From viruses to cancer: exploring the role of the hepatitis C virus NS3 protein in carcinogenesis

Abstract

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide and is a known etiological agent of hepatocellular carcinoma (HCC). The molecular mechanisms of HCV-mediated carcinogenesis are not fully understood. This review article focuses on the oncogenic potential of NS3, a viral protein with transformative effects on cells, although the precise mechanisms remain elusive. Unlike the more extensively studied Core and NS5A proteins, NS3’s roles in cancer development are less defined but critical. Research indicates that NS3 is implicated in several carcinogenic processes such as proliferative signaling, cell death resistance, genomic instability and mutations, invasion and metastasis, tumor-related inflammation, immune evasion, and replicative immortality. Understanding the direct impact of viral proteins such as NS3 on cellular transformation is crucial for elucidating HCV’s role in HCC development. Overall, this review sheds light on the molecular mechanisms used by NS3 to contribute to hepatocarcinogenesis, and highlights its significance in the context of HCV-associated HCC, underscoring the need for further investigation into its specific molecular and cellular actions.

HCV infection

Hepatitis C virus (HCV) is a member of the Flaviviridae family harboring an approximately 9.6 kbp positive single-stranded RNA genome [1]. Upon infection of hepatocytes, the genome is released in the cytoplasm where the single open reading frame (ORF) is transcribed in a polyprotein using an internal ribosome entry site (IRES) sequence [1]. Viral and cellular proteases are responsible for the cleavage and production of 10 viral proteins: structural proteins Core, E1, and E2, and non-structural proteins P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B [1]. Genomic variations within studied HCV genomes have led to the classification of six different genotypes, further divided into subtypes [1]. After acute infection, 75–85% of patients progress to chronic hepatitis, which significantly increases the risk of cirrhosis, fibrosis, and hepatocellular carcinoma (HCC) [2]. The prevalence of HCV infection is high; 170 million people worldwide live with chronic HCV infection [2]. HCV-associated HCC usually develops after the establishment of cirrhosis, and carcinogenesis is tightly connected to immune-induced inflammation [3]. However, recent studies have demonstrated the direct effect of HCV on carcinogenesis, mainly through the Core, NS3 and NS5A viral proteins [4]. This review will focus on the implication of the viral protein NS3 in carcinogenesis, as multiple studies have shown the transformative effects of this protein on cells, although the precise mechanism is still unclear.

HCV NS3 protein

NS3 is a 631-amino acid protein that harbors two distinct enzymatic activities [5]. The first 180 amino acids in the N-terminal region of the protein act as a serine protease which is required for the cleavage of the viral polyprotein at four specific junctions (Fig. 1A) [5]. This NS3 protease domain adopts a β-barrel structure (Fig. 1B) [5]. To achieve its functionality, NS3 needs both a zinc ion and the viral NS4A cofactor, which allows the formation of an eight-stranded β-barrel structure required for the correct orientation of NS3 catalytic triad [5]. The interaction between the two viral proteins is essential for the processing of the polyprotein. NS4A is tethered to the endoplasmic reticulum (ER), and its interaction with NS3 anchors it to the ER [6, 7]. Numerous studies have investigated the co-expression of NS3 and NS4A to unravel their intricate interplay.

Fig. 1

Representation of HCV-NS3 domains. (A) N-terminal contains the protease domain from residues 1–180 represented in purple. Residues His⁵⁷, Asp⁸¹ and Ser¹³⁹ compose the catalytic triad. C-terminal contains a NTPase/helicase domain from 180 to 631, itself composed of three domains represented in different shades of blue. Helicase domain I, Ia and III to VI are shown in domains 1 and 2. (B) 3D representation of NS3 from genotype 1b obtained by X-ray diffraction (PDB: 3O8B). The same color code was used as in (A), residues in the catalytic triad are in orange and represented in their gaussian surface form

