Hepatitis C Virus-Host Interactions and Therapeutics: Current Insights and Future Perspectives E-Book
Imran Shahid
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- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Sprache: Englisch
- Veröffentlichungsjahr: 2008
The burden of hepatitis C virus (HCV) infection on the public health care system continues to remain significant despite the remarkable progress made in HCV therapeutics in the recent past. There are now almost a dozen oral interferon-free direct-acting antivirals available for the treatment of hepatitis C virus infection. Despite advances in the treatment of HCV, therapeutic gaps remain that are yet to be fully explored. Researchers and scientists still strive to understand virus-host interactions to map the disease’s progression along with extrahepatic manifestations and virus invasion strategies impacting the host’s immune system. This book briefly discusses the biology of HCV infection, virus-host interactions, molecular epidemiology of the infection, and the full spectrum of immune responses to hepatitis C. It also provides in-depth information about HCV, clinical diagnostics, and therapeutic knowledge to all stakeholders involved in HCV screening, diagnosis, treatment, and management.
Topics covered in the chapters include 1) HCV-host interactions leading to asymptomatic acute infection, 2) the progression of acute HCV infection to chronic disease and subsequent extrahepatic comorbidities, 3) Innate and adaptive immune responses in HCV infections, 4) Consensus-based Approaches for Hepatitis C Screening and Diagnosis, 5) advances in hepatitis C therapy and global management of HCV, and 6) the outcomes of Oral Interferon-free Direct-acting Antivirals as Combination Therapies to Cure Hepatitis C.
This book is a valuable addition to undergraduate and postgraduate hepatology students and physicians, clinicians, hepatologists, and health care officials involved in HCV clinical diagnosis and therapeutics.
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FOREWORD
Virus-host interactions (dependencies) and their intricate interplay are always phenomenal and of paramount interest to investigate molecular epidemiology and pathogenesis of all viral infections. However; complex cross-talks between hepatitis C virus (HCV) and host cells involving a plethora of genes and cell signaling pathways leading asymptomatic acute hepatitis C infection to chronic hepatitis C (CHC) and further durable, sustained, and insidious extrahepatic manifestations are still mysterious, murky, inconclusive, and even more, remains to elucidate. The world has always seen paradigms in HCV infection biology and therapeutics since its discovery in the late 1980s by gene cloning methods instead of the use of conventional tissue culture, or classical virological techniques. We can say that the world could not be able to see this revolutionary advancement in molecular biology techniques, state-of-the-art diagnostic tools, sophisticated protocols of clinical trials, astute knowledge of molecular drug targets, and serendipitous design and development of drug molecules without HCV discovery that once labeled as the ‘silent epidemic’ afflicting even more than 58 million peoples around the globe nowadays. Before the advent and approval of all oral interferon-free direct-acting antivirals, the gold standard of care, a combination therapy including pegylated interferon plus ribavirin achieved sustained clearance of HCV and subsequent improvements in liver disease in only 60% of treated patients. It has been a long haul, a big haul, but we’ve made it with the current landscape of HCV therapeutics to cure harder-to-treat special HCV subpopulations including cirrhotic, decompensated cirrhotic, HCV/HIV or HCV/HBV co-infections, and liver transplant or post-transplant patients with virus recurrence. Despite oral direct-acting antivirals having started to bear fruit in real-world clinical settings against HCV, unmet challenges, barriers, and hurdles in HCV diagnostics and treatment accessibility still prevail that need to surmount and overcome to reduce the global healthcare burden of HCV and to achieve WHO striving goal of HCV elimination by 2030.
This thought-provoking book tends to cover a comprehensive and up-to-date overview of the latest advancement in HCV infection biology, perplexing molecular clinical pathology, novel diagnostic tools, fortuitous anti-HCV therapeutic options on the horizons, management of difficult-to-treat subpopulations, and imperative compensatory therapies against hepatitis C infection in the late stage of their development. The book intends to cover the latest guidelines issued by the US FDA, AASLD/IDSA (American Association for the Study of Liver Diseases/Infectious Diseases Society of America), ECDC (European Center for Disease Prevention and Control), and NYSDOH-AI (New York State Department of Health Aids Institute) on HCV screening, diagnosis, treatment and management for the clinicians, physicians, infectious disease experts, and primary care providers (nurses and paramedics). The contents of the book not only provide ABC of HCV infection, diagnostic tools, and treatment strategies to common and interested readers but also address current information about some pretty mind-boggling aspects of hepatitis C biology, immunology, pharmacology, therapeutics, and vaccinology to obtain relevant answers with thoughtful explanations of some outstanding problems in the field nowadays. The book also covers up-to-date guidelines about patient screening, treatment protocols, and optimization of therapy in real-world clinical settings.
