viruses Review Hepatitis C Viral Replication Complex Hui-Chun Li 1, Chee-Hing Yang 2 and Shih-Yen Lo 2,3,* 1 Department of Biochemistry, Tzu Chi University, Hualien 97004, Taiwan; [email protected] 2 Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan; [email protected] 3 Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 97004, Taiwan * Correspondence: [email protected]; Tel.: +886-3-8565301 (ext. 2322) Abstract: The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication. Keywords: hepatitis C virus; replication organelles; NS3 to NS5B proteins; direct-acting antivirals 1. Introduction Infection with the hepatitis C virus (HCV) can cause chronic hepatitis C (CHC), Citation: Li, H.-C.; Yang, C.-H.; Lo, liver cirrhosis, hepatocellular carcinoma, and other extra-hepatic manifestations. The S.-Y. Hepatitis C Viral Replication prevalence of CHC patients worldwide was around 71 million in 2017 (https://www.who. Complex. Viruses 2021, 13, 520. int/hepatitis/publications/global-hepatitis-report2017/en/). HCV belongs to the family https://doi.org/10.3390/v13030520 Flaviviridae and genus Hepacivirus. Its genome is a single-stranded RNA with positive polarity. Many different but closely related circulating HCV variants (i.e., quasispecies) Academic Editors: Núria Verdaguer can be detected in CHC patients due to the low fidelity of the HCV RNA polymerase and Diego Sebastian Ferrero (NS5B) and its high replication rate [1]. Thus, HCV genomic RNA sequences are highly heterogeneous among different isolates. At present, HCV is classified into at least six Received: 27 February 2021 major genotypes (GT 1 to 6) [2,3]. The geographic distribution of different HCV genotypes Accepted: 19 March 2021 Published: 22 March 2021 varies [3]. Subtype 1a is found throughout the US and Northern Europe, while subtype 1b is widely distributed throughout the world and is a major subtype in Japan. Genotype 2 is Publisher’s Note: MDPI stays neutral present in the same areas as genotype 1. Subtype 3a is widely distributed in South Asia with regard to jurisdictional claims in and Oceania, while subtype 3b is mainly found in East Asia. Genotype 4 is mainly present published maps and institutional affil- in the Middle East, Northern to Central Africa, and Europe. Subtype 5a is mainly found in iations. South Africa. Genotype 6 is mainly distributed throughout East and South-East Asia. The life cycle of HCV begins with its binding to cells. Numerous cellular factors, including proteins, lipids, and glycans, promote the entry of HCV particles into hepatocytes. HCV initially attaches to the surface proteoglycans, e.g., the scavenger receptor BI, and to the tetraspanin CD81. After lateral translocation to tight junctions, claudin-1 and occludin Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. proteins become essential for HCV entry. HCV particles are engulfed by clathrin-mediated This article is an open access article endocytosis and then fused with endosomal membranes in low-pH conditions. Viral distributed under the terms and genomic RNA is then released into the cytoplasm [4]. Then, the HCV genomic RNA is conditions of the Creative Commons used for both protein translation and viral RNA replication. HCV RNA replication takes Attribution (CC BY) license (https:// place within the replication organelles (RO) in the endoplasmic reticulum (ER). Finally, creativecommons.org/licenses/by/ HCV utilizes the biosynthetic pathway of very-low-density lipoprotein to assemble the 4.0/). viral particles and egress from the cells [5]. Viruses 2021, 13, 520. https://doi.org/10.3390/v13030520 https://www.mdpi.com/journal/viruses Viruses 2021, 13, x FOR PEER REVIEW 2 of 21 Viruses 2021, 13, 520 2 of 22 HCV utilizes the biosynthetic pathway of very-low-density lipoprotein to assemble the viral particles and egress from the cells [5]. The HCV RNA genome (~9600 nucleotides) possesses one open reading frame that is 0 0 flankedflanked by 5’ 5 and 3’ 3 untranslateduntranslated regions (UTRs) (Figure 1 1a).a). TranslationTranslation ofof thethe viralviral RNARNA leads to the synthesis of a polyprotein,polyprotein, which is processedprocessed intointo individualindividual viralviral proteinsproteins via cleavages of both cellular and viral protease proteases.s. The structural proteins (i.e., the core and envelope glycoproteinsglycoproteins