Hepatitis C virus and intracellular antiviral response

Abstract

To establish successful infection in cells, it is essential for hepatitis C virus (HCV) to overcome intracellular antiviral responses. The host cell mechanism that fights against the virus culminates in the production of interferons (IFNs), IFN-stimulated genes (ISGs) and pro-inflammatory cytokines as well as the induction of autophagy and apoptosis. HCV has developed multiple means to disrupt the host signaling pathways that lead to these antiviral responses. HCV impedes signaling pathways initiated by pattern-recognition receptors (PRRs), usurps and uses the antiviral autophagic response to enhance its replication, alters mitochondrial dynamics and metabolism to prevent cell death and attenuate IFN response, and dysregulates inflammasomal response to cause IFN resistance and immune tolerance. These effects of HCV allow HCV to successful replicate and persist in its host cells.

Introduction

Hepatitis C virus (HCV) is one of the most important human pathogens. There are approximately 58 million people worldwide that are chronically infected by HCV, with about 1.5 million new cases every year. People infected by HCV often become chronic carriers of the virus. The chronic infection by HCV may be asymptomatic. However, it can progress into severe liver diseases including cirrhosis and hepatocellular carcinoma (HCC). The development of direct acting antiviral (DAA) drugs in 2011 and their subsequent use greatly improved the therapeutic outcomes for HCV patients [1], achieving a sustained virological response (SVR) rate of >90%. DAA drugs target HCV non-structural proteins including the NS3/4A protease, NS5A, and the NS5B RNA-dependent RNA polymerase [2], all of which are essential for HCV replication. However, the high cost of these DAAs limits their availability for many patients. In contrast to DAAs, the development of HCV vaccines has encountered extreme difficulties due mainly to the high viral mutation rate, which has a frequency of approximately 10⁻³ base substitutions per site per year [3]. The resolution of acute HCV infection requires efficient innate and adaptive immune responses. However, HCV evades the host immune system and establishes persistent infection in approximately 75-85% of patients it infects. Many reports in the past had examined the mechanisms by which HCV interferes with host immune responses. In this article, we will focus on the interaction between HCV and intracellular antiviral responses.

Pattern-recognition receptors and HCV-induced innate immune response

Pattern-recognition receptors (PRRs) sense non-self-molecular signatures called pathogen-associated molecular patterns (PAMPs) to trigger antiviral responses. The three classes of PRRs include the retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs), toll like receptors (TLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). RLRs include RIG-I, melanoma differentiation antigen 5 (MDA5), and the laboratory of genetics and physiology 2 (LGP2) [4]. RIG-I and MDA5 recognize double-stranded RNA (dsRNA), although the specific PAMPs that they detect are different [5]. HCV PAMPs for RIG-I is the 5’-PPP double-stranded RNA (dsRNA) and the 3’ poly U/UC sequence in the genomic RNA [6] (Figure 1). The CARD domain of RIG-I interacts with and activates the mitochondrial adaptor protein MAVS (also known as IPS-1, VISA or Cardif) on the mitochondrial membrane, which then further recruits TRAF3 and TRAF6. This leads to the activation of the downstream TBK1, which then phosphorylates IRF-3 followed by the homo-dimerization and translocation of IRF-3 into the nucleus to induce the expression of the interferon (IFN)-β gene. LGP2, which lacks the CARD domain, binds to RNA and synergizes with MDA5 signaling [4], resulting in the production of IFNs.

Figure 1.

Illustration of interaction between HCV and various cellular pathways including IFN signaling, TLR signaling, apoptosis, autophagy, mitophagy and inflammasomes in cells. Please see text for details.

The HCV-infected liver produces type I and III IFNs and upregulates the expression of IFN-stimulated genes (ISGs). Type I IFN-α binds to the IFN-α receptor (IFNAR) 1 and 2. Type II IFN-γ binds to IFNGR1 and IFNGR2, and Type III IFN-λ binds to IFNLR and IL-10Rβ. Type I and III IFNs share the intracellular signaling molecules including STAT proteins. The antiviral functions of the IFNs are mediated by ISGs, which include ISG15, the GTPase myxovirus resistance 1 (Mx1), ribonuclease L (RNaseL), 2’,5’-oligoA synthase (OAS) and protein kinase RNA-activated (PKR) [7]. HCV NS3/4A, the viral serine protease complex, plays multiple roles in overcoming the antiviral effect of IFNs. NS3/4A blocks the RIG-I signaling by cleaving the adaptor protein MAVS [8]. A point mutation at cysteine-508 (Cys-508) of MAVS, which rendered MAVS resistant to cleavage by NS3/4A, restored the interferon production in cells infected by HCV or harboring an HCV subgenomic RNA replicon [9,10]. The mitochondria-associated membrane (MAM) at the endoplasmic reticulum (ER)-mitochondria contact site serves as the MAVS signaling platform (see below). In another study, NS3/4A was shown to cleave peroxisome-associated MAVS to block the RIG-I antiviral signaling from peroxisomes [11]. The localization of NS3/4A to peroxisomes was not dependent on MAVS, although the presence of MAVS was preferred [11].

