikungunya virus (CHIKV), a positive sense RNA virus that is known to replicate at the plasma membrane, and Zaire Ebola Virus (ZEBOV), a negative sense RNA virus that replicates in cytoplasmic inclusion bodies. The CHIKV replicon screen identified several important host factors involved in CHIKV replication, including CSDE1 and GOLGA7 that were successfully validated in the context of live CHIKV infection. A distinct set of host factors such as Euchromatic histone-lysine N-methyltransferase 1 and 2 (EHMT1, 2), and Ubiquitin specific peptidase 7 (USP7) were identified in the ZEBOV replicon screen and were further validated using independent transient transfection assays. Comparative analysis of the combined hits from all 3 screens reveals distinct subsets of host pathway requirements for each virus. Thus, viral replicons-based screens can be a useful tool that can be extended to diverse classes of viruses to identify host pathways essential for viral replication and to uncover potential novel targets for host-directed antiviral therapeutics.

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The Role of Cholesterol Pathways in Norovirus Replication

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