The book also summarizes unanswered questions currently faced by investigators in the intriguing molecular pathogenesis of CHC in humans, and physicians, clinicians, hepatologists, and healthcare officials are concerned with HCV clinical diagnosis and therapeutics. I hope that this book will also be a valuable addition to hepatology for curious undergraduate and postgraduate students pursuing a new career in HCV medicine or newcomer in hepatitis C research to broaden their knowledge and gain a wide perspective of the field. The multi- and interdisciplinary presentation of information in this book would open a new school of thought for postdoctoral fellows to search for new horizons and milestones in hepatitis C research. The book closes while shedding light on remarkable and amalgamated efforts by the World Health Organization (WHO), health policymakers, and public healthcare providers to a proper ending of this ‘silent epidemic’ from the world by 2030.
Both authors of the book (Imran Shahid and Qaiser Jabeen) are agile, bright, and very active investigators in the concerned field of hepatitis C research with more than 10 and 20 years of experience respectively. I wish that this web-based, as well as hard copy format book, would rapidly become an international referral and guideline in hepatitis C infection biology, diagnostics, and therapeutics.
PREFACE
The public health care burden of hepatitis C virus infection remains significant despite the remarkable progress in HCV therapeutics during the last decade, as around 58 million people are currently having the virus and almost 4,00,000 deaths are reported annually due to hepatitis C-induced hepatocellular carcinoma. The advent and approval of a dozen oral interferon-free direct-acting antivirals have revolutionized the treatment paradigms for hepatitis C infection while achieving higher sustained virologic response rates (>95%) in treated individuals. Analogously, HCV and host interactions to map networks in disease progression, extrahepatic manifestations, and virus invasion strategies to the host immune system are still enigmatic to fully understand. Albeit, ample understanding of HCV life cycle in vitro and transient in vivo replication models, provide valuable insights towards the viral entry, genome replication, virion formation, and host infection involving a highly orchestrated series of molecular and cellular events, including a plethora of genes and cell signaling cascades. However, questions remain to answers regarding the virus clearance without any therapeutic intervention in some HCV-infected populations, molecular pathogenesis of infection transforming the acute hepatitis C infection to end-stage liver disease, post-viral eradication, hepatocellular carcinoma, and the fate of the immune system once the virus eliminates from the body.
This book intends to provide insights into what has been achieved in HCV infection biology, virus-host interactions, molecular epidemiology of the infection, and the full spectrum of immune responses to hepatitis C, either the involvement of innate or adaptive immune responses in the first three chapters. The following chapters 4 and 5 comprehensively illustrate the simplified HCV diagnostic solutions, predictive barriers, and future perspectives for general, as well as vulnerable HCV infected populations, along with the up-to-date procedural and protocol guidelines for HCV diagnosis. Book Chapter 4 also highlights the strides toward a better understanding of HCV screening and diagnostic strategies for high throughput anti-HCV screening and diagnosis, applications for risk prediction tools, and cost-effective analysis of current diagnostic in the hepatitis C cascade of care. The next four book chapters (from chapters 6 to 9) precisely and comprehensively illustrate the current landscape of HCV treatment, their administration, consensus treatment guidelines for their recommendations, and their real-world treat outcomes. These book chapters also provide the probable answers to some fundamental questions like why some HCV-infected populations are nonresponsive to IFN-free DAAs, the phenomena behind viral relapse, and reasons behind treatment failure in harder-to-treat specific HCV subpopulations. Within those book chapters, chapter 9 also emphasizes real-world challenges in HCV therapeutics to unleash certain barriers while making it possible to expand the scale of therapy, and the cascade of care, and bridge existing gaps in hepatitis C care between developed and poor nations. Chapter 10 of the book overviews the emerging anti-HCV treatment strategies in the pipeline and explains the efforts to do while refocusing on anti-mRNA-based treatment strategies and nanomedicine-based approaches used in conjunction with DAAs for hepatitis C. The book chapter also portrays some glimpses into the future for the design of controlled animal and human HCV infection models for the design of an appropriate but effective prophylactic or protective anti-hepatitis C vaccine model. The last two book chapters (chapters 11 and 12) discuss the goals, policy implementation, and progress of the Global Health Sector Strategies (GHSS) 2016-2021 on HCV worldwide in the last decade as well as the cross-cutting barriers to break and actions to be taken in this decade while achieving the global goal of HCV elimination by 2030. The final chapter of the book also describes the strategies to opt at hospitals, community services centers, health care service providers, and government levels to prevent new HCV incidences and reduce HCV-related morbidities and mortalities with concrete efforts, collective will, and public health notes to galvanize the efforts to eradicate this epidemic worldwide by 2030.
Overall, this well-written book provides from simple, accessible introduction to a complex hepatology field, and in-depth information about HCV basics, clinical diagnostics, and therapeutic knowledge to all stakeholders involved in HCV screening, diagnosis, treatment, and management. Without being exhaustive or redundant, it can be easily accessed to concepts by extensive cross-referenced studies. We hope that this book will enhance the knowledge of a common reader about hepatitis C infection, its diagnosis, and treatment, and provide answers to the physicians, clinicians, hepatologists, infectious disease experts, health care providers, and investigators to some of the outstanding problems in the ever-changing field of hepatitis C research.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
ACKNOWLEDGEMENT
Declared none.
