Hepatitis C Virus-Host Interactions and Therapeutics: Current Insights and Future Perspectives

By Imran Shahid and Qaiser Jabeen

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.

• Language: English
• Publisher: Bentham Science Publishers
• Release date: Mar 11, 2008
• ISBN: 9789815123432

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HCV-Host Interactions: A Plethora of Genes and their Intricate Interplay Part 1: Virus Specific Factors

Imran Shahid, Qaiser Jabeen

Abstract

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.

Keywords: Acute hepatitis C, Chronic hepatitis C, Cirrhosis, Cell communication, Cell signaling, Core protein, Envelope glycoproteins, Fibrosis, Genome organization, Hepatocytes, HCV life cycle, Hepatocellular carcinoma, Immunopathogenesis, Molecular pathogenesis, Nonstructural proteins, RNA polymerase, Replication, Structural proteins, Translation, Virus-host interaction.

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].

Table 1 Fluids and tissue specimens capable of transmitting hepatitis C infection [52].

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 JNK (c-Jun N-terminal kinase) and mitogen-activated protein kinase (MAPK) pathway that ultimate initiates anti-apoptotic or hepatocarcinogenesis effects [32]. Likewise, in spontaneous induction and severity of liver steatosis in HCV GT-3 infected patients, a similar correlation has been observed between HCC and CHC 3a subtype-infected patients in Pakistan [31]. Full-length core sequence analysis extracted from the circulating HCV RNA of HCC infected patient’s serum indicates the existence of specific substitutions responsible for HCC induction as compared to patients with CHC infection only [29]. One study analyzed full-length HCV-1b sequences from HCC patients and controls and identified seven polymorphisms (nucleotide) significantly associated with increased risk of HCC (36G/C, 209A, 271U/C, 309A/C, 435A/C, 481A, and 546A/C) [35]. Interestingly, 209A has been associated with IFN resistance and HCC induction suggesting that Core gene sequence data might provide useful information about HCC risk [32]. In one study eight mutations were found in the HCV subtype 1b core gene related to the increased risk of HCC (i.e., A028C, G209A, C219U/A, U264C, A271C/U, C378U/A, G435A/C, and G481A), whereas U303C/A was found to be associated with decreased HCC risk [27]. Overall these mutations bring about four amino acid substitutions: Lys10Glu, Arg70Glu, Met91Leu, and Gly161Ser to induce HCC in CHC-infected patients [27].

Since mutations within HCV core protein or envelope glycoproteins (i.e. E1 and E2) may aggravate or block viral infectivity respectively, it may be assumed that those specific substitutions or mutations could be responsible for the spontaneous induction and severity of HCC in certain HCV GTs [17]. albeit; a lot of viral and host factors may incite and contribute to developing HCC, we only summarized here the polymorphism or mutations in the core gene which are associated with different pathological states leading to HCC and could be used as clinical biomarkers of HCV disease stage or development of HCC [36]. However; complete elucidation to evaluate the importance of these mutations in HCC identification/progression requires further studies. In addition to that, the identification of the molecular pathways underlying the association between certain HCV mutations and hepatocarcinogenesis also demands comprehensive virus-host interaction studies. Consequently, an ample understanding of this relationship would result in a search for novel targets for HCC therapeutic intervention.

HCV Core Protein and Insulin Resistance (IR)

HCV epidemiological data also points out a clear association between HCV infection and the development of type 2 diabetes mellitus due to the development of IR, a common metabolic disorder in the pre-diabetic state in HCV individuals [37]. The ratio of diabetes has been demonstrated more in HCV infections than in other liver diseases (i.e. 20–25% more in patients with HCV vs. 10% of those with hepatitis B) [37]. IR could be promoted by HCV viral proteins in a GT-specific mechanism (Moucari et al., 2008). It has been suggested that HCV core protein leads to IR through the interference of intracellular insulin signaling, mainly by the serine phosphorylation of the insulin receptor substrate (IRS-1) pathway in HCV-infected individuals [37].