The C-terminal region of NS3 houses an essential NTPase/helicase domain. NS3 interacts with nucleotides and utilizes energy from hydrolysis to move along RNA or DNA molecules. This region of the protein is further divided into distinct domains. Within domains 1 and 2, NS3 exhibits the seven canonical SF2 helicase motifs (I, Ia, and II through VI) [8]. Additionally, NS3 also possesses other conserved domains that vary across different HCV genotypes [9]. Two noteworthy features are the Arg-clamp motif, which connects domains IV and V, and the Phe-loop, situated between motifs V and VI. Residues responsible for binding nucleotides include Thr269 in domain 1 and Thr411 in domain 2 [10]. The ATP binding site lies between these two domains, forming two RecA-like domains, and comparative studies suggest that Lys210 may contact ATP [8]. Overall, the helicase activity of NS3 is critical for HCV replication, although its precise function is not fully resolved but likely involves unwinding RNA duplexes or secondary structures within the viral RNA [8].

Since NS3 possesses two enzymatic activities, it has been a target for inhibitor development. The inhibitors targeting NS3 protease are divided into two main categories: first-generation protease inhibitors, which operate through reversible covalent interactions, and second-generation protease inhibitors, characterized by noncovalent binding mechanisms [11]. Treatment strategies vary depending on the HCV genotypes, with protease inhibitors such as Telaprevir and Simeprevir showing efficacy [11]. Despite the presence of helicase inhibitors, their advancement is hindered by the conserved nature of the NS3 helicase domain across various cellular helicases, posing significant challenges in their development [11]. Approaches employing small molecules or nucleic acid-based inhibitors to target RNA or DNA binding sites and disrupt helicase function have been explored, yet inhibitors specifically targeting the helicase activity are still not available for clinical use [11].

NS3 in hepatocellular carcinoma

The correlation between NS3 and cellular transformation was initially highlighted in a seminal study dating back to 1995. In this study, researchers expressed either the 5′ or the 3′ genome segment of NS3 in NIH3T3 fibroblasts and meticulously monitored transformation-associated characteristics such as contact inhibition, doubling time, cloning efficiency, and tumor induction upon injection into mice [12]. Notably, the transforming capacity was unequivocally demonstrated by the NS3 5′ genome segment, corresponding to the N-terminal protease domain [13,14,15]. The helicase domain was also associated with HCC as levels of antibodies that recognize peptides in this region (1381–1403) are lower in HCV patients in chronic stage compared to HCV patients in cirrhotic or HCC stage [16]. Different groups have studied the immunological impact of different peptides spanning NS3 sequence, without studying the association with the stage of the disease [17,18,19]. However, there is an association between viral clearance and immunological response to the NS3 epitope 1248–1261 [18]. Multiple studies have also associated NS3 mutants and polymorphisms with HCC development. For instance, within HCV genotype 1b, the presence of the Tyr1082/Gln1112 polymorphism in NS3 is associated with a heightened risk of HCC [20]. NS3 from genotype 1b is also less immunological, as sera from patients infected with 1b are less reactive than genotypes 1a and 3a [21, 22]. Those observations could indicate that immunological suppression by NS3 is an important feature in HCC development. Moreover, viruses within genotype 1b can be stratified into distinct subgroups based on NS3 secondary structure, with certain subgroups, particularly group B, exhibiting a heightened incidence of HCC [23, 24]. Collectively, these findings underscore the intricate involvement of NS3 in carcinogenesis.

The following paragraphs provide an overview of the mechanisms potentially involving NS3 in carcinogenesis. Cancer emerges when cells divide uncontrollably, seizing control of tissues and organs. It is characterized by various cellular alterations, as reviewed in The Hallmarks of Cancer [25]. For cells to become cancerous, they must acquire the ability to achieve replicative immortality, resist cell death, circumvent growth suppression, instigate angiogenesis, and facilitate invasion and metastasis. Recently, researchers have added two more traits to this list: altering cellular energy processing and avoiding detection by the immune system. Additionally, factors like inflammation and genomic instability also play significant roles in cancer development.