DEDICATION
HCV-Host Interactions: A Plethora of Genes and their Intricate Interplay Part 1: Virus Specific Factors
Imran Shahid,Qaiser JabeenAbstract
Hepatitis C virus (HCV) interaction with host cells is pivotal for natural disease course starting from asymptomatic acute infection to progress into persistent chronic infection and subsequent extrahepatic manifestations, including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The HCV infection biology in infected host cells via virus attachment, virus genome replication, mRNA translation, new virion formation, and egress from infected cells involves highly coordinated participation of the virus- and host-specific proteins, a plethora of genes, and cell signaling cascade. The progression of persistent chronic hepatitis C (CHC) infection to hepatic fibrosis, cirrhosis, and HCC involves viral invasion strategies against host immune system defense mechanisms as well as impeding healthy metabolic and signaling networks of the liver cells. Thereby, HCV-induced liver injury via chronic inflammatory processes that fail to resolve is responsible for decompensated cirrhosis and on occasion, hepatocarcinogenesis in infected individuals. With the latest advancement and rapid expansion of our knowledge in hepatology, the human liver is deciphered as an immunologically distinct organ with its specialized physiological niche. The relationship between human hepatocytes and different components of the immune system is quite complex and dynamic. The immunopathogenesis of various viral infections demonstrates that the immune system plays an essential role to determine the progression of many hepatic diseases through immune cell communication and cell signaling networks. In this book chapter, we overview HCV-host interactions and their intricate interplay with complex crosstalk to propagate less fetal acute HCV infection to CHC and subsequent hepatocarcinogenesis (i.e. HCC) in infected individuals.
INTRODUCTION
Hepatitis C Virus Taxonomy
HCV belongs to the Flaviviridae family of viruses which is divided into three genera: flavivirus, pestivirus, and hepacivirus [1].
Flaviviruses include yellow fever virus, dengue fever virus (DENV), Japanese encephalitis virus, and Tick-borne encephalitis virus [2]. Pestiviruses include the bovine viral diarrhea virus, classical swine fever virus, and Border disease virus [1]. HCV, with 8 genotypes (GTs) and numerous subtypes (around 70), is a member of the hepacivirus genus, which includes tamarin virus and GB virus B (GBV-B) and is closely related to human virus GB virus C (GBV-C) [3]. All currently identified HCV GTs and subtypes are hepatotropic, which means that they preferentially infect hepatocytes and cause liver inflammation, broadly known as viral hepatitis C infection [3]. However, viral infectivity and pathogenicity do vary at GTs and subtypes level, which may variably influence the progression of HCV infection from the acute phase to chronicity and subsequent hepatic-comorbidities in infected individuals [4]. Interestingly, all members of the Flaviviridae family of viruses shares and resembles several basic structural and virological characteristics [1]. At the genome level, all Flaviviridae viruses are single-strand RNA viruses from 9600 to 12,300 nucleotides (nts) in length, with an open reading frame (ORF) encoding a polyprotein of 3000 amino acids (aa) or more [5]. The viral genome is flanked by a 5’ untranslated region (5’ UTR) upstream to the structural part of the ORF of 95-555 nts and a 3’ untranslated region (3’ UTR) of 114-624 nts in length adjacent to nonstructural proteins [5]. Structurally, the HCV virus is enveloped in a lipid bilayer in which two or more envelope glycoproteins (i.e. E1 and E2) are anchored [6]. The virus envelope surrounds the nucleocapsid, which is composed of viral antigen protein (i.e. core or C; multiple copies of a small basic protein) and the viral RNA genome [6]. The viral structural proteins (e.g., C, E1, and E2) are encoded by the N-terminal part of the ORF, whereas the 7 nonstructural proteins (e.g. P7, NS1, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) are constituted by the remaining portion of the ORF [6]. Surprisingly, motifs-conserved across RNA protease helicase (i.e. nonstructural protein; NS3) and RNA replication enzyme (i.e. nonstructural protein NS5B; an RNA-dependent RNA polymerase (RdRp)) are found in similar locations in the polyproteins of all of the Flaviviridae viruses [6]. In addition to that, the hydropathic profile of polyproteins is much closer/identical between flaviviruses and hepaciviruses than pestiviruses [1].
Likewise, with the above-mentioned similarities, HCV exhibits several virological, epidemiological, and pathophysiological differences from the other members of the Flaviviridae genera [1]. Flavivirus translation is cap-dependent, i.e. it is mediated by a type 1 cap structure located in the 5’ UTR (m7GpppAmp), followed by the conserved AG sequence and a relatively short stretch upstream of the polyprotein coding region [7]. In contrast, HCV translation is cap-independent, where the 5’ UTR is uncapped and folds into a complex RNA secondary structure adjacent to a portion of the core-coding domain [7]. Almost all 5’ UTR and a few upstream nucleotides of the core-coding domain are occupied by several stem-loops (SLs) of internal ribosome entry sites (IRES) which span a region of ~341 nts and mediate direct binding of ribosomal subunits and cellular factors and initiate subsequent translation [6]. 3’ UTR of the other members of flavivirus is highly structured, however; HCV 3’ UTR is relatively short, less structured, and contains a poly-uridyl tract that varies in length and plays a central role in HCV replication [6].
