Michael R. Beard School of Molecular and Biomedical Science, University of Adelaide, and Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia
Introduction
Hepatitis C virus (HCV) is classified in the Hepacivirus genus within the family Flaviviridae and is the leading cause of chronic hepatitis and liver disease related morbidity worldwide. With an estimated 170 million people infected worldwide and the ability of the virus to establish a chronic infection in approximately 70% of cases, it is not surprising that HCV represents a major cause of global suffering and morbidity and a burden to many public health systems. Chronic HCV infection is often associated with development of serious liver disease, including cirrhosis, liver failure, and hepatocellular carcinoma. Accordingly, a thorough understanding of the life cycle and molecular biology of HCV and its interaction with the host are essential in the development of treatment and vaccine strategies. Although these studies have been hampered by the lack of a small-animal model and, until recently, a lack of a tissue culture system that accurately reflects the life cycle of HCV, significant progress has been made in the understanding of HCV molecular biology and pathogenesis. In this chapter we discuss recent advances in models to study HCV replication and the HCV life cycle.
The HCV Genome
HCV possesses a 9.6 kb single-stranded, positive-sense RNA genome composed of a 5' UTR (untranslated region), a long open-reading frame (ORF) encoding a polyprotein of approximately 3000 amino acids, and a 3U TR (Figure 1.1). The polyprotein can be divided into three segments based on the functional aspects of the proteins: the NH2 terminal region comprises the structural proteins (core, E1, and E2); a central region consists of two proteins (p7 and NS2) that are not involved in HCV replication or are structural components of the virus, but probably play a role in virion morphogenesis; and the COOH-terminal proteins (NS3, NS4A, NS4B, NS5A, and NS5B) that are required for HCV replication (Figure 1.1). A detailed description and function of the HCV proteins can be found in an excellent review from Moradpour and colleagues. After release of the HCV genome into the cytoplasm the genome is exposed to the host cellular machinery for translation of the viral polyprotein. The 5' and 3' UTRs are highly conserved and critical to viral genome replication and translation of the viral polyprotein. The 5' UTR is approximately 341 nucleotides long and contains a highly structured RNA element known as the internal ribosome entry site (IRES) that is recognized by the cellular 40S ribosomal subunit to initiate translation of the RNA genome in a cap-independent manner. The importance of the secondary and tertiary structure of the IRES domain for initiation of translation has been demonstrated by mutational analysis. However, the primary sequence, particularly in stem-loop IIId and IIIe, is also critical for efficient HCV IRES activity. Recently, the structural nature of HCV IRES interactions with the 40S ribosomal subunit and the eIF3 complex has been revealed by cryo-electron microscopy. Preceding the IRES at the extreme 5' end are elements required for viral replication that overlap partially with the IRES region (domain II), leading to speculation that this region is involved in regulation of a viral translation to replication switch. Consistent with this speculation is the recent observation that a short highly conserved RNA segment at the 5' end of the HCV genome binds a liver-specific microRNA, miR-122, that is required for efficient HCV replication in cultured hepatoma cells. However, in the HCV-infected liver no correlation was noted between miR-122 abundance and levels of HCV RNA in the liver or serum, which suggests that the impact of miR-122 may be less prominent in vivo than in vitro. miR-122 may impact HCV replication indirectly through stimulation of translation of the viral polyprotein by enhancing association of ribosomes with the IRES. Although more work is required to determine its role in the HCV life cycle and pathogenesis, modulation of miR-122 expression and activity presents as an attractive target for future therapy.
Like the 5' UTR, the 3' UTR of the HCV genome contains a high degree of secondary structure. This region is 200–300 nucleotides in length and is comprised of three major elements involved in replication: (i) a variable region (30–50 nucleotides), which directly follows the NS5B stop codon; (ii) a polyuridine (U/C) tract (20–200 nucleotides); and (iii) a highly conserved region (98 nucleotides), known as the 3' X region, which forms a three stem-loop structure. Mutational analysis has revealed that the poly-U/C tract and the 3' X region play a more important role than the variable region in the synthesis of negative-strand RNA.
