Hepatitis C is a life-long disease that begins when a virus infects the cells of the liver. Although the infection is curable, it is expensive to treat, and there is not yet a vaccine to prevent the disease. This is largely because the virus that causes hepatitis C, also known as HCV, naturally only infects humans and chimpanzees, which has made it difficult to generate an effective animal model for developing a vaccine.

Mice are frequently used as a model for studying disease and can be genetically altered to allow HCV to enter their liver cells. However, once HCV enters mouse cells, it struggles to replicate. As a result, an infection does not develop, and the immune system’s response to the virus cannot be fully explored. Replication of HCV in humans relies on a protein called cyclophilin A, or CypA for short. Now, Gaska et al. have set out to improve current animal models for HCV by investigating whether HCV can also use CypA from other species, including mice, to replicate.

Gaska et al. showed that the mouse form of CypA could help HCV replicate in human liver cells with lower than normal levels of CypA, but only very poorly. Editing the mouse gene for CypA to be more like the human version resulted in higher HCV replication. Putting variants of CypA into the liver cells of mice, which do not normally replicate HCV at high levels, led to an increase in HCV replication. However, replication of HCV in mice was still far lower than in human liver cells, suggesting that the mouse model system could be improved by learning more about which proteins interact with CypA.

Injection drug use – one of the main ways hepatitis C spreads – is becoming increasingly common because of the growing opioid epidemic in many countries. A clinically relevant animal model that supports hepatitis C virus infection would be an important milestone towards a vaccine that could prevent the continued spread of this disease.

Every year, an estimated 3–4 million individuals become newly infected with hepatitis C virus (HCV) (Westbrook and Dusheiko, 2014), with 60–80% going on to join the population of approximately 71.1 million who are chronically infected (Blach et al., 2017). A hepatotropic virus, HCV has only been shown to robustly infect in vivo human and chimpanzee hepatocytes. This limited host tropism has proven problematic in developing an animal model that is not only ethically and financially sound but also immunocompetent. Such a model would allow the study of the underlying immunopathogenesis of HCV infection as well as the development and testing of vaccine candidates. Most efforts to generate such an in vivo model have focused on mice, which are amenable to genetic manipulation and have many well-established research tools built around their use.

Overcoming the natural imperviousness of murine hepatocytes to HCV has required adjustments at multiple stages of the virus life cycle. Barriers at the level of entry (McCaffrey et al., 2002; Park et al., 2009) in murine cells could not be overcome until the identification of the four canonical HCV human entry factors – claudin-1 (CLDN1; Evans et al., 2007), occludin (OCLN; Liu et al., 2009a; Ploss et al., 2009), CD81 (Pileri et al., 1998) and scavenger receptor class B member 1 (SCARB1; Scarselli et al., 2002). Of these, human CD81 and human OCLN were the minimal factors needed for viral entry (Ploss et al., 2009). Alternatively, infecting with HCV adapted to utilize murine CD81 could also successfully overcome this initial obstacle (Bitzegeio et al., 2010). Once inside murine cells, HCV faces another block at the level of replication, which initial studies circumvented through the use of selectable subgenomic HCV replicons (McCaffrey et al., 2002; Park et al., 2009; Uprichard et al., 2006). Murine cells did appear to support assembly and release of infectious particles, albeit at low levels, once NS2 and the structural HCV proteins were provided in trans and the murine or human ortholog of the apolipoprotein ApoE was overexpressed (Long et al., 2011). Further efforts to improve HCV replication in a murine context have relied on disruption of innate immune responses (Aly et al., 2011; Anggakusuma et al., 2015; Chang et al., 2006; Frentzen et al., 2014; Lin et al., 2010; Nandakumar et al., 2013), with robust replication and completion of the HCV life cycle proving difficult following infection unless a selectable genome is used (Vogt et al., 2013).

These poor levels of replication could be due to the lower compatibility of murine orthologs of vital replication host factors with the viral replication machinery or the absence of proteins that normally facilitate these interactions in human cells. Although numerous intracellular host factors have been implicated in HCV replication, extensive experimental evidence has only been provided for three host factors: cyclophilin A (CypA) (Kaul et al., 2009; Yang et al., 2008a), phosphatidylinositol four kinase IIIα (PI4KA) (Berger et al., 2009; Borawski et al., 2009; Reiss et al., 2011; Tai et al., 2009; Trotard et al., 2009) and microRNA-122 (miR-122) (Jopling et al., 2005; Lanford et al., 2010; Machlin et al., 2011). miR-122 is highly conserved between humans and other species, including mice, making its expression alone unlikely to explain the weak replication of HCV in murine cells. In an effort to systemically dissect the impact of such replication co-factors during infection, we focused in this study on CypA and how it may contribute to the restricted host range of HCV.

