8.1 Introduction: Hepatitis C virus (HCV) infection was first suspected in the 1970s when most blood transfusion infections were associated with either hepatitis A or hepatitis B virus. The viral genome was identified in 1989 and named hepatitis C. HCV belongs to the Hepacivirus, Flavivridae family, and has six major genotypes, and more than 70 subtypes. The HCV genome encodes a single precursor polyprotein of about 3,000 amino acids which is cleaved into co- and post-translationally into functional structural and non-structural proteins by host and viral protease including three structural proteins: the core proteins forming the viral nucelocapsid and two envelope glycoproteins: E1 and E2. HCV particle have a size of about 55-60 nm in diameter. The E1 and E2 are type I transmembrane glycoprotein composing of up to 6 and 11 potential glycosylation sites respectively as well as forming noncovalent heterodimers. The nucelocapsid is probably made of multiple copies of core protein in complex with the viral genome and lies beneath the envelope. The nonstructural proteins are NS2, NS3, NS4A, NS4B, NS5A, and NS5B. HCV infection is a major public health issue with an estimated 170 million people infected globally and approximately 3 to 4 million new infections yearly. Most of the patients infected are unaware of their status and remain asymptomatic; however majority will progress to chronic HCV. The infection by HCV maybe unresolved in about 85% of the infected individuals which represents an important cause of liver cirrhosis and hepatocellular carcinoma (HCC). HCV is the most common cause of chronic liver disease and cirrhosis in the world as well as the major cause of liver transplantation in USA, Australia, and Europe. The chronic infection is evidenced by the presence of HCV-RNA in the blood at least 6 months after viral contamination. HCV infects the hepatocytes and other different cells such as leukocytes, and epithelial cells of different organs. However it does not cause cytotoxicity which suggests that both the hepatitis injury and extra-hepatic clinical manifestations caused by HCV infection are probably mediated by the immune evasion of cryoglobulinemia, and complex persistence and autoimmune recognition. This means that the pathogenesis of HCV infection involves a complex virus/host interaction which is the focus of this chapter.
8.2 Association of HCV and HCC: HCC accounts for 85 to 90% of all cases of primary liver cancer. Chronic hepatitis and cirrhosis constitutes most of the tumor preneoplastic condition in majority of HCC. The risk of developing HCC for patients with HCV-associated cirrhosis is about 2-6% per year. HCC risk increases to 17-fold in HCV-infected patients compared to HCV-negative patients. In general, HCC develops after 2 or more decades of HCV infection and the increased risk is restricted to patients with cirrhosis or advanced fibrosis. It has been explained earlier that multiple steps are needed for the induction of all cancers. For hepatocarcinoma, it is mandatory that genetic mutation should accumulate in the hepatocytes. In HCV infection, however, it has been found that some of these steps might be skipped in the development of HCC in the presence of the core protein. The overall effects achieved by the expression of the core protein are the induction of HCC, even in the absence of a complete set of genetic changes required for carcinogenesis. HCC is among the leading cause of cancer-associated mortality, and the third most common cause of cancer deaths in the world. In Japan, for example, unlike other Asia countries, there are high incidences of HCC caused by HCV infection accounting for 80 to 90% of all cases, while in the Western countries, HCC is known to complicate cirrhosis secondary to hepatitis C in 2-6% per year. Currently there is no evidence to show that HCV by itself is oncogenic; however HCC may rarely develop in non-cirrhotic HCV-infected individuals. This means a direct oncogenic effect cannot be ruled out. However, in the pathogenesis of HCV-associated HCC, it remains controversial whether the virus plays a direct or indirect role. Studies using transgenic mouse models in which the core protein of HCV has oncogenic potential showed that HCV is directly involved in hepatocarcinogenesis, although other factors such as continued cell death, and regeneration associated with inflammation may have a role. HCV causes HCC via an indirect pathway by causing chronic inflammation, cell death, proliferation, and cirrhosis. HCV genome has been detected in the tumor and surrounding liver tissue. Regarding association with the genotype of HCV and HCC, the incidence of genotype 1b has been shown to be markedly high among the patients where it is associated with a more rapid determination of the liver histology in chronic hepatitis. However some studies have argued against this proposition. Prospective studies undertaken to establish whether inflammation with specific HCV genotype was associated with an increased risk of development of HCC in cirrhosis reported that cirrhotic patients infected with HCV ib carry significant higher risk of developing HCC than patients infected by other HCV types. There is the suggestion that the presence of HBV gene in patients with chronic HCV-associated liver injury appears to promote hepatocarcinogenesis.