E1E1 and and E2) E2) are are the the main main constituents constituents of HCVof HCV particles, particles, whereas whereas the theviroporin viroporin p7 andp7 and nonstructural nonstructural protein protein 2 (NS2) 2 (N areS2) involvedare involved in virion in virion assembly assembly [6]. The[6]. Theremaining remaining nonstructural nonstructural proteins proteins (i.e., NS3,(i.e., NS3, NS4A, NS4A, NS4B, NS4B, NS5A, NS5A, and NS5B; and NS5B; NS3-NS5B) NS3- NS5B)that have that specific have specific roles in roles viral in genome viral geno amplificationme amplification form theform replication the replication complex complex [7–9]. [7–9].The roles The ofroles different of different viral proteins viral proteins in HCV in replicationHCV replication are summarized are summarized in Table in1 .Table 1. Figure 1. The synthesis of the hepatitis C virus (HCV) proteins. ( a) The start and stop codons for protein translation were marked by black circles, while two recognition sites on the 5’ UTR for miR-122 were marked by black rectangles. (b) The marked by black circles, while two recognition sites on the 50 UTR for miR-122 were marked by black rectangles. (b) The polyprotein is co- and post-translationally cleaved by cellular or viral proteases to yield the structural proteins (core, E1 polyprotein is co- and post-translationally cleaved by cellular or viral proteases to yield the structural proteins (core, E1 and E2) and the nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins). The core, E1, and E2 are andprocessed E2) and by the cellular nonstructural signal peptidase proteins (filled (p7, NS2, arrowhead). NS3, NS4A, A ma NS4B,ture core NS5A protein and NS5B will be proteins). generated The after core, further E1, and cleavage E2 are processedby signal peptide by cellular peptid signalase peptidase(empty arrowhead). (filled arrowhead). The NS2/NS3 A mature junction core site protein is cleaved will be by generated the NS2-NS3 after furtherauto-protease, cleavage and by signalthe remaining peptide nonstructural peptidase (empty proteins arrowhead). are processed The NS2/NS3 by the NS3/4A junction proteinase. site is cleaved (c) Allby of thethe NS2-NS3HCV proteins auto-protease, are directly and or theindirectly remaining associated nonstructural with the proteins endoplasmic are processed reticulum. by the Curr NS3/4Aently used proteinase. anti-HCV (c) Alldirect-act of theing HCV antivirals proteins (DAAs) are directly target or indirectlyNS3, NS5A, associated and NS5B, with respectively. the endoplasmic NS3, NS4A, reticulum. NS4B, Currently NS5A, and used NS5B anti-HCV proteins direct-acting will form antiviralsthe replication (DAAs) complex. target NS3, NS5A, and NS5B, respectively. NS3, NS4A, NS4B, NS5A, and NS5B proteins will form the replication complex. Viruses 2021, 13, 520 3 of 22 Table 1. Hepatitis C virus (HCV) proteins play different roles in viral replication. Viral Protein Role in HCV Replication Package HCV genomic RNA to form nucleocapsids and also Core involve in lipid synthesis E1, E2 Responsible for the entry of virions to cells p7 Ion channel NS2 Auto-protease to cleave the junction between NS2 and NS3 NS3 contains an amino-terminal protease domain responsible for the HCV polyprotein processing and a carboxy-terminal NS3 DExD-box helicase domain responsible for HCV RNA replication through unwinding RNA secondary structures NS4A Cofactor for NS3 protease To serve as a scaffold for the viral replication complex and to NS4B induce the rearrangements of membrane vesicles To interact with a large number of cellular proteins that are NS5A important for viral assembly and function of the replication complex HCV RNA-dependent–RNA-polymerase responsible for HCV NS5B RNA amplification Over thirty years of research on the mechanisms of HCV replication has led to the successful development of direct-acting antivirals (DAAs) targeting the replication com- plex [10]. We summarize the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs inhibiting HCV replication in this review article. 2. Viral Replication Organelles (RO) HCV induces cellular membrane alterations referred to as the membrane web (MW) for viral RNA replication [11]. Different types of membrane alterations induced by HCV were observed [12,13]. Among these membrane alterations, double-membrane