The Role of Cholesterol Pathways in Norovirus Replication

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. To identify host-factors associated with norovirus replication, we have performed DNA microarray analysis of replicon-bearing cells (HG23) and parental Huh-7 cells using a Affymetrix GeneChip. One- or two-day old semi-confluent Huh-7 cells and HG23 cells grown in T-75 flasks were prepared to do DNA microarray analysis. Total RNA which was isolated using the RNeasy kit (Qiagen, Valensia, CA) was used on the DNA array. At least 3 independent cell cultures and RNA preparations were supplied for statistical analysis. The synthesis of cDNA, labeling and hybridization were done using Affymetrix regents according to the instructions from the company. We used Affymetrix GeneChip Human Genome U133 Plus 2.0 (a whole genome array covering 47,000 transcripts) for the DNA array. The entire process was done in the DNA array facility at Kansas State University. The genes either up- or down-regulated in HG23 cells compared to Huh-7 cells were analyzed with the Student’s t test of the three independent samples. The genes showing at least 1.5- or 2-fold difference with p<0.05 were further analyzed.