NS3 molecular mechanisms of carcinogenesis

Sustaining proliferative signaling

Cell division is intricately regulated by multiple extracellular signals. These signals culminate in proliferation through the mitogen-activated protein kinases (MAPK) signaling pathway, among others. Extensive research has explored the role of NS3 in MAPK signaling. Across various studies, NS3 has been implicated in the activation of multiple MAPK pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 [26,27,28,29,30,31]. This activation, as elucidated by different research groups, triggers distinct patterns of MAPK phosphorylation, consequently modulating the activity of activator Protein-1 (AP-1) transcription factor involved in cell growth regulation [26,27,28,29]. Some of those studies have shown that NS3-mediated MAPK activation is required for the viral protein’s effect on cell growth [26, 29]. In HepG2 and HeLa, NS3 expression mediates cell growth through activation of JNK, leading to AP-1, activating transcription factor-2 (ATF-2), and c-jun expression [26]. One of AP-1 transcriptional targets is cyclin D1, which is involved in the progression of G1/S phase transition of cell cycle. Moreover, NS3 expression in QSG7701 cells induces an increase in cyclin D1 levels through the activation of ERK and nuclear factor-κB (NF-κB) pathways [29]. However, it’s crucial to note the inconsistency among studies regarding which specific MAPK pathway NS3 activates, highlighting the lack of consensus in the literature on this subject.

Other cell-growth pathways, such as NOTCH and epidermal growth factor receptor (EGFR), are also modulated by NS3. However, direct involvement of NS3 in cell proliferation via these pathways remains unconfirmed. In HEK 293 cells, NS3 was shown to interact with Snf2-related CBP activator protein (SRCAP), a mediator of NOTCH activation [32]. Interaction between SRCAP and NS3 activates NOTCH-dependent transcriptional activity in Hep3B cells [32]. NS3-mediated NOTCH activation also requires the presence of another protein possibly interacting with NS3, namely p400 [32]. Despite reports of NOTCH upregulation in HCC, a pathway known for its dual roles as both an oncogene and tumor suppressor, the exact impact of NS3-mediated NOTCH activation on proliferation necessitates further investigation [33, 34]. NS3/4A is also responsible for the cleavage and inhibition of T-cell protein tyrosine phosphatase (TC-PTP), a phosphatase involved in regulation of EGFR signaling [35]. A negative correlation between NS3 and TC-PTP was confirmed in liver biopsies from patients infected with HCV, supporting the evidence of cleavage by NS3 [36]. This cleavage results in the activation of EGFR and protein kinase B (Akt), with Akt activation proving pivotal for both viral replication and resistance to apoptosis in HCV-infected cells [35, 37]. However, the involvement of this pathway’s activation on NS3-mediated cell growth needs further studies. Furthermore, NS3 may activate EGFR through an alternate mechanism involving neuregulin 1 (NRG1). NS3 was shown to cleave NRG1, which could explain the upregulation of EGFR pathway in cells infected with HCV (Fig. 2, Table 1) [38]. However, the physiological implications of this mechanism remain largely uncharacterized, warranting further investigation into its role in cellular physiology.

Fig. 2

Visual summary of the different mechanisms by which NS3 could contribute to carcinogenesis

Table 1 Summary of the effects of NS3 on different pathways involved in carcinogenesis

The co-expression of NS3 with its cofactor NS4A appears to modulate NS3’s ability to enhance cell growth. While various studies have demonstrated NS4A’s inhibitory effect on NS3-mediated growth promotion, this inhibition does not extend to the activation of MAPK pathways, as detailed previously [26, 39]. Intriguingly, certain research groups have also noted the antiproliferative properties of NS3 [39, 40]. In hepatic cell lines, NS3 expression inhibits cell cycle progression either in G0/G1 or G2/M phases, depending on the study [39, 40]. However,  the absence of p21 cell cycle inhibitor expression in the cell lines was noted, potentially explaining the disparate findings [40]. Moreover, in one particular report, despite the inhibitory effect of NS3 on cell growth in vitro, mice inoculated with HepG2 cells expressing NS3 exhibited augmented proliferative capacities [39]. Further investigation is warranted to elucidate the precise mechanisms underlying these observations.