HCV Envelope Glycoproteins (E1 and E2)

HCV E1 and E2 Morphology

HCV encodes two envelope glycoproteins (E1 and E2) which are released from viral polyproteins by signal peptidase cleavage [38]. Both glycoproteins are type 1 transmembrane proteins (i.e. the proteins which are anchored to the lipid membrane with a stop-transfer anchor sequence and have their N-terminal domains targeted to the ER lumen during synthesis) with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor [38]. Both proteins associate with each other as noncovalent heterodimers after their synthesis. This E1-E2 heterodimer complex is necessary for HCV entry into host cells where the transmembrane domain plays an essential role in E1-E2 heterodimer assembly and subcellular localization. The transmembrane domain may also play a coordinate role to reorganize the E1-E2 protein complex for the fusion process to occur. Both proteins are retained in ER, signals for which are present in the transmembrane domains. Similarly, It has been found that the E1-E2 complex is present at the surface of HCV particles, and therefore it is an obvious candidate ligand for host cellular receptors (i.e. CD81[Cluster of Differentiation 81], SR-BI [Scavenger receptor class B type 1], and CLDN1 [Claudin-1]). E1 and E2 envelope glycoproteins exhibit the highest degree of genetic heterogeneity as compared to other HCV proteins, where E2 protein contains a hypervariable region 1 (HVR1), which is located at the N- terminus [38].

HVR1 of E2 Glycoprotein Modulates the Immune System

An important correlation has been found between E2 glycoprotein HVR1 sequence variation and the intensity and cross-reactivity of humoral immune responses [39]. It strongly supports the contention that HCV variant selection is driven by immune pressure. In one study, monoclonal antibodies (mAbs) generated after immunization of mice with peptides derived from the natural HVR1 sequence of HCV E2 protein were noticed to generate a neutralization response for other several HVR1 sequences, predicting the existence of conserved amino acid motifs among different HCV variants [40, 41]. This mechanism of inducting a broadly cross-reactive immune response to envelope glycoprotein E2 HVR1 could be used to generate protective immunity [41]. As the E1-E2 protein complex is exposed at the surface of the virion, this protein complex is a target for neutralizing antibodies. Antibodies to HVR1 may block viral infection by inhibiting virion binding or membrane fusion [41]. It has been attested in one study that rabbit hyperimmune serum generated against the HVR1 can neutralize the ability of HCV to infect chimpanzees [42]. Furthermore, in a lymphoid cell line, neutralizing antibodies (nAbs) were also identified by their ability to prevent HCV replication [43]. It suggests that an HCV clone lacking E2 HVR1 is infectious and can be attenuated in an in vivo model (e.g. chimpanzees), however; HCV-infected strains in humans always contain E2 HVR1, which predicts that human intact HCV strains have HVR1 with a significant survival advantage [43]. Characterization of HCV envelope glycoproteins entry function was initially studied by the development of the HCV pseudo particle (HCVpp) models and provided useful information related to the role of this heterodimer protein complex in viral entry [43]. However, the development of a full-length HCV infectious clone (e.g., JFH-1) in cell culture provided a new platform for a new generation of HCV investigations. The efficient, reproducible, and robust expression with viral infectivity of this infectious clone in permissive cell lines (e.g., Huh7.5 [Human hepatoma cell line] and Huh7.5.1]) further expedited the cell culture experiments in molecular virology [44]. Consequently, the ample understanding of HCV biology in infected hosts from virus entry to new virion secretions, as well as to the development of effective antivirals was made possible and is continued.

HCV Nonstructural Proteins and their Role in Hepatitis C Pathogenesis

HCV nonstructural proteins play essential regulatory roles in viral replication, translation, posttranslational protein modifications, and to development of antiviral drug resistance [14]. Some nonstructural proteins like NS3, NS5A, and NS5B are the potential antiviral drug targets for newly developed direct-acting antivirals (DAAs) and other anti-mRNA based treatment strategies (e.g., oligonucleotides and small interference RNA, etc.) as well as some are acting as emerging drug targets (e.g. NS2 and NS4B) for the DAAs in clinical trials [14]. We briefly overview HCV nonstructural proteins' role in the viral life cycle and molecular pathogenesis of HCV infection, although most of the nonstructural proteins play regulatory roles in the viral life cycle than molecular pathogenesis of the infection.

