VIRAL HEPATITIS FORUM Getting Close Viralto Eradication Hepatitis Forum I. Basic Getting Research Close to Eradication I. Basic Research Entry of Hepatitis B and C Viruses Seungtaek Kim Severance Biomedical Science Institute, Institute of Gastroenterology, Department of Internal Medicine, Yonsei University Col- lege of Medicine, Seoul, Korea B형과 C형 간염 바이러스에 대한 최근의 분자, 세포생물학적인 발전은 간세포를 특이적으로 감염시키는 이들 바이러스에 대한 세포 수용체의 발굴과 더불어 그들의 작용 기전에 대해 더 자세한 정보들을 제공해주고 있다. 특히 C형 간염 바이러스의 경우, 간세포의 서로 다른 곳에 위치한 세포 수용체들이 바이러스의 세포 진입시에 바이러스 표면의 당단백질과 어떤 방식으로 서로 상호 작용하며 세포 내 신호 전달 과정을 거쳐 세포 안으로 들어오게 되는지 그 기전들이 서서히 드러나고 있다. 한편, B형 간염 바이러스의 경우, 오랫동안 밝혀내지 못했던 이 바이러스의 세포 수용체인 NTCP를 최근 발굴하게 됨으로써 세포 진입에 관한 연구에 획기적인 계기를 마련하게 되었으며 동시에 이를 저해할 수 있는 새로운 항바이러스제의 개발도 활기를 띠게 되었다. 임상적으로 매우 중요한 이 두 바이러스의 세포 진입에 관한 연구는 앞으로도 매우 활발하게 이루어질 것으로 기대된다. Keywords: B형 간염 바이러스, C형 간염 바이러스, 세포 진입, 신호 전달, NTCP There are five hepatitis viruses although their classes, genomes, and modes of transmission are different from each other. Of these, hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most dangerous, life-threatening pathogens, which are also responsible for 80-90% of hepatocellular carcinoma. HBV belongs to hepadnaviridae (family) and it has double-strand DNA as its genome, however, its replication occurs via reverse transcription like retrovirus replication. In contrast, HCV belongs to flaviviridae (family) and has positive-sense, single-strand RNA as its genome. Most of HCV infection becomes chronic (~70%) compared to that of HBV infection (5-10%). Hepatitis C Virus HCV is an enveloped virus coated with two kinds of glycoproteins (E1 and E2) and contains RNA genome within the viral capsid. One of very interesting features of HCV is its close association with lipoproteins in virion assembly and entry (for a recent review, see Lindenbach and Rice.1 Depending on how lipoproteins associate with virus, two different models were proposed (two-particle and single-particle models). The viral life cycle of HCV is similar to that of other members of flaviviridae and there has been a substantial increase in the knowledge of HCV life cycle since development of cell culture-infectious HCV clones.2-5 HCV enters hepatocytes via receptor-mediated endocytosis. After membrane fusion and uncoating, the viral genome is released into the cytosol and the released RNA functions as mRNA for subsequent translation reaction. Nonspecific proteins translated from this viral RNA then make RNA replication complex within the “membraneous web” structure near ER, where virus genome replication occurs. Structural proteins and viral RNA that is synthesized in replication complex are assembled near lipid droplets to make HCV virions. And finally, virions are secreted using the VLDL secretory pathway. The Liver Week 2014 283 Viral Hepatitis Forum Getting Close to Eradication I. Basic Research The size of virus genome RNA is 9.6 kb and this RNA has a single ORF flanked by 5’ and 3’ UTRs. IRES-dependent translation generates a single polyprotein, which is co- and post-translationally processed by host and viral proteases to generate 10 viral proteins. Proteins at the N-terminus are structural proteins (core, E1, and E2), and the remainders at the C-terminus are nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), most of which are involved in viral RNA replication as components of replication complex. Entry of HCV is a complex process involving interactions with numerous cellular (co)receptors. CD816 and SR-BI7 are well-known HCV receptors and both are located at the basolateral membrane of hepatocyte. SR-BI is also the antiviral target of ITX-5061,8 which is currently at phase 2 clinical trials. However, additional host factors (CLDN19 and OCLN10) for HCV entry were discovered at tight junction structure. Seemingly two different locations of these (co)receptors raised a question: how virus can use all these host factors to enter the hepatocyte. In this regard, a previous study on the entry of coxsackievirus provides some clues on the mechanism of HCV entry.11 Receptors for this virus are also located at two different places of intestinal cell membrane (DAF, apical membrane; CAR, tight junction), but their communications are carried out by several kinases-mediated signaling, thus DAF-bound viruses can move to the tight junction for entry. Prior to describing HCV entry, it is necessary to explain experimental systems for studying HCV replication, entry, and infectivity. Replicon system was first developed by Lohmann et al.,12 in which viral RNA replication is carried out by nonstructural proteins from NS3 to NS5B with a selectable marker. Although this system allows for genome replication, it does not produce infectious particles, but it is a useful system to test numerous direct-acting antivirals (DAAs) targeting viral RNA replication in cell culture. HCV pseudoparticle (HCVpp) system was developed to study viral entry.13 This system employs 293T cells to express HCVpp by co-transfecting