HCV: Route of Transmission
The major route of HCV transmission is exclusively through direct blood-to- blood contact between humans which signifies its narrow tissue tropism and host specificity for HCV infection [8]. However, some other body fluids and tissue specimens are also capable of transmitting HCV infection (Table 1). Flaviviruses principally infect a broad range of vertebrate animals by using mosquitoes or ticks as a virus vector, and humans are dead-end host that does not participate in the perpetuation of virus transmission [9]. Pestivirus can rarely infect humans and no known insect vector has been identified until now [10]. Most of the infections caused by flaviviruses are acute-limited in vertebrate animals whereas HCV in humans has a high chronicity rate (50%-80%) depending on the age at infection [11]. Protection and recovery of flavivirus and pestivirus infections are entirely dependent on strong and adapted humoral and cellular immune responses [10]. However; in CHC and subsequent extrahepatic manifestations of HCV infection (i.e. cirrhosis, HCC), virus-induced host-immune responses fail to confer protection and prevention to reinfection with homologous and heterologous strains of HCV in the infected individuals [11].
HCV Genome Organization
HCV is a positive-sense single-stranded RNA virus of approximately 9600 nucleotides in length [7]. The viral genome flanks by highly conserved 5’ and 3’ UTRs along with a single ORF of 3010 to 3037 amino acids polyprotein [7]. Both 5’ and 3’ UTRs are pre-requisite for virus replication and mRNA translation and signals required for both these phenomena are coordinated in a highly orchestrated manner within viral and cellular proteins and between these two regions of the HCV genome (e.g., molecular interaction of IRES domain IIId, stem-loop (SL) -II and SL-III of 3’ UTR and cis-acting replication elements (CRE)/region at 3’ end of NS5B is key for genome replication)) [5, 6]. The precursor polyprotein is post-translationally processed by cellular and host proteases into an initial structural region encoding three proteins including core (C), E1, and E2, and a nonstructural region of 4 nonstructural proteins (NS2 to NS5) after a cap-independent IRES translation mechanism mediated by an IRES structure within the 5’ UTR (Fig. 1) [12].
These secondary and tertiary structures (i.e. 5’ UTR, IRES, 3’ UTR) are also essential for the proper binding and positioning of HCV RNA within the host cell’s protein translation machinery [5]. The 3’ UTR is a tripartite structure located at the end of the viral genome and is indispensable for HCV replication [5] (Fig. 1).
HCV Molecular Biology
HCV replicates in the cytoplasm of hepatocytes with a very high replication rate producing approximately 1012 virions daily with a short turnover reaching a maximum time of three hours [13]. The host cells' immune responses are triggered to neutralize viral translation and polyprotein processing, however; the extraordinary ability of the virus to mutate because of poor fidelity and error-prone nature of RdRp enzyme during virus replication as well as lack of RNA repair mechanism, those mutations are accumulated within the HCV genome [14]. Consequently, HCV circulates as a population of closely related but diverse viral sequences in infected individuals referred to as quasispecies [14, 15]. Persistent infection, resistance to therapy, and variable treatment outcomes are believed to be attributed to the quasispecies dynamics of HCV isolates circulating in an infected individual [13]. Viral genetic heterogeneity is also a consequence of those accumulated mutations by which different HCV isolates display significant nucleotide variability within different genome regions [6]. The envelope glycoproteins (i.e., E1 and E2) and some nonstructural proteins (e.g., NS3, NS5A, and NS5B) are significantly variable, whereas 5’ UTR, Core, and 3’ UTR are highly conserved regions [6]. The evolution of six HCV GTs and more than 70 subtypes is also based on the accumulation of mutations in the HCV genome over time (Fig. 2) [7]. HCV replication and translation both occur simultaneously in the hepatic cytoplasm, which makes it an ideal candidate to elucidate HCV infection biology and molecular pathogenesis mechanisms [13]. However, HCV-host intricate interplay involving many viral and host-specific proteins and their overlapping signaling cascade during viral replication is not fully understood [7]. Surprisingly, much is known about virus-host interactions during viral translation and polyprotein processing. The lack of an inefficient cell culture system also hampers carrying on HCV infection biology studies in the long-term perspective of the molecular pathogenesis of the infection [16].
Fig. (1)) Genome organization of HCV. HCV genome is a linear, positive-sense single-strand RNA(+ssRNA) of 9.6 kb in size. The viral genome is flanked by 5’ and 3’ untranslated regions (UTRs). The genomic mRNA encodes for three structural and seven nonstructural proteins upon primary translation, which is post-translationally modified by viral proteases and host cellular peptidases into mature proteins. Each viral protein is specific for its role (indicated in a red rectangular box with its estimated weight) in the virus life cycle to produce new viral progeny. Generally, HCV structural proteins are essential for viral infectivity and the production of new virions, while viral nonstructural proteins are pivotal for HCV replication and translation. UTR; untranslated region, IRES; internal ribosome entry site, kD; kilo Dalton. Fig. (2)) Global distribution of HCV genotypes [51].HCV Molecular Virology
HCV-host interactions start as the virus attaches to the host cell surfaces (mostly hepatocytes; albeit HCV also infects some dendritic and lymphocytic cells, please see chapter 2 for more details) while binding to one or more cellular receptors organized as a receptor complex [17] (Fig. 3). The cellular membranes of envelope glycoproteins E1 and E2 support the deliverance of nucleocapsids to the cytoplasm during pH-dependent virion fusion into hepatocytes. After decapsidation the viral genome is translated, leading to the production of a precursor polyprotein [18].