Models to Study HCV Replication
HCV Replicons
The development of the subgenomic HCV replicon system, first reported in 1999, significantly enabled the study of HCV replication in cultured cells for the first time. Replicons represent autonomously replicating HCV RNAs, and typically contain an inframe insertion of a selectable antibiotic marker (e.g., neomycin phosphotransferase: G418) within the amino terminal HCV core sequence, followed downstream by a heterologous IRES from encephalomyocarditis virus (EMCV), a picornavirus, to drive internal translation of the downstream HCV open reading frame (NS2 to NS5B) (Figure 1.2). The minimal requirements for a viable HCV replicon are HCV-derived 5' and 3' termini and the non-structural proteins (NS3 to NS5B) that form the replication complex, however, replication-competent HCV replicons encoding the complete HCV polyprotein are viable. Transfection of Huh-7 (hepatoma-derived) cells with synthetically derived transcripts followed by selection with G418 results in the establishment of cell lines that harbor autonomous replication of the virus. HCV RNA isolated from cell lines under antibiotic selection often contains cell-culture adapted mutations that greatly enhance replication, although the molecular basis for this increased replication is unclear. These adaptive mutations often map to the NS5A protein and may potentially influence phosphorylation, resulting in a hypophosphorylated state and increased replication. Adaptive mutations have also been mapped to NS3, NS4A, NS4B, and NS5B. Interestingly, these replication-adaptive cell-culture mutations have been shown to reduce in vivo infectivity in chimpanzees, highlighting the adaptive nature of these viruses derived from cell culture. HCV replicons are not restricted to Huh-7 cells, and other cell lines such as HeLa and cells of murine origin have also yielded selected clones of replicating HCV, highlighting that HCV replication is not restricted to liver-derived cells of human origin.
The antibiotic selection process not only selects for HCV genomes with high replication capacity but also clones of Huh-7 cells that are highly permissive for HCV infection. One such cell line, Huh-7.5, has been "cured" of HCV by treatment with low doses of interferon, is hyperpermissive for HCV replication, and is clearly enriched for factors that promote replication and/or defects in innate viral sensing pathways. For example, Huh-7.5 cells have a spontaneous knockout of the dsRNA cellular sensing protein RIG-1 and do not mount a robust antiviral response to viral infection that allows for HCV replication. This highlights the importance of innate immune sensing in HCV infection and is consistent with the ability of the HCV NS3/4A protein to cleave IPS-1, which is integral to the innate immune RIG-I pathway.
HCV replicons have been valuable tools for studying numerous aspects of the HCV life cycle and interaction with the host cell. However, their major limitation has been inability to produce infectious virus particles even when the complete complement of HCV proteins is expressed, for reasons that are not entirely clear. The original replicon concept has undergone evolution and replicons are now available that contain various markers (e.g., GFP, luciferase) that allow quantitative assessment of HCV replication and have been useful in high-throughput screening of antiviral compounds.
Productive Viral Infection in Cell Culture
The recent identification in 2005 of a cloned HCV genome (genotype 2a), known as JFH-1, that is capable of initiating high-level replication in cell culture and production of infectious virus particles represents a major breakthrough in the pursuit of a cell-culture model for HCV. In contrast to HCV replicon systems, transfection of Huh7 cells with RNA synthesized in vitro from the cloned JFH-1 cDNA genome and a related genotype 2a chimera, FL-J6/JFH replicate efficiently in Huh-7 cells without the need for cell-culture adaptive mutations. Moreover, virus particles produced by these cells are infectious in chimpanzees and can be serially passaged in vivo while the FL-J6/JFH virus can infect mice containing human liver grafts. Interestingly, virus produced in vivo has a lower buoyant density than virus produced in cell culture, suggesting association with low-density lipids. This system represents a major advance in the study of virus-host interactions and the virus life cycle, all in the context of replicating virus. Similarly, the highly adapted genotype 1a HCV isolate known as H77-S (derived from the H77 isolate) is also capable of instigating HCV RNA replication and production of infectious virus particles. This represents another breakthrough in the generation of tools to study the HCV lifecycle, particularly because this genotype is more prevalent worldwide and is associated with more significant liver disease. Intrageno-typic and intergenotypic chimeras of HCV that contain the non-structural protein-encoding regions of JFH-1 and the structural protein-encoding regions of other HCV genomes may help define regions of structural proteins that influence the efficiency of virus particle synthesis and secretion. This relatively new cell-culture model system will be invaluable in the study of many aspects of virus-host interaction, including viral entry, and assembly and release, which were previously inaccessible to manipulation.