Resisting cell death

NS3 interacts with p53, a transcription factor involved in the control of cell cycle progression, via specific amino acids (Leu106 and Phe43), effectively inhibiting p53’s function [41,42,43,44]. In cells expressing NS3, both p53 and its target gene p21 exhibit reduced mRNA levels [39]. Although another study found that NS3 does not affect p53 stability or transcriptional activity, NS3 inhibits p21 transcription in a manner depending on the number of p53 binding domains in the p21 promoter [45]. The authors hypothesized that p21 expression could be repressed by the interaction between NS3 and p53, which could explain the increased cell growth observed in NIH 3T3 cells. Additionally, another group has linked the NS3-p53 interaction to the upregulation of ubiquitin D (UBD), which correlates with proliferative effects [46].

Beyond its pivotal role in cell cycle regulation, p53 is intricately linked to apoptosis. NIH 3T3 cells expressing the N-terminal region of NS3 are more resistant to actinomycin D-induced apoptosis and present reduced protein levels of p53 [47]. However, the impact of NS3 on apoptosis remains a subject of debate. While some studies find no discernible effect of this viral protein on apoptosis, others reveal a pro-apoptotic influence of NS3 [48]. First, a study demonstrated the apoptotic effect of NS3 on mature dendritic cells, which correlated with a decrease in p21 expression [49]. Subsequently, NS3 was found to interact with caspase-8, an apoptosis effector, thus promoting this form of cell death [50, 51]. Intriguingly, NS3-mediated apoptosis in phagocytes may contribute to carcinogenesis. Exposure of phagocytes to NS3 activates NADPH oxidase, resulting in heightened oxidative stress and subsequent apoptosis [52]. Consequently, NS3-induced apoptosis impairs phagocyte response, a process crucial for eliminating infected cells.

Genome instability and mutation

Genomic instability is a hallmark of cancer cells [25]. Mutations in DNA are usually repaired by different mechanisms. However, when those processes fail, mutations accumulate, leading to genomic instability. Therefore, genes involved in DNA repair play a critical role as tumor suppressors [25]. The expression of NS3 has been shown to induce cellular DNA damage which could mediate carcinogenesis. In Raji cells expressing NS3, inducible nitric oxide synthase (iNOS) expression was upregulated [53, 54]. Remarkably, this upregulation correlates with the number of NF-κB copies in the iNOS promoter, suggesting that NF-κB acts as a mediator in this process [53]. Notably, iNOS generates nitric oxide (NO), a genotoxic molecule that inflicts various types of DNA damage [55]. Furthermore, in NS3-expressing cells, double-stranded breaks occur, further emphasizing the intricate interplay between NS3, DNA damage, and potential carcinogenic pathways [54].

The HCV NS3 protein plays a significant role in promoting DNA damage and disrupting DNA repair through various mechanisms. Notably, the NS3/4A complex interacts with key DNA repair proteins such as ataxia telangiectasia mutated (ATM), Ku70, and Werner syndrome helicase (WRN), thereby affecting the cellular response to DNA lesions. ATM is essential for recognizing double-stranded DNA breaks and for the phosphorylation of histone H2AX on serine 139 (γ-H2AX), a marker of DNA damage [56]. The interactions of NS3 and NS3/4A with ATM do not lead to cleavage of ATM but result in its relocation to the cytoplasm, impairing the phosphorylation of γ-H2AX [57, 58]. This mislocalization correlates with ineffective DNA damage repair, as observed in U2OS cells expressing NS3 [58]. Additionally, the NS3/4A complex promotes the degradation of WRN through proteasomal pathways, thus interfering with WRN’s role in DNA repair. NS3 also binds to Ku70, a protein crucial for non-homologous end joining (NHEJ), diminishing its ability to bind to DNA breaks [59]. This interaction significantly reduces the effectiveness of NHEJ, as demonstrated in studies involving NS3 and NS3/4A [59]. These findings collectively highlight the complex role of NS3 in hindering DNA repair processes.

Activating invasion and metastasis

Migration and invasion are key aspects of cancer cell transformation [25]. The NS3 protein significantly influences these processes through multiple mechanisms. Firstly, NS3 interacts with protein phosphatase 1A (PPM1A) and promotes its ubiquitination and subsequent degradation. PPM1A is a phosphatase that plays a role in several cellular pathways, and its degradation has been associated with increased cell migration and invasion in Huh-7 cells [60]. Additionally, NS3 expression is linked to changes in key molecules involved in epithelial-mesenchymal transition (EMT). This includes increased levels of vimentin and N-cadherin, along with decreased expression of E-cadherin, further facilitating the migration and invasion capabilities of cancer cells [60].