The precursor polyprotein is post-translationally processed by both cellular and viral proteases into 3 structural (C, E1, and E2) and 6 nonstructural proteins (NS2-NS5B) [18]. HCV replication takes place in the endoplasmic reticulum (ER)-derived membrane spherules (i.e. membranous web) in the cytoplasm via the synthesis of full-length negative-strand RNA intermediates [19]. During the assembly of progeny virions from cytoplasmic vesicles, the core (C) protein is released and transferred from the lipid droplets to form nucleocapsids that with the assistance of a nonstructural protein (i.e. NS5A) are loaded with RNA [19]. While the replicase proteins can bind to the HCV genomic RNA during replication and protein assembling process in close proximity through intracellular membranes of the membranous web (Albeit; replicase proteins are removed from maturing nucleocapsids, the intracellular sites of which might converge) [19]. HCV mature virion morphogenesis is coupled to the VLDL (very low-density lipoproteins) pathways, and new HCV particles are produced as lipoviral particles (LVPs). Mature full-length HCV virions are released into the extracellular milieu by exocytosis [18].
Fig. (3)) HCV life cycle in host cells. HCV binds to host cell receptors (i.e. GAG, LDL-R, CD81, SR-B1) and with the help of tight junction-forming proteins (CLDN, OCLN) penetrates and fuses at the cellular or endosomal membrane of host cells. Following receptor-mediated endocytosis and uncoating of the virion, the +ssRNA genome is released into the cytoplasm where it is subjected to immediate translation and replication. Synthesis and post-translational modifications of structural and nonstructural proteins are processed by viral proteases and host signal peptidases that further translocate into the endoplasmic reticulum (ER) membranes. An intermediate (complement) negative-strand RNA is synthesized by viral RNA-dependent RNA polymerase (RdRp) that acts as a template for the synthesis of new viral mRNAs/new +ssRNA genomes. From ER, HCV structural and nonstructural proteins translocate to the Golgi complex. Virion assembly/packaging and budding originally initiates at the ER and completes to Golgi complex where the interaction of newly synthesized genomic RNA with viral core protein encapsidate viral genome and results in budding into the lumen of secretory vesicular compartments. Newly produced virions are released/excreted from the infected host cells by exocytosis. PM; plasma membrane, GAG; glycosaminoglycan, LDL-R; low-density lipoprotein receptor, SR-B1; scavenger receptor B type 1, CD; cluster of differentiation, CLDN; claudin, OCLN; occludin. NPCIL1; Niemann-Pick C1-like protein 1.Hepatitis C Proteins in Molecular Pathogenesis of Infection
Almost every region of the HCV genome is crucial to play an active role in viral translation and replication regulation as well as in disease progression from asymptomatic acute infection to CHC and subsequent extrahepatic HCV manifestations [20]. Furthermore, some genome regions elicit humoral and cellular adaptive immune responses against HCV to protect infected host cells and prevent further infection transmission [18]. Some viral proteins specifically regulate host cell machinery as well as impede metabolic networks and cell signaling cascade to propagate HCV infection severity from chronicity to end-stage liver disease (ESLD) by developing cirrhosis and hepatocarcinogenesis [19]. All this happens due to an intricate interplay between hepatitis C and host cell interactions, including a plethora of genes, intracellular host cell factors regulation by viral proteins, and cell-signaling communication between infected and non-infected host cells as well as cell signaling pathways to overlap or impede between viral and host factors [21]. In the following section, we overview the contribution of viral-specific factors to disseminate HCV infection intensity from the self-limited acute phase to evolve chronic HCV infection followed by progression to severe hepatic co-morbidities (e.g. cirrhosis & HCC). Meanwhile, (Fig. 4) depicts the structures of HCV structural and non-structural proteins and membrane association.
