HCV infects approximately 170 million people, including 2 million in Japan, and it is estimated that 3-4 million people are infected with HCV each year. HCV induces serious chronic hepatitis that results in the development of steatosis, cirrhosis and ultimately hepatocellular carcinoma (HCC). HCV core protein is well known to be the viral capsid protein as well as the pathogenic factor that induces steatosis and HCC without apparent inflammation in transgenic mice model. The functions of the core on the modulation of cellular events have been extensively examined and characterized. Recently, quantitative

mitochondrial proteomics show promising results for exploring the mechanism of HCV pathogenesis at the protein level. In this review, we summarize the current status of our knowledge regarding the pathogenicity of HCV core protein and the findings from recent proteomic surveys of mitochondrial proteins using HCV core gene transgenic mice. 12.1 HCVWith an estimated 3% of the world’s population currently infected with hepatitis C, and approximately 170 million persons at risk of hepatitis disease, the World Health Organization (WHO) recognizes hepatitis C as a global health problem [1]. Many HCV-infected individuals develop chronic hepatitis, which eventually progresses into liver cirrhosis and hepatocellular carcinoma. The shape of HCV is enveloped in a lipid bilayer in which envelope proteins (Envelope) are anchored. The envelope surrounds the nucleocapsid, which is composed of multiple copies of a small basic protein (core), and contains the RNA genome. HCV envelope proteins interact with several host receptors. The receptor proteins for HCV entry into cells are identified: CD81, scavenger receptor class B type I or SCARB1, and claudin-1. A fourth cellular protein, occludin, is found to be essential for HCV entry into cells [2]. Recently, liver microRNA miR-122 [3, 4] has been thought to be essential for viral infectivity. Gene expression profiling and proteomic approaches have led to the identification of several host-viral interactions [5]. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Recently, the development of HCV strain JFH-1, which generates infectious HCV in culture, has made a contribution to the study of the HCV life cycle [6]. 12.2 HCV GenomeHepatitis C virus (HCV) is an enveloped RNA virus of the Flavivirus family, in which a positive-sense, single-stranded RNA genome of approximately 9600 nucleotides (nt) is contained within the nucleocapsid (Fig. 12.1). The genome consists of a large translational open reading frame (ORF) encoding a polyprotein of approximately

3010 amino acids (aa) . The ORF is flanked by highly conserved untranslated regions (UTR) at both the 5¢ and 3¢ termini. The complete 5¢ UTR consists of 341 nt and acts as an internal ribosomal entry site. This feature leads to the translation of the RNA genome using a cap-independent mechanism, rather than ribosome scanning from the 5¢ end of a capped molecule. The polyprotein is processed by both the cellular and viral proteases to generate the viral gene products, which are subdivided into the structural and non-structural proteins. The structural proteins, which are encoded by the NH2-terminal quarter of the genome, include the core protein and the envelope proteins, E1 and E2. The E2 has an alternative form, E2-p7, though it is not clear whether the p7 composes the viral particle. The NS2, NS3, NS4A, NS4B, NS5A, and NS5B are the non-structural proteins that are coded in the remaining portion of the polyprotein. These include serine protease (NS3/4A), NTPase/helicase (NS3) and RNA-dependent RNA polymerase (NS5B). 5’UTR

Figure 12.1 Hepatitis C virus structure. Map of the HCV genome. The 9500-nucleotide-long positive-strand RNA of HCV is shown. It encodes a single 3000-amino-acid polyprotein that is proteolytically cleaved into mature proteins by virally encoded proteases. 12.3 HCV Core ProteinThe core protein of HCV occupies residues 1-191 of the precursor polyprotein and is cleaved between the core and E1 protein by host signal peptidase. The C-terminal membrane anchor of the core protein is further processed by host signal peptide peptidase [7]. The mature core protein is estimated to consist of 177-179 amino

acids and shares high homology among HCV genotypes. The HCV core protein possesses the hydrophilic N-terminal region “domain 1” (residues 1-117) followed by a hydrophobic region called “domain 2,” which is located from residue 118-170. The domain 1 is rich in arginine and lysine, and is implicated in RNA-binding and homo-oligomerization. The amphipathic helices I and II spanning from residue 119-136 and residue 148-164, respectively, in domain 2 are involved in the association of HCV core protein with lipid [8]. In addition, the region spanning from residue 112-152 is associated with membranes of the endoplasmic reticulum and mitochondria [9]. The core protein is also localized into the nucleus [10, 11] and binds to the nuclear proteasome activator PA28g/REGg, resulting in PA28g-dependent degradation of the core protein [12]. A recent report suggests that ubiquitination and adenosine triphosphate (ATP) are not required for PA28g-dependent proteasome activity [13]. HCV core protein is also known to be ubiquitinated by E3 ligase E6AP and degraded in the ubiquitin/ATP-dependent pathway [10]. In summary, the HCV core protein-unglycosylated protein-is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm and also detected in the nucleus. It has been reported to interfere with cell signaling by modulating mitogen-activated protein kinase (MAPK) signaling [14], interacting with STAT3 and RXR and modifying the expression of cellular protooncogenes such as c-myc and tumor suppressor genes (p53, p21, and pRb) [15]. In cooperation with H-ras, the HCV core has been reported to transform both immortalized and primary rat fibroblasts [16]. Our transgenic mice carrying HCV core gene develop HCC [17, 18]. This information implies that the core protein has a direct effect on the pathogenesis of diseases caused by HCV. 12.4 Transgenic Mice ModelWe have engineered transgenic mouse lines carrying the HCV genome by introducing the genes from the cDNA of the HCV genome of genotype 1b (Fig. 12.2) [17, 18]. Established are three different kinds of transgenic mouse lines, which carry the core gene, envelope genes or non-structural genes, respectively, under the same

transcriptional regulatory element. Among these mouse lines, only the transgenic mice carrying the core gene developed HCC in two independent lineages. The envelope gene transgenic mice do not develop HCC [19, 20]. Interestingly, the envelope gene transgenic mice develop an exocrinopathy in the salivary and lachrymal glands resembling Sjögren syndrome occurring in cases with human chronic HCV infection. The transgenic mice carrying the entire non-structural genes have developed no HCC.