NS3 enhances cell migration through several mechanisms. One such mechanism involves increasing the expression of gelatinase-B (MMP-9), an enzyme that degrades collagen-IV and promotes tumor invasion. In various cell lines, NS3 activates the transcription of MMP-9 by triggering the ERK1/2 and p38 MAPK pathways [61]. This activation leads to the translocation of NF-κB to the nucleus, where it binds to the MMP-9 promoter [61]. Additionally, NS3 further boosts MMP-9 levels through expression of cyclooxygenase-2 (COX-2), an enzyme involved in inflammation and other pathways important for carcinogenesis [61, 62].

Moreover, NS3/4A increases invasion by promoting invadopodia formation, cell protrusions involved in matrix degradation and invasion [63]. As previously described, NS3 achieves this by cleaving TC-PTP, which activates the EGFR pathway, a critical process in invadopodium assembly [63]. Furthermore, a protein network analysis revealed that proteins associated with focal adhesion interact with NS3, influencing cellular dynamics [64]. Cells expressing NS3 or NS3/4A exhibit a significantly reduced ability to bind to fibronectin in vitro [64], a key component of the cellular matrix. Additionally, upregulation of UBD in HepG2 cells expressing NS3 also leads to increased cell migration, reinforcing NS3’s role in enhancing invasive properties [46].

Tumor-related inflammation

Chronic inflammation is tightly linked to cancer as it creates a niche favorable for the malignant transformation of cells [65]. Multiple processes induced by chronic inflammation are associated with cancer, such as proliferation, invasion, and fibrosis [65, 66]. Since viruses such as HCV create a hepatic cancer niche from chronic inflammation, it was suspected that viral proteins participated in inflammation induction [65]. NF-κB is involved in inflammation by regulating the expression of different genes, including tumor necrosis factor α (TNF-α), an inflammatory cytokine [27, 67,68,69]. It was demonstrated that NS3 promotes the activation of IκB Kinase α (IKKα) in HepG2 and Huh7 [27]. IKKα is responsible for the degradation of NFKB inhibitor α (IκB-α), a negative regulator of NF-κB [27]. NS3 activation of IKKα is mediated through IKKα/NF-κB but also requires JNK/AP-1 signalling [27]. Others have reported that activation of NF-κB, and other signalling pathways such as JNK and ERK, in HepG2 leads to an upregulation in COX-2 expression [30].

NS3 has also extracellular inflammatory effects: a study showed that monocytes are activated in response to NS3 exposition, which leads to the expression of TNF-α and interleukin-10 (IL-10) [68]. A downregulation of IκB-α was also observed in the same study, suggesting the involvement of NF-κB [68]. Furthermore, exposing myeloid dendritic cells to NS3 reduces their allostimulatory capacity by inhibiting their differentiation resulting in irregular cytokine expression [68]. The same group demonstrated the capacity of NS3 exposition on HEK 293 cells, mouse macrophages, and human monocytes to activate toll-like receptor 2 (TLR2) [69]. In this case, TLR2, ERK, and JNK/AP-1 pathways were necessary to induce TNF-α [69]. Further studies highlighted the importance of TLR2, TLR1, and TLR6 in cytokine expression mediated by NS3 exposition in human monocytes [70]. Similar results were obtained in microglia cell line CHME3 exposed to NS3 in which inflammatory cytokine expression, such as IL-8, IL-6, and IL-1β, was induced through TLR2 or TLR6, but not TLR1 [71].

Furthermore, co-expression of HCV nonstructural proteins in hepatic stellate cells (HSC) promotes the expression of pro-inflammatory molecules such as RANTES, monocyte chemoattractant protein-1 (MCP-1), IL-8, and intercellular Adhesion Molecule 1 (ICAM-1) through a mechanism involving upregulation of intracellular Ca²⁺ and reactive oxygen species (ROS) formation [28]. Those results were obtained in HSC expressing NS3-NS5 with an adenovirus system. While it was shown that HSC express receptors necessary for HCV infection, the capacity of the virus to infect this cell type was not characterized [28]. Nevertheless, NS3 does have extracellular effects on HSC by activating pro-inflammatory signaling pathways such as c-Jun, p38, AP-1, and NF-κB [28].