5’ Untranslated Region (5’ UTR)
The HCV 5’ UTR spans from 332 to 343 nucleotides and contains up to 5 AUG codons depending on HCV GTs and subtypes. It is a highly conserved sequence among all HCV GTs (> 90% conservation) that folds into a complex of secondary and tertiary structures encompassing multiple SLs and two RNA pseudoknots of IRES structure [6]. However; with minor variants, it expresses quasispecies distribution in the infected populations. Quasispecies kinetics of all HCV GTs is widely based on nucleotide substitutions/mutations in one of the most conserved regions of the HCV genome like 5’ UTR during viral replication [6]. These mutations provide a survival advantage or disadvantage to the mutated HCV genome in infected individuals [6]. The existence of naturally occurring variants (i.e. nucleotide signature sequence) within the 5’ UTR region of HCV generates distinct subtype sub-populations and HCV diversification in certain HCV strains in infected human populations [5, 6]. A study demonstrates the existence of a distinct subtype 1a subpopulation in South American HCV strains which were identified by the presence of nucleotide signature sequence within the 5’ UTR region of isolated strains [6]. The co-existence of multiple HCV subpopulations may occupy different regions on a fitness landscape to allow the virus to adapt rapidly to the changes in the landscape topology, which may enhance the virus settlement in its human host populations [5]. Virus particles isolated from infected host serum are likely to be released from the hepatocytes but also migrate from other cells like lymphocytes or dendritic cells, which indicates that nucleotide substitutions or sequence diversity found in 5’ UTR-IRES may reflect tropism and translational activity for these compartments as well [6]. Intergenotypic and intragenotypic recombinants have also been demonstrated in some studies reported from St. Peters-burg (2k/1b), Philippines (2b/1b), Vietnam (GT-2/6), Ireland (2k/1b), France (GT-2/5), and in Peru (1b/1a) [6]. Recombination is an influencing mechanism to cause genetic diversity in some RNA viruses’ families including HCV, with a potential impact on epidemiological studies, molecular pathogenesis, diagnosis, and treatment outcomes [5].
Fig. (4)) HCV proteins structure and their membrane association [22]. Scissors indicate cleavage of HCV structural and nonstructural proteins by the endoplasmic reticulum (ER) signal peptidase, except on the cytosolic side where it indicates the processing of HCV core protein (antigen) by signal peptide peptidase. The cyclic arrow indicates the cleavage of HCV nonstructural protein NS2 by the NS2 protease. Black arrows indicate the cleavage of HCV polyprotein at four sites by the NS3/4A serine proteinase. The known protein structures are depicted as ribbon diagrams. HCV protein structures and the membrane bilayers are shown at the same scale. HCV proteins or protein segments whose structures are unresolved are represented as colored spheres or cylinders with their approximate sizes. HCV structural and nonstructural proteins are shown from left to right in the figure. 1) HCV core protein (red) comprises the N-terminal natively unfolded domain (D1) and two amphipathic α-helices connected by a hydrophobic loop (D2 domain) [52]. The core-E1 signal peptide (PDB entry 2KQI) is cleaved by SPP [53]. 2) HCV envelope glycoproteins (E1 and E2) heterodimer are associated by the C-terminal transmembrane domains. Green spots indicate glycosylation of the E1 and E2 envelope proteins. 3) Oligomeric model of HCV nonstructural protein “p7” based on the structure of the monomer solved by nuclear magnetic resonance [54] and molecular dynamics simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid bilayer [55]. 4) The catalytic domain (dimer subunits) of HCV nonstructural protein NS2 are depicted in blue and magenta [56] which are connected to their N-terminal membrane domains constituted of three putative transmembrane segments [57, 58]. The active site residues of NS2 protein (His 143, Glu 163, and Cys 184) are shown as spheres. 5) NS3 serine protease domain (cyan) associated with the central protease activation and the N-terminal transmembrane domains of NS4A are depicted in yellow. The catalytic triad of NS3/4A serine protease (His 57, Asp 81, and Ser 139) is shown as spheres (magenta). NS3 helicase domains 1,2, and 3 are represented in silver, red, and blue respectively. The current NS3 protein structure present in this figure [59] illustrates that the NS3 helicase domain can no longer interact with the NS3 protease domain when the latter is associated with the membrane through its amphipathic α-helix 11–21 (green) and the transmembrane domain of NS4A [60]. 6) The N-terminal part of NS4B includes two amphipathic α-helices of which the second has the potential to traverse the membrane bilayer [61]. The C-terminal cytosolic part includes a predicted highly conserved α-helix and an amphipathic α-helix interacting in-plane with the membrane [62]. 7) NS5A domain 1 dimer (D1) [63]; subunits colored in magenta and ice blue), as well as intrinsically unfolded domains 2 and 3 (D2D3) [64-67], are shown in the figure. The N-terminal amphipathic α-helix of NS5A protein in-plane membrane anchor (Penin et al. 2004; helices colored in red and blue) are shown in relative position to the phospholipid membrane (adapted from [63]. (8) NS5B RNA-dependent RNA polymerase (RdRp) catalytic domain [68] associated with the membrane via its C-terminal transmembrane segment (F.P. et al. unpublished data) is shown in the figure. The fingers, palm, and thumb subdomains of the catalytic domain are colored blue, red, and green, respectively. The catalytic site of the NS5B lies within the center of the cytosolic domain and the RNA template-binding cleft is located vertically on the right along the thumb subdomain β-fap (orange) and the C-terminal part of the linker segment (silver), connecting the cytosolic domain to the transmembrane segment (magenta). The cell membrane is represented as a simulated model of a POPC bilayer (http://moose.bio.ucalgary.ca/). Polar heads and hydrophobic tails of phospholipids (stick structure) are colored light yellow and light gray, respectively. The positions of the NS5A in-plane membrane helices at the membrane interface as well as that of the transmembrane domain of NS5B were deduced from molecular dynamics simulations in POPC bilayer (F.P., D.M. et al. unpublished data). The positioning of the NS3-4A membrane segments and of the amphipathic α-helices in the core and NS4B are tentative.Being the most conserved region of the HCV genome, 5’ UTR is mostly suited for amplification methods and contains specific sequence motifs for HCV GTs/sub-types identification [23]. Many diagnostic laboratories and most commercially available HCV typing assays target 5’ UTR due to higher genotype-based assay sensitivity. However, mounting evidence suggests that direct sequencing of 5’ UTR does not identify all existing HCV GT 1 subtypes in 20% of infected cases [6]. Other methods based on more variable regions of the HCV genome (e.g. NS5B) could be relied upon for the accurate identification of subtypes [6]. A study describes the sequencing and phylogenetic analysis of the NS5B region as the first step in molecular epidemiological studies to recognize the route of HCV transmission [14]. NS5B is an extremely preferred region for HCV subtyping, but it is not always accurately amplified because of primer-target mismatch to the highly variable nucleotide region of NS5B [14].