While TLR1, TLR2, TLR4, TLR5 and TLR6 are expressed on the cell surface and recognize PAMPs from bacteria, fungi, or viruses, TLR3, TLR7, TLR8 and TLR9 reside in endosomes and recognize intracellular viral nucleic acids (Figure 1). TLR3 recognizes dsRNA, TLR7 and TLR8 recognizes single-stranded RNA (ssRNA), and TLR9 detects DNA with unmethylated CpG sites. Most TLRs signal through MyD88 while TLR3 signaling is mediated by Toll/interleukin-1 receptor/resistance domain-containing adaptor-inducing IFN (TRIF). TLR signaling activates nuclear factor-κB (NF-κB) and IFN regulatory factors (IRFs). NF-κB induces inflammatory cytokines, and IRFs induce IFN production. The induction of IFN-β after the activation of TLR7 could suppress HCV replication [12]. Our lab also found that HCV induced the expression of tumor necrosis factor-α (TNF-α) via TLR7 and TLR8 in hepatoma cells and primary human hepatocytes [13]. This induction of TNF-α was biphasic, with the initial phase peaking at 2 hours post infection and subsiding at about 8 hours, followed by the second-phase induction at around 24 hours post-infection. TNF-α is required for type I interferon signaling in HCV-infected cells, as the silencing of either TNF-α or its receptor TNFR1 abrogated the expression of IFN-α receptor 2 (IFNAR2) and desensitized HCV-infected cells to type I IFNs. It should be noted that TNF-α had also been shown to promote HCV infection through a paracrine mechanism to disrupt the tight junctions and promote the entry of the virus into polarized hepatocytes [14]. HCV could also induce the autophagic degradation of IFN-α receptor, IFNAR1, to disrupt the IFN signaling [15].

Although host cells could produce TNF-α via TLR7/8 to support IFN signaling [13], HCV could also disrupt the TLR signaling. HCV could induce autophagic degradation of TRAF6 [16,17], which is an important adaptor molecule for TLR7/8 signaling. The HCV protease complex NS3/4A could also cleave TRIF, an important adaptor in the TLR3 signaling pathway [18]. Similarly, the HCV protein NS4B could disrupt TLR3-mediated interferon signaling by inducing the degradation of TRIF via caspase-8 [19]. NS5A could also bind to MyD88 and inhibit TLR signaling [20]. In 293T cells, NS3/4A bound to TBK1 and interfered with its association with IRF-3 [21].

Protein kinase R (PKR), a dsRNA-activated kinase, is an antiviral protein induced by IFNs. Viral infections and dsRNA can also activate PKR, which will then phosphorylate and inactivate eukaryotic translation initiation factor 2 (eIF2) α, a protein factor required for the cap-dependent translation of mRNAs. Although HCV E2 and NS5A by direct binding were implicated in the inhibition of PKR [22,23], others found that HCV infection or the expression of NS5B alone activated PKR and eIF2α phosphorylation, resulting in the suppression of expression of IFNs, ISGs, and MHC class I [24–27]. Meanwhile, the translation of HCV RNA genome was not affected, as it is cap-independent and mediated by the Internal Ribosome Entry Site (IRES) located in the 5’ untranslated region (UTR) of the genome.