Fibrosis is an inflammatory response where cells deposit extracellular matrix to repair the tissue [72]. It is a mechanism correlated with cancer as they both arise from chronic inflammation. However, fibrosis can lead to cytokine production, creating a positive feedback of inflammation response [72]. Non structural proteins (NS3–NS5) expression in HSC leads to fibrosis induction by secretion of transforming growth factor β1 (TGFβ1) and increased procollagen α1 expression [28]. Another study found that NS3 binds the type I subunit of the transforming growth factor receptor (TβRI) by mimicking its ligand TGFB2 transforming growth factor beta 2 (TGF-β2) [73]. In LX-2 HSC cells treated with NS3 but not normal hepatic cells, this interaction results in the expression of collagen and TGFβ1, a cytokine promoting fibrosis [73]. Those results suggest once again that NS3 has extracellular effects on HSC. Dysregulation of micro-RNA (miRNA) expression by NS3 is also involved in fibrosis. Human monocytes stimulated with NS3 show an increase of miR-155, responsible for activating inflammatory signaling pathways [74, 75]. Furthermore, NS3 expressing cells show a downregulation of miR-335, miR-150, and miR-122, with anti-fibrotic effects, and an upregulation of miR-27a, with pro-fibrotic effect [76,77,78].

Avoiding immune destruction

The progression of HCV infection into chronic infection is a risk factor for HCC development. Indeed, most HCV-induced HCC occur after the onset of inflammation and cirrhosis, although cases that defy that statement have been reported [79,80,81]. Immune system evasion contributes to chronic infection, which can lead to carcinogenesis. Avoiding immune destruction is also an important cancer hallmark. As mentioned in the last section, many studies have demonstrated the activation of NF-κB and the upregulation of TNF-α expression in the presence of NS3. However, it was shown that NS3/4A expressed in mice hepatocytes in vivo confers resistance to TNF-α treatment [82]. Another study showed a downregulation of NF-κB activation following TNF-α stimulation in HEK 293T and Huh-7.5.1. This effect was attributed to NS3 and its interaction with HOIL-1-interacting protein (HOIP), a member of the linear ubiquitin chain assembly complex (LUBAC), competing for NF-κB essential modulator (NEMO), both involved in NF-κB signalling [83]. In Huh-7.5.1, perturbation of this interaction by NS3 inhibits polyubiquitination of NEMO, which is important in the activation of NF-κB [83].

HCV can be recognized by different pathogen recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and TLRs [84]. After viral RNA sensing by RLRs, the caspase recruitment domain (CARD) of RLRs interacts with the same domain on the mitochondrial antiviral-signaling protein (MAVS) present in the mitochondrion and peroxisome membrane [85]. MAVS then activates signalization, leading to interferon (IFN) expression [85]. NS3 impairs this pathway through cleavage of MAVS at Cys-508, inhibiting IFN production [86,87,88,89,90,91,92,93]. Another cleavage target of NS3/4A in this pathway is Riplet, which is necessary for the ubiquitination and activation of RIG-1 [94]. Another study revealed that residue Y16 of NS4A is essential for NS3/4A Riplet inhibition in Huh7 cells [95].

In addition to RLR dysregulation, NS3 controls TLR signaling. Indeed, in vitro assays demonstrated the capacity of NS3/4A to cleave TIR domain-containing adapter inducing IFN-β (TRIF), a downstream effector of TLR3, involved in the recognition of dsRNA [96]. TRIF cleavage was also shown in osteosarcoma cells, impairing IFN response [97]. However, another team observed no cleavage of TRIF in HEK 293T expressing NS3 or NS3/4A [98]. Still in the TLR pathway, TANK-binding kinase 1 (TBK1) was demonstrated to be an interactor of NS3 [98]. In HEK 293T, NS3 competitively binds TBK1, inhibiting its interaction with interferon regulatory factor 3 (IRF-3), a transcription factor responsible for IFN expression [98]. Concordantly, another study observed lower levels of phosphorylated IRF-3 in UHCV11 cells expressing NS3/4A following IRF-3 activation by Sendai virus [99]. Upon IFN secretion, the molecule binds to its receptor, activating a signaling cascade involving signal transducer and activator of transcription 1 (STAT1). In Huh-T7 cells expressing NS3/4A, lower levels of STAT1 are observed, although the two proteins don’t interact [100].