8.3 HCV/ Host Interaction: Just like other viruses, HCV absolutely depends on the host cell for their replication. Studies of HCV life cycle (figure 1) has been hampered by lack of efficient cell culture systems to generate infectious viral particles in vitro. However using a number of model systems such as recombinant HCV envelope glycoprotein, HCV-like particles (HCV-LPs), HCV cell culture (HCVcc), and retroviral HCV pseudotypes (HCVpp), it was shown that E1 and E2 are critical for host cell entry. Studies using monoclonal or polyclonal antibodies targeting both the linear and conformational epitopes of E2 showed that there was inhibition of cellular binding of HCV-LP binding, entry of HCVpp and infection of HCVcc which suggest that E2 has essential role for host cell surface interaction. Within E2 envelope glycoprotein sequence are hypervariable regions. These amino acids differ by up to 80% among the HCV genotypes, and even differ among subtypes of the same genotypes, The N-terminal 27 residues of E2 show high degree of variation and this portion of the sequence is termed hypervariable regions 1 (HVR-1). This region plays a crucial role in HCV interaction with the host cell.
Figure 1: HCV Life cycle
A number of studies have demonstrated the role of HVR-1 in HCV infectivity. Studies have shown that antibodies targeting regions within HVR-1 inhibit cellular recombinant E2, HCV-LP binding, and HCVpp entry into target cells. The exact role of E1 remains unknown but has been proposed that E1 may directly interact with cell surface molecules and/or contribute to proper folding and processing of E2. It has been shown that antibodies targeting the N-terminal region of E1 inhibit HCV-LP binding and HCV infection of B-cell derived cell line which suggests that E1 cell surface interaction may contribute to viral binding and entry. Furthermore, it has been shown that HCV envelope E1 and E2 induce fusion between the viral envelope and host cell membrane. HCV entry into hepatocytes is a multistep process which involves the utilization of multiple host molecules such as low density lipoprotein receptor (LDLR), tetraspanin CD81, scavenger receptor class B type I (SR-BI), tight junction (TJ) proteins, Claudi-1 (CLDNI), the cholesterol uptake receptor Niemann-Pick C1-like1 (NPC1L1), and occludin (OCLN). In addition, receptor tyrosine kinase epidermal growth factor receptor and ephirin receptor AZ have been identified as HCV entry cofactors. Martin and Uprichard also identified transferring receptor 1 as a HCV entry factor. Several studies analyzing interaction of HCV proteins with host cellular proteins utilizing proteomics analysis showed that at least 420 host proteins interact with Jak/STAT, insulin, TGF β, and focal adhesion molecules pathways with majority of these interacting with core proteins, NS3, and NS5A. It is interest to note that NS5A interacts with many proteins implicated in signal transduction, cell growth and death, and in cancer. Data shows that viral proteins plays central role in regulating metabolic processes, cell-to-cell adhesion, and cytoskeletal organization, leading to HCV pathogenesis. Some of these interactions plays essential role in viral replication, eliciting strong cellular and humoral immune response, developing strategies to evade immune recognition, and in the induction of hepatitis leading to cirrhosis and HCC. HCV have developed different mechanisms of hijacking protein -1 and its associated proteins, DEAD box helicases DDX3 and DDX6 as well as heteronuclear protein A1 which are all associated with viral replication, assembly and virus egress. The viral proteins core, NS2, NS3/4A, and NS5A have been shown to be associated with interacting with key oncoproteins and contribute to the development of HCC in HCV-infected patients; although understanding the mechanism of such interaction remains elusive. After the establishment of persistence infection, some of the viral proteins interact with the host cellular proteins resulting in change of their properties and functions. Due to this, the cells and extracellular matrix components change over a period of time leading to the remodeling of the tissue and loss of control as well as regulation of cell proliferation. Marked induction of ROS in infected cells leads to oxidative stress and suppression of host immunity by viral proteins as reported by some studies. Also HCV proteins interacts with the cellular molecules regulatory gene YB-1, p53, and cyclin D1 resulting in the induction of liver cancer. HCV constantly evolves new variants during persistent infection with the host been constantly subjected to episodes of hepatitis. These variants evade immune recognition, alter interactions with the host cell proteins, and induce chromosomal abnormalities which result in liver cancer. Asialoglycoprotein receptor has also been suggested to play a role in HCV entry based on interaction between this protein and baculovirus-expressed HCV structural proteins. However, the importance of this data in the context of functional HCV entry has not been elucidated.