plasmids for E1/E2 glycoproteins, HIV gag-pol, and a reporter gene such as GFP or luciferase. The particles that are generated and secreted by the transfected 293T cells are retroviruses coated with HCV glycoproteins, which allow for infection of Huh7 cells. Finally, discovery of JFH1 genotype 2a isolate from a Japanese fulminant hepatitis C patient2 has enabled studying entire viral life cycle of HCV in cell culture (HCVcc). This clone, for the first time, recapitulated entire HCV life cycle both in vivo and in vitro. The prior result about the mechanism of coxsackievirus entry has led to screening of host factors to identify kinases involved in HCV entry and discover the relevant mechanism. Such screening effort has found receptor tyrosine kinase EGFR as a novel host factor in HCV entry.14 Silencing EGFR specifically inhibited HCV entry (both HCVpp and HCVcc) regardless of viral genotypes and FDA-approved EGFR inhibitor, erlotinib, also inhibited HCV entry in dose-dependent manner. Since EGFR starts a signaling cascade, downstream targets of this signaling cascade were subsequently identified.15,16 Specifically, the inhibitors of Ras-Raf-MEK-ERK pathway were shown to block HCV infection in contrast to those of PI3K- Akt pathway.15 And recently, MKNK1, a downstream target of ERK, was also shown to be involved in HCV entry.16 Thus, our current understanding of HCV entry can be summarized as follows. Lipoprotein-associated HCV virion attaches to the cell surface by interacting with heparan sulfate proteoglycan (HSPG) and low-density-lipoprotein receptor (LDLR). A subsequent interaction of the virus with SR-BI is thought to delipidate lipoprotein and induce conformational changes in E2 glycoprotein, thus facilitating interaction with another receptor, CD81. Interaction between E2 and CD81 then triggers signaling cascade through EGFR, HRAS, and RHO GTPases. This signaling event also induces lateral movement of virion-CD81 complex to the tight junction area, where CD81 interacts with CLDN1 and virus is internalized by clathrin- mediated endocytosis. The lateral movement of virion-CD81 complex is facilitated by actin filament polymerization which is mediated by RHO signaling. Within the cell, viral membrane fusion occurs by low pH of the endosomal compartment, thus releasing viral genome RNA in the cytosol. 284 The Liver Week 2014 Viral Hepatitis Forum Getting Close to Eradication I. Basic Research Hepatitis B Virus Compared to HCV entry, the mechanism of HBV entry is still not very well known except for recent identification of a novel entry receptor, NTCP.17 HBV is an enveloped virus coated with large (L), middle (M), and small (S) envelope glycoproteins. In addition to the infectious virion (Dane particle), HBV-infected cells secrete a lot of empty subviral particles, either spherical or filamentous form. HBV enters hepatocyte via NTCP-mediated entry pathway. In contrast to HCV, HBV viral genome DNA enters nucleus, where relaxed circular DNA (rcDNA) is repaired to covalently closed circular DNA (cccDNA). In the nucleus, cccDNA plays a role of transcription template to generate pregenomic and subgenomic RNAs. Viral core and polymerase are translated from pregenomic RNA (pgRNA) and the interaction between viral polymerase and ε structure at the 5’ terminus of pgRNA initiates encapsidation by viral core proteins. Within the viral capsid, viral genome replication occurs first by (-) DNA synthesis followed by (+) DNA synthesis. Mature capsids containing rcDNA have two alternative pathways; either travel to ER-Golgi intermediate compartment for envelopment and secretion or go back to the nucleus to repeat the genome replication cycles. HBV genome has a very complex organization. Although its length is only 3.2 kb, it encodes a total of 7 viral proteins. As explained above, core and polymerase are translated from 3.5 kb pgRNA. Other proteins are expressed from subgenomic RNAs (sgRNAs); large (L) protein from 2.4 kb RNA, middle (M) and small (S) proteins from 2.1 kb RNA, and X protein from 0.7 kb RNA. In addition, precore is expressed from preC RNA and secreted out of the hepatocyte. Discovery of NTCP (sodium taurocholate cotransporting polypeptide) as a cellular receptor for HBV infection has opened a new era in HBV research.17 Expression of NTCP by transfection in hepatoma cell lines has allowed HBV infection and this infection was demonstrated by HBsAg and HBeAg expression, viral RNA transcription, and cccDNA detection, etc. when the transfected cells were inoculated by HBV. Thus, we now have one more antiviral target of HBV infection in addition to the currently available RT inhibitors (e.g., entecavir, tenofovir, etc.). Since identification of NTCP as a cellular receptor for HBV, cyclosporin A and other NTCP inhibitors have been found to inhibit HBV entry.18-20 And in the near future, more intensive research on HBV entry would be able to find more antiviral candidates to inhibit this first step of viral life cycle.