CypA is a cytosolic 18 kDa peptidyl-prolyl cis-trans isomerase (PPIase) and a part of the biologically ubiquitous cyclophilin enzyme family (Fischer et al., 1989), the members of which were first characterized in mammals by their common ability to bind the immunosuppressive drug cyclosporin A (CsA) and their shared cyclophilin-like domain (CLD) which catalyzes the cis-trans isomerization of proline residues (reviewed in Marks, 1996). CypA overexpression has been implicated in a wide variety of human diseases, ranging from cancer to atherosclerosis (reviewed in Nigro et al., 2013), and it has a demonstrated role in the life cycles of multiple viruses besides HCV (de Wilde et al., 2018; Frausto et al., 2013; Li et al., 2016; Phillips et al., 2015; Tian et al., 2010; von Hahn and Ciesek, 2015; Watashi and Shimotohno, 2007; Zhou et al., 2012). Early work showed that CsA had an inhibitory effect on HCV in chronically infected chimpanzees, but it was not until subsequent in vitro CypA knockdown experiments and dose-response assays with CsA derivatives that CypA was specifically recognized as critical to HCV replication (Chatterji et al., 2009; Ciesek et al., 2009; Coelmont et al., 2009; Kaul et al., 2009; Liu et al., 2009b; Yang et al., 2008b).

These studies showed that CypA’s relevance to HCV replication was intimately linked to its PPIase activity, as the introduction of point mutations in the PPIase active site led to impaired viral replication (Chatterji et al., 2009; Kaul et al., 2009; Liu et al., 2009b). Individuals exhibiting an HCV non-permissive phenotype were shown to express a rare homozygosity at any of three SNP sites in the coding region of CypA – but not in the enzymatic active site – that subsequent in vitro work showed resulted in markedly decreased levels of intracellular CypA (von Hahn et al., 2012). Despite its known importance, the exact mechanism by which CypA facilitates HCV replication remains poorly characterized. Interactions between CypA and several HCV proteins have been demonstrated, including the RNA-dependent RNA polymerase (RdRP) NS5B (Chatterji et al., 2009; Fernandes et al., 2007; Robida et al., 2007; Yang et al., 2008b), NS5A (Anderson et al., 2011; Coelmont et al., 2010; Foster et al., 2011; Grisé et al., 2012; Hanoulle et al., 2009; Nag et al., 2012; Verdegem et al., 2011) and NS2 (Ciesek et al., 2009; Kaul et al., 2009), but how CypA’s binding, PPIase activity and the viral polyprotein are precisely intertwined remains to be understood. The specific impact that cross-species differences in CypA might have on HCV replication and the restricted host tropism of this virus remains an open question and one the present study sought to address. Here, we examined the ability of CypA from diverse species, some of which could serve as feasible small animal models for HCV, to facilitate HCV replication. We found that murine CypA, relative to human CypA, is less proficient at facilitating HCV replication due to differences at the amino acid level and that overexpression of human CypA can increase replication in an engineered murine hepatoma line.

Although advances in available therapies, even cures, for HCV are striking, preventive measures, such as a vaccine, have still not been developed. In the United States alone over the past decade, there has been a more than 133% increase in acute HCV incidence, which is strongly associated with the domestic opioid epidemic (Zibbell et al., 2018; Zibbell et al., 2014; Zibbell et al., 2015). Even with treatment, which has its own financial and logistical limitations, reinfection can still occur and is likely among high-risk individuals such as injection drug users and HIV-positive men who have sex with men (Falade-Nwulia and Sulkowski, 2017; Hill et al., 2017; Salazar-Vizcaya et al., 2018). Thus, HCV research is still greatly needed and immunocompetent models necessary more than ever for vaccine development.