Enabling replicative immortality

Cells have a limited division potential according to the length of their telomeres framing chromosome ends, after which, apoptosis or senescence occurs. To evade this unfortunate destiny, cancer cells reactivate telomerase (TERT), an enzyme catalyzing the lengthening of telomeres. In normal cells, telomere shortening limits cellular replication by inducing senescence or apoptosis. Many different proteins from oncogenic viruses control TERT activity [101]. This prevents telomere shortening and allows continuous cell division, effectively enabling cells to bypass replicative limits. However, very few studies have focused on the role of NS3 in replicative immortality. Still, NS3/4A was found to interact with C-terminal region of TERT and increase its activity in HEK 293 expressing NS3/4A [102]. Considering the crucial role of TERT in cancer development, further studies on the relationship between NS3 and replicative immortality are warranted.

The Flaviviridae NS3 protein

The Flaviviridae family contains four genera: Flavivirus, Hepacivirus, Pestivirus, and Pegivirus [103]. Members of the first two genera cause disease in humans [103]. Viruses in the Flaviviridae family have a similar genome organization and possess a conserved NS3 protease-helicase protein that requires a viral co-factor (NS2B for Flavivirus and NS4A for Hepacivirus) [103]. However, HCV, the only human virus in the Hepacivirus genus, is one of the only Flaviviridae associated with cancer. Furthermore, attenuated forms of Flavivirus have been proposed as oncolytic therapies [104]. Reasonably, HCV-NS3 is far more studied for cell transformation than other Flaviviridae. Nonetheless, some studies have shown similar roles of NS3 from other Flaviviridae to those presented in this review.

First, Flaviviridae NS3 has a role in intrinsic immunity and inflammation, as reviewed in Latanova et al. [105] and Chen et al. [106]. It was observed that Kunjin virus (KUNV) and Dengue virus (DENV) NS2B/NS3 inhibit IFN response [107,108,109]. DENV-NS2B/NS3 targets STING, involved in pathogen-associated molecular pattern recognition, for cleavage and impairs IFN expression [109]. DENV-NS3 also targets mediator of IRF3 activation (MITA) for cleavage [110]. Moreover, DENV-NS3 interacts with 14-3-3ε, leading to inhibition of RIG-1 translocation to MAVS [111]. Zika virus (ZK) NS3 also interacts with 14-3-3ε and 14-3-3η, inhibiting RIG-1 and melanoma differentiation-associated protein 5 (MDA5) translocation to mitochondria [112]. Furthermore, ZK-NS3 induces MAVS and MITA degradation leading IFN-β expression inhibition [113]. ZK-NS3 was also associated with the degradation of nucleotide-binding oligomerization domain-like receptor 3 (NLRP3), involved in activation of the inflammasomes [114].

However, contrary to some reports on HCV-NS3, multiple studies reported the apoptotic effect of NS3 from different Flaviviridae. First, DENV-NS2B/NS3 was shown to induce apoptosis [115, 116]. DENV-NS2B/NS3 cleaves IKKα/β, leading to activation of NF-κB and promotion of apoptosis through extrinsic pathways [116]. West Nile Virus (WNV) NS3 cleaves procaspase-8 triggering apoptosis [117]. Japanese encephalitis virus (JEV) was also shown to induce apoptosis through the caspase and mitochondrial pathways [118, 119]. Finally, Langat virus NS3 triggers apoptosis through caspase-8 binding [120].