8.4 Entry Inhibitors: As outlined earlier, HCV is a global public health issue associated with chronic hepatitis, liver cirrhosis, and HCC. The only approved treatment is a combination therapy with IFN-α and ribavirin which targets cellular pathway. However, sustained virologic response is achieved only in about half of patients treated. These are the urgent need for the identification of novel drug targets against HCV. Although most of the research focuses on the development of HCV-specific antivirals such as protease and polymerase inhibitors, cellular targets could be ideal for the development of broad spectrum of antivirals. One strategy is silencing of cellular proteins. In a study it was found that by silencing some proteins using RNA interference (RNAi) decreased HCV infectious titers by 42-fold which means that the cellular host proteins could be associated with HCV replication, release, or viral entry. Also targeting same interaction patterns as small molecules inhibitors or specific antibodies might increases antiviral efficacy. A study showed that antibody-mediated blocking of HCV co receptor CD81 prevented infection with HCV. A study by Randell et al showed that RNAi machinery exerted a proviral effect on HCV life cycle because by silencing of Dicer and components of RNA-induced silencing complex RISC (Argonaute proteins EIF2C1-4) decreased HCV infectious titer. Another novel approach is using potential inhibitors of viral attachment. As found in HIV infection, lactin cynanovirin-N (CV-N) is an active compound and was shown to possess antiviral activity against other envelope viruses. The antiviral ability is due to interaction between CV-N and high mannose oligosaccharides in the viral envelope glycoprotein. It has been shown that the envelope of HCV is highly glycosylated containing oligomannose glycans. Oligomannose glycans interact with CV-N leading to HCV antiviral activity by blocking HCV entry into target cell. Because HCV glycosylate sites are highly conserved, drugs that target glycans on HCV glycoprotein may not lead to the rapid development of viral escapes or resistance as found in other carbohydrate-binding agents such as plant lectins, mAb, and other specific-non-peptide antibiotics such as Predimicin A which is a HCV infectivity inhibitor. These substances can be used efficiently against viruses that need glycosylated envelope for entry into target cells. Another potential approach is the utilization of heparin-derived molecules because heparin has been shown to potently inhibit HCV E2, HCVpp, HCV-LP and HCVcc binding to hepatoma cells. Already inhibitors of EGFR (erlotinib), EphA2 (dasatinib), and NPCILI (ezetimibe) have been licensed and shown to inhibit HCV entry in vitro. In addition, a small inhibitor of SR-BI (ITX 5061) is in advanced clinical study as HCV entry inhibitor. There is the need to develop our understanding of the complex viral entry process to enable us develops new therapeutic targets to prevent the virus from reaching its site of replication. Both the viral and host cell components involve in virus entry may serve as targets for the development of HCV entry inhibitors. However, targeting viral proteins rather than host cell proteins will be more ideal because of potential adverse events resulting from interference with normal cell functions. Future anti-HCV therapies should be combination of drugs that target distinct steps of HCV infection (Figure 2)
Figure 2: Targets of HCV infectivity inhibition
One of the major limitations of studying HCV-host interaction is the availability of limited number of HCV strains and cell types used to characterize them. There are seven known HCV genotypes with a nucleotide sequence diversity of about 30-35%. But the available viral infection clones are for only four genotypes 1a, 1b, 2a, 3a, and 4a. Of these only genotypes for 2a JFH1 and adapted genotypes 2a J6, 2b J8, and 1a H7 33 complete the viral life cycle in cell culture. An advanced is the development of Chimeric HCV which contains JFH1 nonstructural genes that has been fused to structural genes for all seven HCV genotypes. It allows for cross-genotyping to compare virus-host interaction with HCV core, E1, E2, p7, and NS2. For example it has been found that infection with all seven genotypes chimers can be neutralized with antibodies to CD81 and SR-BI. A number of in vitro studies have been undertaken to validate host cell factors important for HCV replication mostly in single human hepatoma cell line, Huh-7 and other sublines termed Huh-7.5, Huh-7.5.1, and Huh-7 lunet cells. There has been some progress in developing polarized cell models for HCV entry, such as Caco-2 colorectal adenocarcinoma cells that develops columnar polarity and the HepG2 hepatoma cells that develop complex hepatic polarity. Primary hepatocytes from patients can be productively infected with HCV but high variability between patients and access to fresh hepatocytes are some of the limitations. An ideal approach is using pluripotent stem (iPS) cells. Numerous HCV-host interaction factors have been identified despite of the experimental hurdles but still new model for studying HCV-host interaction are needed to address some of the intriguing host factors which will lead to better understanding of this interaction.
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