Autophagy and HCV-induced innate immune response

Autophagy is a catabolic process by which damaged organelles and protein aggregates are removed. This process involves the formation of a double-membrane vesicle termed autophagosomes, which sequester part of the cytoplasm. Autophagosomes mature by fusing with lysosomes. The cargos of autophagosomes are then digested by lysosomal enzymes for recycling. Autophagy is also used by cells to remove intracellular microbial pathogens in a process known as xenophagy. The specific removal of viruses by autophagy is termed virophagy [28]. HCV infection induces autophagy [17,29]. However, it was able to subvert this intracellular antiviral response and use it to support its replication. HCV-induced autophagy is dependent on its induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which is triggered by three different ER stress sensors known as the activating transcription factor 6 (ATF6), the inositol-requiring enzyme 1 (IRE1), and the double-stranded RNA-activated protein kinase-like ER kinase (PERK) [17]. The silencing of ATF6, IRE1 or PERK to disrupt UPR or the silencing of autophagy-related proteins Atg5, Atg7, and Atg12 suppressed the replication of HCV [17,30–32]. HCV uses autophagy to benefit its replication in several ways. First, HCV utilizes the autophagic membranes as the sites for its RNA replication [17,33] (Figure 1). Second, HCV delays the maturation of autophagosomes early in infection to maximize its RNA replication. Third, HCV uses autophagic membranes to facilitate the interaction between its E2 envelope protein and apolipoprotein E (ApoE) for the production of infectious HCV particles [34]. Fourth, autophagy regulates type I interferon response (Reviewed in [35]), and the disruption of the UPR-autophagy pathway by silencing UPR- or autophagy-related genes inhibited HCV replication with a concurrent increase in ISG expression [30]. Finally, HCV-induced autophagy also impedes the innate immune response by stimulating autophagic degradation of TRAF6, an important signal transducer that activates NF-κB and the expression of pro-inflammatory cytokines [16,17].

Mitophagy and HCV-induced innate immune response

Mitochondria play an important role in the control of viral infections. They produce reactive oxygen species (ROS), which was shown to suppress HCV replication [36,37], and trigger apoptosis, which eliminates HCV-infected cells [38]. A low Ca²⁺ level in the mitochondrial matrix is needed to maintain the activity of matrix dehydrogenases and oxidative phosphorylation for the production of ATP, and Ca²⁺ overload can lead to mitochondrial fragmentation, disruption of ATP synthesis, and apoptosis [39,40]. HCV causes Ca²⁺ overload inside the mitochondria, which is followed by an enhanced production of ROS and the formation of mitochondrial permeability transition pore (MPTP), leading to apoptosis [38].

HCV promotes mitochondrial fission via upregulation of dynamin-related protein (Drp1) and the Drp1 receptor mitochondrial fission factor (Mff) [41]. Fragmented and dysfunctional mitochondria are removed by mitophagy, a mitochondria-selective autophagy, which is also induced by HCV [41] (Figure 1). Mitophagy is initiated by the stabilization of PINK1, a protein kinase, on the outer mitochondrial membrane and the recruitment of the E3 ubiquitin ligase Parkin by PINK1. Parkin ubiquitinates outer mitochondrial membrane proteins to trigger mitophagy. The silencing of PINK1 or Parkin decreases HCV replication, indicating a positive role of mitophagy in HCV replication [42]. The role of mitophagy in HCV replication was further supported by the observation that the inhibition of mitochondrial fission and mitophagy by Drp1 silencing suppressed HCV secretion with a concomitant increase in innate immune response and apoptosis [41].

MAM is a distinct membrane compartment that links mitochondria to the ER. The HCV NS3/4A protease complex targets MAVS at this synapse, not on mitochondria, to disrupt the RIG-I signaling [43]. Pila-Castellanos et al. recently demonstrated that the infection by influenza virus induced mitochondrial fusion, which was beneficial for the viral replication [44]. The authors argued that mitochondrial fusion resulted in the loss of MAMs where MAVS relayed the RIG-I signaling, and failure in maintaining MAMs following the fusion compromised innate immunity. With the use of Mito-C, a novel pro-fission compound, they were able to restore MAMs, increase IFN production, and enhance viral clearance [44]. Similarly, the silencing of mitofusin 2 (MFN2), which mediates mitochondrial fusion [45], enhanced the IFN-β promoter activity up to 24 hours post infection and reduced the permissiveness of cells to HCV infection [43].