Interestingly, the 5’ UTR role in HCV translation regulation has been studied in detail, however; its significance for RNA replication so far is not fully elucidated [6]. Previous studies demonstrate that for plus-strand RNA virus replication, signals are located in 5’ UTR of the template strand where they act as promoter elements for RNA replication initiation of both minus and plus-strand RNAs [5]. But how they interact with NS5B replication machinery and 3’ UTR CRE is still poorly understood. Although, RNA secondary and tertiary structure stability is regarded as a significant factor for virus genome stability, but not essential to predict virus stabilization and response to pegylated interferon (PEG-IFNα) therapy [20]. Either nucleotide variations in 5’ UTR of HCV-infected treatment-naive individuals may affect viral translation or response to therapy is still unclear [20]. Previous studies show that no correlation exists between treatment outcomes and the number of viral strains and HCV genome heterogeneity in treatment-naïve patients or before initiating treatment in such populations [19]. Equivocally, treatment-experienced patients with SVRs against PEG-IFN plus RBV (ribavirin) exhibited a significant decrease in the number of viral strains as well as overall genetic diversity [19]. A significant decrease in genetic diversity was evident with increased homogenous virus population and viral clearance in patients with higher SVR rates to PEG-IFN/RBV therapy and was independent of HCV GTs [18]. Another plausible justification is the correlation between increased genetic diversity and acute hepatitis C which leads to chronic hepatitis; whereas decreased genome variations were made explicit to resolve acute hepatitis before viral clearance [18].
HCV Antigen (Core;C) Protein
HCV core protein is the first protein to be synthesized during viral translation and is considered to be the most conserved part of HCV polyprotein [24]. It is also the first protein to contact HCV-infected host cell cytoplasm contents during viral entry and nucleocapsid release [25]. In the later phase of HCV biology, it plays an important role in mature virion formation and viral assembly in the ER [26]. The core protein amino acid composition is highly conserved among different HCV GTs as compared to other HCV proteins, however; sequence variations have been reported within different domains of the protein in different HCV-induced pathological states [26]. The key function of core protein is RNA binding to encapsidation with associated membranes and with lipid droplets (LDs) to produce virus-like particles (VLPs) and infectious virion progeny [27]. However; core protein directly or indirectly interacts with host cellular factors to play an essential role in virus-mediated pathogenesis (i.e. oxidative stress, steatosis, insulin resistance, and hepatocarcinogenesis) [27].
HCV Core Protein Morphology
The intracellular posttranslational processing of core protein is mediated by an intramembrane protease; the signal peptide peptidase (SPP) that cleaves an immature p23 core protein (191-aa polypeptide) at the C-terminal signal peptide and releases N-terminal 173-179 aa p21 mature core protein [28]. SPP is also required to translocate E1 glycoprotein to ER during the HCV life cycle in infected host cells. The mature core protein p21 then interacts with LDs on the ER membrane (a major site of HCV particle assembly) and thus influences mature virion infectivity and morphogenesis of the viral particles [29]. p21 is found in secreted viral progeny as determined by the analysis of serum samples of HCV-infected patients [29]. The X-ray crystallography predicts that core protein consists of three domains; D1: a basic hydrophilic N-terminal region of 1-118 aa, D2: a central hydrophobic domain of 119-173 aa, and D3; the last hydrophobic signal sequence domain of 174-191 aa containing SPP [29]. The mature core protein is a 177 aa dimeric α-helical protein and acts as a membrane protein. The domain D1 is mainly involved in viral RNA binding and encapsidation of the viral genome. Domain D2 is required for proper folding and stability of D1 and association of core with ER and outer mitochondrial membranes. D3 domain along with SPP is involved in stable and infectious viral particle formation [29, 30]. HCV core protein itself is capable and sufficient to induce lipid accumulation and elicit or propagate other HCV-induced pathogenesis in infected hepatocytes that’s why also named HCV ‘core antigen’ [30]. As a protein-protein interaction with other HCV proteins, it interacts with a non-structural NS5A phosphoprotein to form the bridge between LDs and the sites of viral RNA replication. Furthermore, it recruits other HCV non-structural proteins on LDs and this LDs and NS proteins arrangement as membrane spherules play a crucial role in viral replication and HCV particle morphogenesis [30].
