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Hepatitis C virus (HCV) infection is a major health problem world-wide. HCV infection causes chronic hepatitis in up to 60-80% of infected adults and is associated with steatosis, cirrhosis and hepatocellular carcinoma[8, 10]. HCV is an enveloped, positive-sense RNA (~9.6kb) virus of the family Flaviviridae. It has a central ORF flanked by the 5' and 3' noncoding regions. The ORF encodes one large polyprotein that can produce different proteins: the core protein, the envelope proteins (E1, E2, p7), and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B)[2, 6]. Our understanding of the biology of HCV RNA replication has been facilitated by the development of full-length HCV replicons that produce active HCV virions.
Chronic HCV infection is characterized by several histological features of the liver such as bile duct damage, lymphoid follicles, low cholesterol levels and steatosis[18]. During the early stages of HCV infection, host genes involved in lipid metabolism are differentially affected. Recently, genomic analysis of the livers of HCV-infected chimpanzees revealed that the transcriptional levels of the genes involved in the lipid metabolism and homeostasis are altered in vivo and in vitro[3, 25]. Francis V. Chisari et al also demonstrated that several cellular genes involved in lipid metabolism, such as ATP citrate lyase, clathrin light polypeptide, lipase A and GlcT-1, are differentially up-regulated during the early acute infection of HCV in chimpanzees[25]. Furthermore, it has been demonstrated that HCV genomic RNAs induce the expression of the elevated levels of ATP citrate lyase which is involved in cholesterol biosynthesis[15]. On the other hand, Ceramide glucosyltransferase (GlcT-1), plays an important role in the infection of some RNA viruses such as HIV-1[11]. GlcT-1, which catalyzes the first glycosylation step in glycosphingolipid biosynthesis, the transfer of glucose to ceramide, is a pivotal enzyme in the GSL synthesis. Therefore, it is important to investigate the relationship between HCV and host lipid metabolism by exploring the activity change of GlcT-1 caused by HCV proteins.
In this study, using the HCV full-length replicon system, we validated a positive correlation between HCV replication and the expression of genes involved in the fatty acid synthesis[9]. Our replicon system was constructed by the transfection of pHCV-WHU-1 to the Vero cells. Furthermore, we constructed ten stable Vero cell lines to carry individual HCV proteins. Study of these cell lines revealed that HCV NS5A and NS5B may regulate the transcription of GlcT-1.
HCV NS5A and NS5B Enhance Expression of Human Ceramide Glucosyltransferase Gene
- Received Date: 11 October 2011
- Accepted Date: 13 December 2011
Abstract: Host genes involved in lipid metabolism are differentially affected during the early stages of hepatitis C virus (HCV) infection. Here we demonstrate that artificial up-regulation of fatty acid biosynthesis has a positive effect on the replication of the HCV full-length replicon when cells were treated with nystatin. Conversely, the HCV RNA replication was decreased when fatty acid biosynthesis was inhibited with 25-hydroxycholesterol and PDMP(D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol). In agreement with these results, the expression level of GlcT-1(ceramide glucosyltransferase), a host glucosyltransferase in the first step of GSL (glycosphingolipid) biosynthesis, was found to be closely associated with the expression and replication of HCV RNA. On the other hand, the viral RNA can also activate GlcT-1 in the early stage of viral RNA transfection in vitro. To identify viral factors that are responsible for GlcT-1 activation, we constructed ten stable Vero cell lines that express individual HCV proteins. Based on the analyses of these cell lines and transient transfection assay of the GlcT-1 promoter regions, we conclude that HCV proteins, especially NS5A and NS5B, have positive effects on the expression of GlcT-1. It is possible that NS5A and NS5B stimulate transcription factor(s) to activate the expression of GlcT-1 by increasing its transcription level.