NS2 Metalloprotease

NS2 protein along with NS3 constitutes a highly hydrophobic protease responsible for the cleavage of viral polypeptide between non-structural proteins NS2 and NS3 [45]. Three residues H143, E163, and C184 (while H952, E972, and C993 when numbered in polyprotein) constitute a catalytic triad of NS2/NS3 protease and are considered to be conserved among all HCV GTs [46]. However, many aspects of NS2/NS3 protease’s role in HCV infection biology and viral life cycle are still controversial or unknown [45]. Some studies project its role as either cysteine or metalloprotease, albeit; its sequence homology is not significant to proteases of known function. Similarly, some studies demonstrate its pivotal role for persistent infection in a chimpanzee, however; not fully elucidated in an in vitro system to study all aspects of HCV replication. Interestingly, some recent studies have alluded to the possible role of cleaved NS2 protein in the modulation of host cell gene expression and apoptosis [45].

NS3/4A Serine Protease/Helicase

NS3 acts as a serine protease as well as an RNA helicase/NTPase during HCV polyprotein processing and viral replication [47]. It is a non-covalent heterodimer consisting of a catalytic subunit (the N-terminal one-third of NS3 protein) and an activating cofactor (NS4A protein) and is responsible for cleavage at four sites of the HCV polyprotein. The NS3-4A protease activity involves a catalytic triad of three motifs including Ser-139, His-57, and Asp-81, and an oxy-anion hole comprising backbone amides of Gly-137 and Ser-139. In addition to that, NS3-4A acts as a helicase to unwind viral RNA by integrating into the HCV replication complex, although not fully understood [47]. It is an essential protease for viral replication in cell culture as well as in chimpanzees and has been considered one of the most pioneering, attractive, and well-studied drug targets for the design and development of anti-HCV therapies (e.g., oral IFN-free DAAs, siRNAs, etc.). However, its shallow substrate-binding groove and lack of an efficient in vitro viral replication model hampered the researchers and investigators for decades to discover, design, and developing small and selective inhibitors against HCV NS3-4A protease as oral drug candidates. However, during the last decade, rapid advancement in molecular biology techniques, the introduction of novel technologies in molecular medicine, and drug designing strategies made it possible to overcome these two obstacles and provided a full understanding of this protease's biological functions, biochemistry, and three-dimensional structures. It culminates the efforts to demonstrate proof-of-concept anti-HCV activities of newly developed IFN-free DAAs against NS3/4A protease in treated patients. Some studies explicitly the role of NS3 in the progression of persistent HCV infection by inhibiting the host's innate immune defense mechanisms by blocking retinoic acid-inducible gene I (RIG-I) and toll-like cell signaling receptors (TLRs). It suggests that NS3-4A protease inhibition may restore the host's innate immune mechanism against viral infection in the host cells [47]. NS4A is the smallest known non-structural protein of the HCV genome and is essential as a co-factor for serine protease activity of the NS3-4A complex. It has been shown that in its absence only one cleavage site (i.e. NS5A/NS5B) is partially processed by NS3 protease alone. The central region of the protein (i.e. from residue 21 to 34) is important and sufficient for the enzymatic co-factor function [47].

NS4B

NS4B for many years was characterized mainly as a protein of unknown function [48]. However; the latest studies predict NS4B's potential role in the modulation of RdRp enzyme activity and regulation of many host signal transduction pathways leading to HCV carcinogenesis, impairment of ER function, and regulation of both viral and host mRNA translation [14]. The most significant role found recently to be involved in the formation of a novel intracellular membrane structure, known as membranous web or membrane spherules (i.e. a platform upon which viral replication occurs). Furthermore, specific domains within NS4B including an amphipathic helix and nucleotide-binding motif also act as attractive targets for the design and development of novel IFN-free DAAs nowadays [48].

NS5A: An Interferon Resistance Protein

NS5A is a phosphoprotein that acts as a multifunctional regulator of cellular pathways and virus replication as well as is involved in conferring resistance against IFN therapy [48]. The protein first time grabbed the attention due to its role in modulating the host cell IFN response. A domain within the protein known as the interferon-α sensitivity determining region (ISDR) was found to confer resistance against IFN treatment in HCV-infected patients [49]. It is also an essential component of the HCV replicase enzyme and exerts its wide range of effects on host cell processes including innate immunity, host cell growth, and cell proliferation [50]. In the last decade, the protein has been widely searched, investigated, and targeted to design and develop IFN-free DAAs, some of which have been approved by the US FDA to use in real-world clinical settings with promising SVR rates in treated individuals.