HCV core interaction with host cellular factors may impede metabolic pathways of infected hepatocytes leading to hepatosteatosis, insulin resistance (IR), varied IFN response as well as affecting the transcription of cellular proto-oncogenes and other tumor suppressor genes which may progress CHC to apoptosis and hepatocarcinogenesis (i.e. HCC in CHC patients) [30]. However; the intensity of viral infectivity and pathogenesis may vary among different HCV strains which mainly relate to amino acids sequence variations of core protein and other viral/host factors [30]. As mentioned earlier, intergenotypic core sequences are similar, however; at subtype and quasispecies levels, the amino acid mutations or single amino acid polymorphism can modulate core protein interplay with other HCV genome proteins and host cell factors and tamper their stability to regulate normal cellular functions and metabolic pathways [30].
HCV Core Protein and Hepatic Steatosis
HCV core protein-induced hepatosteatosis (also known as liver steatosis) and oxidative stress are two preliminary pathological states which are raised due to enhanced accumulation of triglyceride-rich lipids in hepatic cells and could be used as a prognosis biomarker of CHC infection progression to hepatic fibrosis, cirrhosis, and subsequent hepatocarcinogenesis [30, 31]. Core protein causes lipid accumulation as a part of the HCV life cycle to assemble infectious virion and steatosis is the result of this lipid accumulation. Core protein upregulates the promoter activity of two major lipid synthesis enzymes in HCV-infected hepatocytes including fatty acid synthase (FAS) and sterol regulatory element-binding protein-1 (SREBP-1). The Core protein activates transcription factors, SREBP-1c4 and RXRa (Retinoid X receptor alpha), leading to enhanced activity of various enzymes involved in cellular lipid biosynthesis, and down-regulates PPAR-a (peroxisome proliferator-activated receptors alpha) and mitochondrial carnitine palmitoyl transferase-1, resulting in the reduction of fatty acid oxidation activity [32]. Hepatic cell oxidative stress is associated with core protein upregulation of the production of reactive oxygen species (ROS) from induced nitric oxide species (iNOS) by activating cyclooxygenase-2 (Cox-2) expression in hepatocyte-derived cells [33]. Another potential mechanism of HCV core-induced oxidative stress damages the mitochondrial electron transport system in core protein-expressing cells which further leads to HCC [34]. HCV GT-3 core protein has been demonstrated to induce more lipid accumulation in vitro than GT-1 core protein expression because of the difference of a single amino acid substitution in the D2 domain of the core protein [31]. Furthermore, core protein expression in hepatocytes of transgenic mice also resulted in steatosis and HCC development as one study expressed [33]. Very interestingly, steatosis is found more spontaneously and severely in HCV GT-3 infected patients because of the presence of specific steatogenic sequences in the core genome of this HCV GT which directly correlates to the burden of HCV viral load in hepatic cells [33]. Surprisingly, this association of high viremia to steatogenic severity is not observed in other HCV GTs-infected patients [33].
HCV Core Protein and Hepatocarcinogenesis
HCV core protein is capable to regulate the growth of hepatic cells by affecting the transcription of cellular proto-oncogenes and other tumor suppressor genes [1]. Single amino acid polymorphism or mutations within the core sequence may alter the predicted HCV RNA structures of new virion formation and can differentially regulate cellular pathways and processes that can contribute to oncogenesis [29]. In domain D3 of core protein, aa positions 70 and 91 are very much associated with anti-HCV treatment response to IFN/RBV, to induce insulin resistance (IR), apoptosis, and HCC [29]. It is beyond the scope of this book chapter to discuss in detail the intricate interplay of viral and host factors and cellular pathways involved in the pathogenesis of these hepatic comorbidities, hence we briefly overview such interactions here [32].
As previously mentioned, different domains of core protein have specific roles in HCV infection biology, therefore every domain is different to induce hepatic cell apoptosis [33]. For example, the N-terminal D1 domain is likely to be involved with a high percentage of apoptosis and necrosis than the D3 C-terminal domain, while the middle D2 domain with least induced effects [29]. The previous studies have alluded that binding interactions between HCV core and p53 protein (a tumor suppressor protein) either activation or inhibition of p53 expression resulted in consecutive anti- or pro-apoptotic effects [33]. Similarly, another study demonstrates that core protein suppresses the activation of a transcription factor NFkB (nuclear factor-kB; an inducible transcription factor and regulator of many genes involved in inflammation, immune responses, cell proliferation, and apoptosis) by inhibiting the degradation of IkBα (nuclear factor of kappa B), and activating the transcription factor activator protein-1 (AP-1) via