The Pegivirus genus, to which HCV bears more resemblance than the Flavivirus genus, contains viruses that infect humans (human pegivirus: HPgV) [121]. Besides, HCV-NS3 shares more similarities with HPgV-1-NS3 than other Flaviviruses NS3, such as WNV, KUNV, ZK, and DENV (Fig. 3). Considering this resemblance, it could be hypothesized that HPgV-NS3 has oncogenic functions, as does HCV-NS3. HPgV is a lymphotropic virus, but its pathogenicity is unclear; it is not considered a causal agent of any disease but is associated, among others, with the development of non-Hodgkin lymphoma (NHL) [122]. The association between HPgV infection and NHL is not understood mechanistically, but it has been hypothesized that reduced immune surveillance in chronic HPgV infection could be in cause [122, 123]. Nevertheless, HPgV-1-NS3/4A/B was shown to cleave MAVS, as HCV-NS3, leading to impaired IFN response [124, 125].

Fig. 3

Percent identity matrix of NS3 alignment from different Flaviviridae. Alignment and analysis were done with Uniprot Align tool with the Clustal Omega program. HCV genotype 1b isolate BK (P26663); WNV strain NY99 (Q9Q6P4); KUNJIN strain MRM61C (P14335); ZIKA strain MR 766 (Q32ZE1); DENV type 2 strain IQT2913 (Q9WDA6); HPgV-1 (Q69422)

Conclusion

The HCV NS3 protein disrupts a variety of cellular pathways, potentially contributing to the development of HCC. Additionally, NS3 may influence other cancer-related characteristics that are not discussed in this review. Indeed, other oncogenic viral proteins are known to promote angiogenesis, such as the human papillomavirus E6 protein, which increases the expression of vascular endothelial growth factor (VEGF) [126]. Furthermore, oncogenic viruses can disrupt cellular metabolism; for example, HCV infection alters glucose metabolism through several mechanisms, including increased glycolysis [127, 128]. Moreover, oncogenic viruses like Epstein-Barr virus and Hepatitis B virus are known to modify alternative splicing (AS) [129,130,131]. Interestingly, the interaction between NS3 and PKC-δ leads to its activation. PKC could then phosphorylate human antigen R (HuR), allowing its relocalization to the cytoplasm, where it plays a role in viral replication. HuR is also involved in pre-mRNA splicing through its binding of AU-rich sequences [132]. HuR is involved in many viral infections; for example, in Sindbis virus infection, HuR interacts with viral RNA 3’-untranslated regions, which mediates its translocation to the cytoplasm [133, 134]. HuR sequestration in the cytoplasm leads to changes in AS [133]. AS alterations are frequent in cancer, and some even consider it as a hallmark [135, 136]. Given these observations, it is plausible to hypothesize that oncogenic viral proteins, such as HCV-NS3, may contribute to cancer development through modulations in AS.

Despite the numerous pathways altered by the HCV NS3 protein, there is no consensus on the exact mechanism by which it contributes to cell transformation, as findings from various studies often contradict each other. These discrepancies could stem from several factors, including differences in the expression systems used, the types of cells studied, and whether other co-factors are co-expressed. Additionally, the protease activity and localization of NS3 are influenced by its interaction with NS4A, and co-expression of these two proteins can alter the outcomes when protease activity is a factor. The validity of results derived from ectopic expression of viral proteins is also debatable. Issues such as the level of protein expression and the absence of other viral proteins create conditions that do not fully mimic natural viral infections. For instance, other HCV proteins like E2 and Core have been shown to inhibit NF-κB activity, which contradicts many studies focused solely on NS3 [137, 138].

Understanding the interaction between the NS3 protein and its host cell is crucial for effective HCV diagnosis and treatment [139, 140]. NS3 participates in various cellular signaling pathways, which may influence the success of therapies. For instance, cancer cells often upregulate COX-2 expression in response to chemotherapy drugs, a behavior also observed in cells expressing NS3 [141]. Consequently, inhibiting COX-2 has been shown to enhance the effectiveness of chemotherapy [141]. Additionally, evidence suggests that COX-2 inhibitors can slow the progression of hepatocellular carcinoma (HCC) in various models [142].

In conclusion, while NS3 has the potential to transform cells and various mechanisms have been proposed to explain its effects, further research is necessary to clarify these mechanisms and reconcile the conflicting results.