NS5B: RNA Dependent RNA Polymerase (RdRp)

NS5B protein also known as the RdRp enzyme, is a catalytic subunit of the replicase enzyme complex consisting of virally encoded and host proteins, which is responsible for viral RNA replication [14]. The NS5B protein sequence motifs are highly conserved among all the known RdRps and contain discrete fingers, palm, and thumb subdomains [14]. A unique feature of RdRp is an enriched active site developed due to extensive interactions between the finger and thumb subdomains. Sequence motif residues D220, D319, and D318, are actively involved in transferring nucleotide to an active catalytic subunit of the NS5B protein [14]. Being the viral RNA replication enzyme, NS5B is a potential target to develop novel nucleoside and non-nucleoside NS5B inhibitors (NIs and NNIs respectively) among which the sofosbuvir (SOF) and dasabuvir (DSV) are clinically used to treat different HCV GTs and subtypes infected individuals with promising SVR rates [14]. Nowadays some other NNIs (e.g., radalbuvir) are also in the pipeline and clinical trials as an emerging regimen with increased anti-hepatitis C clinical efficacy.

3’ UTR

The 3’ UTR of the HCV genome is a tripartite structure located at the 3’ end of the viral genome and is predominantly involved in HCV replication [6]. This region is vital for viral RNA replication, either in an in vitro cell culture system transfected with HCV replicon or in vivo chimpanzee model inoculated with full-length HCV infectious clone [51]. The 3'UTR genome contains a variable region of 26-70 nucleotides in length, a poly U/UC tract of variable length and sequence, and the X-tail of 98-100 nucleotides which is conserved among various HCV GTs [51]. The 3'UTR indispensable signals for viral RNA replication and translation while interacting with other viral genome regions in a highly orchestrated manner [14]. The sequence and secondary structure of 3’UTR are somehow conserved among different HCV GTs and subtypes. The interaction among 5'UTR, 3'UTR, and NS5B is mandatory for viral replication, particularly in the initiation of minus-strand RNA [51]. In addition to that, 3'UTR binding with NS5A is assumed to mediate genome circularization and switch on the viral translation. Recent studies predict that the viral translation and replication signals overlap among 5’ UTR, 3’ UTR, and other viral proteins, and studies are in progress to fully elucidate these mechanisms. Furthermore, some studies suggest that sequence and structural variability of the 3’ UTR regulatory elements (e.g. variable region and poly U/UC tail) found in inter-genotypic and intra-genotypic isolates could be associated with the differential prevalence of HCV across the world and variable treatment responses of HCV subtypes to the same therapy [51].

CONCLUSIONS

The intricate interplay of HCV pathogenesis, epidemiology, and therapeutics is always remaining a big challenge to investigators, researchers, and physicians. Despite the revolutionary advancement in molecular biology techniques, as well as the introduction of state-of-the-art diagnostic procedures, and imperative development in current molecular medicine, the public healthcare burden of hepatitis C and associated hepatic comorbidities are still significant around the world. The multifaceted, perplexing, and intermingled interplay between hepatitis C and host cellular factors provides the survival advantage to the virus as well as progress from acute hepatitis C disease to CHC infection and subsequent other hepatic comorbidities. HCV structural and nonstructural proteins, as well as host cell regulatory factors, cell signaling, and metabolic networks assist, modulate, or impede the natural physiological role of liver cells to lead to hepatosteatosis, hepatic fibrosis, cirrhosis, and hepatocarcinogenesis in infected patients. A better understanding of all these regulatory pathways involved in the molecular pathogenesis of HCV infection could be useful to cure harder-to-treat hepatitis C-infected specific populations. The advent and approval of IFN-free DAAs to treat CHC infection has provided clinical promise in real-world clinical settings, however; their therapeutic benefits in complicated hepatitis C-induced co-morbidities (e.g., decompensated cirrhotic patients, HCC, HCV/HIV, or HCV/HBV co-infected patients) and in heart, kidney, and liver transplant donors and recipients infected with HCV are remained to investigate. All these demands refocus the search for novel roles of hepatitis C-host interactions, the meaningful and smart dissection of regulatory pathways, and cell signaling networks to identify new drug targets ligands and prognosis biomarkers for the clinical diagnosis of these comorbidities. Furthermore, pharmacogenomics studies and pre-clinical trials of the new regimens in the pipeline should be considered to prevent any hepatic or systemic toxicity which may further exacerbate hepatitis C-induced extrahepatic manifestations in infected patients as well as in pre-and post-treated individuals with IFN-free DAAs.

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