Due to the limited host range of HCV, research has focused on finding an immunocompetent small animal model without the financial and ethical restrictions associated with chimpanzees. At the heart of such research is understanding the basic virology and host restrictions of HCV, chipping away at the known barriers to the viral life cycle in a murine context such as at the level of entry (Ding et al., 2017; Dorner et al., 2011; Ploss et al., 2009) and viral particle assembly and release (Frentzen et al., 2014; Long et al., 2011; Vogt et al., 2013). Viral replication in murine cells has consistently been low, and increasing replication has relied on selectable subgenomic replicons (Long et al., 2011) or disrupting cell-intrinsic antiviral responses (Aly et al., 2011; Anggakusuma et al., 2015; Frentzen et al., 2014; Long et al., 2011; Nandakumar et al., 2013; Vogt et al., 2013). Similarly, to observe even low levels of HCV replication in vivo using transgenic mouse models, blunting of innate immune signaling is also needed (Dorner et al., 2013; Vogt et al., 2013). Although the entire HCV life cycle can be completed with robust replication in xenorecipient mice engrafted with human or stem cell-derived hepatocytes (Bissig et al., 2010; de Jong et al., 2014; Kosaka et al., 2013; Mercer et al., 2001; Meuleman et al., 2005; Tesfaye et al., 2013; Washburn et al., 2011), the immunocompromised nature of these mice precludes studying immune responses.

It may not be possible to completely close this gap between levels of HCV replication in human versus other small animal model hepatocytes, but we sought to determine how species-specific differences in CypA – one of the few host factors experimentally validated as essential for viral replication – might contribute to these observed differences. We argue that such work has an impact on the development of an immunocompetent small animal model for studying HCV infection, which remains a critical need.

In the present study, we assessed the ability of CypA orthologs from a variety of species to support HCV replication in a human context. Besides chimpanzee, bonobo, gorilla, olive baboon and rhesus macaque, which are 100% identical to human CypA at the amino acid level, all the remaining orthologs tested varied from hCypA by no more than six amino acids. Recent work has shown that single nucleotide polymorphisms in the hCypA gene can abrogate HCV infection in primary human hepatocytes (von Hahn et al., 2012). The ability of these single missense mutations in the hCypA coding sequence to dramatically reduce HCV replication lent credence to the possibility that the small number of amino acid differences that differentiate hCypA from the orthologs we tested might have important functional significance in the context of HCV replication. In our system, the only other unique CypA ortholog able to rescue HCV replication during infection with Jc1-Gluc to the same extent as human was orangutan, which differs by only one amino acid.

Tree shrew, pigtailed macaque and squirrel monkey CypA did not significantly increase replication compared to the non-transduced Huh7.5-shRNA CypA cells. Although we were not able to robustly detect squirrel monkey CypA and observed no signal for the pigtailed macaque TRIM5-CypA fusion protein by western blot, these two orthologs could still be expressed as commercial antibodies with confirmed specificity for these two orthologs are not available. This is especially pertinent as the antibodies we used had listed specificity for human and/or mouse. Compared to CypA from these two species, the pigtailed macaque and squirrel monkey orthologs differed by multiple residues that could impact antibody binding. Tree shrew CypA was readily detected with either of the two antibodies tested, so its failure to rescue HCV replication is more conclusive. It is important to underscore that our observations for tree shrew (and for squirrel monkey and pigtailed macaque if they are in fact expressed) do not mean that this species could not support HCV replication if its CypA ortholog was expressed in its native cellular environment. The amino acid differences in CypA might be best suited for interaction with species-specific factors for which the human ortholog is not an apt replacement. Indeed, recent work has demonstrated that HCV can replicate efficiently in pigtailed macaque stem cell-derived hepatocyte-like cells after overcoming the CD81- and OCLN-related barriers to viral entry (Sourisseau et al., 2013), although whether the TRIM5-CypA fusion tested here was expressed in these particular cells is unknown. Likewise, rhesus macaque primary hepatocytes were also shown to support HCV infection in vitro and in vivo, albeit only at low levels (Scull et al., 2015). Of course, it is still possible that a given CypA ortholog, even in its native context, would fail to properly interact directly with the necessary HCV proteins to facilitate replication. For example, the T73I difference between human and squirrel monkey CypA (if indeed expressed) could have a meaningful impact on such interactions as it is adjacent to G72, one of the CsA-binding residues.

