Role of Cholesterol Pathways in Norovirus Replication

Abstract

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.

The Feline Calicivirus Leader of the Capsid Protein Is Associated with Cytopathic Effect

“…The function of the LC protein is not clear, but cleavage of the precursor to release LC and VP1 is essential for the recovery of infectious virions (5). Transient expression of the LC was reported to enhance replication of a human norovirus RNA replicon (23), and an increase in the level of mRNA for the low-density lipoprotein receptor (LDLR) was observed (24). We showed previously that the FCV LC can tolerate the insertion of foreign proteins such as green fluorescent protein and DsRed between amino acids 88 and 89, and recombinant viruses expressing fluorescent markers were used to visualize a calicivirus infection in real time (1).…”

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs

“…Unlike statins, treatment with ACAT inhibitors such as Cl-976, Sandoz 58-035, YIC-C8-434 and pyripyropene resulted in reduced levels of Norwalk replication and interestingly, in reduced levels of LDLR (Chang, 2009). This data indicate that ACAT may be a novel target for inhibiting norovirus replication and its inhibitors could be further developed as anti-norovirus drugs.…”

“…A study using Norwalk replicon system demonstrated that cholesterol pathways were also important in the replication of norovirus (Chang, 2009). Statins, such as www.intechopen.com simvastatin and lovastatin, are well known drugs that interfere with cholesterol pathways by inhibiting de novo synthesis of cholesterol through inhibition of HMG-CoA (3-hydroxy-3-methyl glutaryl-coenzyme A), reducing plasma cholesterol levels by upregulating low density lipoprotein receptor (LDLR) and promoting the uptake of LDL bound cholesterol to cells.…”

“…It has been demonstrated that the use of statins resulted in a reduction of HCV replication through blockage of protein geranylgeranylation and the proper formation of viral replicase complexes (Ye, 2007;Ye et al, 2003). On the contrary, the inhibition of cholesterol biosynthesis using statins significantly enhanced the replication of Norwalk virus which was correlated with an increased expression of LDLR (Chang, 2009). It was postulated that LDLR could play an important direct role in virus replication such as participating in viral replication complexes as an essential cofactor (Chang, 2009).…”

“…On the contrary, the inhibition of cholesterol biosynthesis using statins significantly enhanced the replication of Norwalk virus which was correlated with an increased expression of LDLR (Chang, 2009). It was postulated that LDLR could play an important direct role in virus replication such as participating in viral replication complexes as an essential cofactor (Chang, 2009). …”