The HCV Virion and Entry
The relatively low levels of HCV in plasma samples have hampered visualization of viral particles; however, virus-like particles have been identified by electron microscopy, which has indicated that infectious HCV virions are roughly spherical particles of diameter 5565 nm with fine projections of approximately 6 nm (and references therein). The major protein constituents of the host-derived lipid bilayer envelope are the highly glycosylated HCV envelope glycoproteins E1 and E2 that surround the viral nucleocapsid, composed of many copies of the HCV core protein and the genomic HCV RNA (Figure 1.3). HCV from serum and plasma fractionates with a wide range of buoyant densities that can be attributed to association of the virus with lipoproteins, in particular apolipoprotein-B (Apo-B) and apolipoprotein-E (ApoE), which are components of host low density lipoprotein (LDL, Apo-B) and very low density lipoprotein (VLDL, Apo-B, Apo-E) and suggest a close association with circulating LDL/VLDL. The physiological association of HCV with LDL/VLDL remains unexplained mechanistically; however, it could be involved in viral uptake (see below), or alternatively the association of Apo-B with HCV virions may indicate a role for the hepatic LDL/VLDL secretory pathway in release of the virus.
Hepatocytes are the main target for infection with HCV; however, identification of the cellular receptors responsible for HCV entry has proven difficult due to the lack of appropriate model systems. However, using a combination of HCV pseudotyped particles (HCVpp) and cell-culture-derived HCV (HCVcc), the complement of HCV receptors now seems complete.
The 25 kDa tetraspanin molecule CD81 and the human scavenger receptor class B type I (SR-BI) both bind HCV E2 and are necessary but not sufficient for HCV entry. For example, CD81 ectopic expression in hepatocyte-derived cell lines that are negative for CD81 confers susceptibility to HCVpp and HCVcc; however, expression of both factors in non-hepatocyte-derived cell lines does not concur infectivity. Clearly additional hepatocyte factors are required for HCV entry. Using an interactive cloning and expression approach, the tight junction protein claudin-1 (CLDN1) was recently identified as an HCV co-receptor. CLDN1 was found to be essential for entry into hepatic cells and rendered non-hepatic cells susceptible to infection. However, despite the identification of CD81, SR-B1, and CLDN1 as essential HCV entry co-factors, a number of human cell lines and those of nonprimate origin remained resistant to HCV infection, suggesting an additional entry factor. Using a cyclic lentivirus-based screen of a cDNA library derived from a highly HCV-permissive hepatocarcinoma cell line (Huh-7.5) for genes that render the non-permissive CD81+, SR-BI+ 293T cell line infectable with HCVpp, the remaining crucial factor was recently identified as occludin (OCLN),also a tight junction protein [34]. Although expression of all four entry factors (CD81, SR-B1, CLDN1, OCLN) renders mouse cell lines susceptible to HCVpp infection, these cells could not support HCVcc infection. This is not surprising given past reports of inefficient replication of HCV RNA in mouse cell lines, and suggests that specific hepatocyte factors are crucial for efficient HCV replication. The identification of CD81 and OCLN as the minimal humanspecific entry factors (HCV can bind to murine SR-B1 and CLDN1) not only significantly advances our understanding of the molecular mechanisms of HCV entry but also provides important steps for the development of a mouse model of HCV infection and provides an attractive target for the development of novel antiviral strategies.
Other molecules have been suggested to be involved in HCV entry. The association of HCV virions in serum with LDL and VLDL suggests that the LDL receptor (LDLR) may be an attractive candidate receptor. However, its precise role remains to be determined. LDLR is not sufficient itself for entry and it does not bind directly to HCV E2. Together with the glycosaminoglycans, the LDLR in concert with other cell-surface proteins may serve to collect HCV at the cell surface and facilitate binding with receptors crucial for HCV entry. Consistent with this, a role has been proposed for L-SIGN and DC-SIGN in HCV attachment although they do not seem to mediate cell entry of HCV and their role is unclear.
The precise molecular events underlying HCV binding and entry are not well understood. However, HCV binding to the cell surface is thought to occur in a stepwise process by binding to several receptors followed by transfer to the tight junction proteins CLDN1 and OCLN that may facilitate cellular uptake (Figure 1.4). Similar to other flaviviruses, HCV entry is thought to be mediated by clathrin-mediated endocytosis with delivery of the nucleocapsid from the endosome in a pH-dependent manner. Furthermore, the E1 and E2 proteins are class II fusion proteins that result from the production of a fusion pore in the endosome membrane that facilitates genome release to the cytoplasm.
HCV Replication
The HCV replication process is summarized in Figure 1.5. After translation of the HCV proteins from the positive-sense RNA genome by direct interaction of the host 40S ribosomal subunit with the IRES within the 5' UTR of the genome, HCV replication begins. This IRES-directed translation is cap-independent and enables virus translation/replication to continue even after host cell cap-dependent translation has been shut down in response to viral infection.
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Excerpted from Advanced Therapy for Hepatitis C Copyright © 2012 by Blackwell Publishing Ltd. Excerpted by permission of John Wiley & Sons. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
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