Inflammasomes, HCV-induced innate immune response and IFN resistance

Inflammasome is a multiprotein complex composed of a NOD-like receptor (NLRs) such as NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the effector protease caspase-1. PAMPs, toxins, environmental irritants, and damage-associated molecular patterns (DAMPs) can activate inflammasomes [46]. The assembly of inflammasomes leads to the activation of caspase 1, which then cleaves and activates IL-1β and IL-18 for their release from cells, and gasdermin D (GSDMD) to induce pyroptosis [46,47]. In response to HCV infection, the NLRP3 inflammasome is activated in hepatocytes and macrophages to induce the release of IL-1β [48,49]. In chronic HCV patients, the IL-1β level is elevated [50]. Further analysis indicated that Kupffer cells were the major source of IL-1β, and HCV could enter macrophages to trigger TLR7 signaling to activate inflammasomes and the release of IL-1β [50]. The HCV core protein was necessary and sufficient for IL-1β production from macrophages, and this process required Ca²⁺ mobilization and phospholipase C activation [50,51]. Interestingly, HCV-infected cells displayed apoptosis as well as pyroptosis, the caspase-1/GSDMD-mediated cell death [52]. Daussy et al. examined the role of inflammasome components on the HCV-induced Golgi fragmentation [49], typically found in apoptotic cells [53]. They found NLRP3 silencing inhibited the HCV-mediated Golgi fragmentation while ASC silencing triggered Golgi fragmentation even in the surrounding non-infected cells [49]. Inefficient pyroptosis possibly led to apoptosis of infected cells.

Type I IFNs could repress the NLRP1- and NLRP3-dependent IL-1β production via IL-10 production [54]. Aarreberg et al. showed that IL-1β induced the release of mitochondrial DNA (mtDNA) into the cytoplasm, which is then detected by cyclic GMP-AMP synthase (cGAS), a cytosolic DNA senser (Figure 1). Subsequent generation of the second messenger cyclic GMP-AMP (cGAMP) led to STING activation followed by the activation of IRF-3 and NF-κB, which mediated the production of IFNs and other cytokines [55]. Thus, the ongoing interplay between IL-1β and IFN in the microenvironment of HCV-infected cells may contribute to an extended inflammation state, immune tolerance, IFN resistance and the development of liver cirrhosis and HCC [56,57]. In addition, the expression of HCV proteins, lipid accumulation, ROS, ER stress and other organelle stresses may exacerbate the inflammatory response induced by HCV. Dysfunctional lipid metabolism associated with HCV infection leads to steatosis and detrimental liver damages [58]. The interaction between HCV NS5A and the α and β subunits of the mitochondrial trifunctional protein (MTP), which catalyzes the last 3 steps of mitochondrial lipid β-oxidation, was shown to be associated with the reduction of lipid β-oxidation and the suppression of MTP expression [59]. This depletion of MTP reduced the responsiveness of HCV-infected cells to IFN-α [59]. HCV core had also been shown to increase lipid accumulation and activate the NLRP3 inflammasome [51,60,61].

Conclusion

An effective host innate immune response plays a critical role in the clearance of viral infections. During the past decades, studies provided mechanistic insights on how HCV evades the host innate immune response to establish chronic infection in patients. Cleavage of TRIF and MAVS by the HCV NS3/4A protease complex interferes with TLR3 and RIG-I signaling pathways, respectively, hindering the production of pro-inflammatory cytokines and IFNs (Figure 1). Since these cytokines and IFNs are important antiviral factors, the suppression of their expression is critical for HCV to persist inside its host cell. In addition, autophagy, which is supposed to control viral infection, is usurped and used by HCV to promote its replication. Similarly, mitophagy removes dysfunctional mitochondria, thereby preventing premature apoptotic cell death and promote HCV persistence. Mitochondrial fusion-induced loss of MAMs and its associated MAVS is yet another way to suppress the RIG-I-dependent induction of IFNs.

HCV can induce inflammasomes for the production of IL-1β, and Kupffer cells appear to be the major source of IL-1β in HCV patients. IL-1β can induce the expression of IFNs, which in turn can inhibit the formation of inflammasomes to repress the production of IL-1β. The prolonged interplay between IL-β and IFN can lead to chronic liver inflammation and severe liver diseases as well as IFN resistance. HCV can also perturb mitochondrial metabolism to desensitize HCV-infected cells to IFN-α. These activities of HCV provide an explanation to why most patients infected by HCV failed to clear this viral infection and ultimately develop severe liver diseases including steatosis, cirrhosis and HCC, and why most of HCV patients are not responsive to IFN therapies.

Highlights.

• HCV disrupts signaling pathways initiated by pattern recognition receptors.

• HCV subverts and uses the antiviral autophagic response to support its replication.

• HCV perturbs mitochondrial dynamics to suppress apoptosis and interferon response.

• HCV dysregulates inflammasomal response to cause interferon resistance.