Role of Cholesterol Pathways in Norovirus Replication

Norwalk virus (NV) is a prototype strain of the noroviruses (family Caliciviridae) that have emerged as major causes of acute gastroenteritis worldwide. I 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 the present study, I 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, I found that mRNA levels of hydroxymethylglutaryl-coenzyme A (HMG-CoA) synthase, squalene epoxidase, and acyl-CoA:cholesterol acyltransferase (ACAT), ACAT2, small heterodimer partner, and low-density lipoprotein receptor (LDLR)-related proteins were significantly changed in the cells. I also found that the inhibition of cholesterol biosynthesis using statins (an HMG-CoA reductase inhibitor) significantly increased the levels of NV proteins and RNA, whereas inhibitors of ACAT significantly reduced the replication of NV in replicon-bearing cells. Up- or downregulation of virus replication with these agents significantly correlated with the mRNA level of LDLR in replicon-bearing cells. Finally, I found that the expression of LDLR promoted NV replication in trans by transfection study with pNV-GFP. I conclude that the cholesterol pathways such as LDLR expression and ACAT activity may be crucial in the replication of noroviruses in cells, which may provide potential therapeutic targets for viral infection.Human noroviruses are now the leading cause of food- or waterborne gastroenteritis illnesses responsible for more than 60% of outbreaks (10). It has been estimated that noroviruses cause 23 million cases of illness, 50,000 hospitalizations, and 300 deaths each year in the United States alone (19). Molecular epidemiological studies have confirmed a global distribution of these viruses (13). The major public health concern with human noroviruses is their ability to cause large outbreaks in group settings such as schools, restaurants, summer camps, military units, hospitals, nursing homes, and cruise ships. Human noroviruses are currently classified as NIAID category B priority pathogens (category B bioterrorism agents). Noroviruses generally cause mild to moderate gastroenteritis, but the disease can be severe to life-threatening in the young, the elderly, and immunocompromised patients. During the last decade, noroviruses have gained media attention for causing large-scale outbreaks of gastroenteritis on cruise ships, in nursing homes, etc. Although noroviruses do not multiply in food or water, they can cause large outbreaks because as few as 10 to 100 virions are sufficient to cause illness in a healthy adult (12). Recent reports of noroviral gastroenteritis outbreaks among hurricane Katrina evacuees underscores the importance of preventive and therapeutic measures for noroviruses to promote public health (32). However, no vaccines or antivirals are currently available for the prevention or treatment of norovirus disease in humans, which is largely due to the absence of a cell culture system for human noroviruses. The recent development of replicon-bearing cells for Norwalk virus (NV) (7) has made possible the study of NV replication in cells and the discovery of antivirals. We recently demonstrated that the system provides an excellent platform for screening small molecules for antivirals (3, 7). We also reported another NV replicon system with reporter genes (green fluorescent protein GFP] or luciferase) to study virus replication (4).As a component of membrane structures and a precursor for the steroid hormones and bile acids, cholesterol is one of the most essential biological molecules in the body (8). Cholesterol levels are maintained by controlling both de novo synthesis (major) and dietary uptake (minor) of cholesterol (8). De novo synthesis of cholesterol is subject to complex regulatory controls by various enzymes such as 3-hydroxy-3-methyl glutaryl-coenzyme A (HMG-CoA) reductase and acyl-CoA:cholesterol acyltransferase (ACAT) (1, 8, 21). The synthesis of bile acids from cholesterol is also tightly controlled and represents an important factor the cholesterol homeostasis (14, 22, 23). In the present study, I first performed DNA microarray analysis of replicon-bearing cells to identify cellular factors associated with NV replication. Analysis showed genes in lipid (cholesterol) or carbohydrate metabolic pathways were significantly (P < 0.001) changed by the gene ontology analysis. Because it has been shown that bile acids are essential for the replication of porcine enteric calicivirus (PEC) in cells (6) and important natural modulators of cholesterol pathways, I was particularly interested in potential regulation genes in the cholesterol pathways. I demonstrate here that the modulation of the cholesterol pathways via inhibitors of HMG-CoA reductase or ACAT led to either up- or downregulation of the replication of NV. I also show that the expression level of low-density lipoprotein receptor (LDLR) was positively correlated with NV replication in cells. These studies suggest that the cholesterol pathway is crucial for norovirus replication and provide potential therapeutic targets for noroviral infection.