In contrast, murine CypA was the only other ortholog that demonstrated some ability to rescue HCV replication. This was especially striking as mouse was the most evolutionarily distant species we examined and the murine CypA ortholog had the most differences in amino acid sequence compared to human. As we tested, this was not due to a dominant negative effect of murine CypA on replication as HCV could still replicate efficiently in the presence of both human and murine CypA. Thus, we created a series of mutants where we interchanged the six differing residues between human and mouse CypA. We were able to confirm expression of these mutants by western blot with at least one of the antibodies we tested. We cannot adequately control for the varying antibody reactivity, so comparing the expression of different CypA constructs with each other is difficult as lower detected expression could indicate lower antibody reactivity. This is highlighted by the differences between the two antibodies we tested, with one indicating a more than 100-fold higher expression for a CypA variant compared to the non-transduced Huh7.5-shRNA CypA cells while the other antibody reached only 20-fold. Since we could detect all of the CypA mutants by western blot, expressed them all in the same backbone construct, and observed high transduction efficiency, conceivably all the transduced cells would be able to express at least some level of the CypA variant.

Of the six amino acids differing between the two orthologs, single ‘humanization’ of mCypA residues 12, 14, 52, and 76 had the greatest impact, resulting in increased rescue. Conversely, ‘murinization’ of hCypA residues led to significant decreases in rescue ability across all residues tested. Strikingly, simultaneously altering the three residues clustered together that differed between human and mouse (positions 11, 12 and 14) resulted in a gain-of-function phenotype for murine CypA and a loss-of-function phenotype for human CypA. The gain-of-function phenotype was not further exacerbated in the triply humanized murine CypA by introducing the S52C mutation as well, which as a single mutant had shown rescue efficiency similar to the individual mutants at positions 12 and 14. None of the differences between human and mouse CypA, or indeed for any of the CypA orthologs, overlapped with residues of the active site/CsA-binding site of CypA, which as further described below has been characterized as important for interaction with HCV proteins. This raises the intriguing possibility that this cluster of three residues plays a role in maintaining these interactions with the HCV replication machinery and/or associating with host factors that help facilitate such interactions. The location of the three residues on the face of the protein opposite the CsA-binding/active site makes it unclear whether changing these residues leads to a loss of PPIase activity that would explain the accompanying change in phenotype. Identifying these potential conformational changes and/or host factors would provide novel insights into the still unclear mechanism of action by which CypA promotes HCV replication.

Furthermore, we tested in our system the impact of mouse, human or triply humanized mouse CypA on infectious particle production and viral spread over time. Even though spread over time was not detectable by NS5A staining in the murine CypA rescue cells, the more sensitive luminometry assay still demonstrated infectious particles were being released into the supernatant. Thus, in our system, the HCV life cycle is still being completed and the virus able to propagate beyond the cells initially infected with the inoculum.

Although informative, these findings were all in the context of a human cell and thus whether or not this ‘humanization’ of mCypA would have the same effect in murine cells was unclear. In the same vein, it was not known if exogenous expression of human CypA in a murine context would boost HCV replication. As briefly alluded to above, there have been numerous efforts to engineer murine cells for studying HCV. Previous efforts using subgenomic replicons indicated replication could occur in murine embryonic fibroblasts (MEFs) at least to some extent and could be enhanced with the addition of miR-122 (Jopling et al., 2005; Lin et al., 2010) and deletion of IRF3 (Lin et al., 2010). Hep56.1D cells also support replication of subgenomic replicons and infectious particle release was achieved by using a selectable replicon with HCV core, E1, E2, p7 and NS2 provided in trans (Long et al., 2011). The low levels of infectious particle release initially observed could be further increased by expressing either murine or human ApoE (endogenous expression in Hep56.1D cells is low). However, in this same study, replication of transfected full-length genomes (selectable or non-selectable) was poor. Replication of subgenomic replicons was also enhanced in murine liver tumor (MLT) cells with the addition of miR-122 and disruption of IFN receptors (Anggakusuma et al., 2015) or in MEFs with blunted innate immune responses (Nandakumar et al., 2013). Similarly, replication of full-length HCV RNA following transfection in primary murine hepatocytes remained low unless MAVS, IRF1 or IFNAR — all important players in cell-intrinsic antiviral responses — were knocked out (Aly et al., 2011; Nandakumar et al., 2013). Transfecting full-length genomes into MAVS^-/- MLT cells expressing miR-122 and the human HCV entry factors resulted in robust replication and infectious particle production; however, in performing infections, replication declined and infectious particle release fell below background levels (Frentzen et al., 2014). The only instance of viral infection in murine cells where strong replication and particle production were demonstrated relied on the use of a blasticidin-selectable genome (Jc1-bsd) in immortalized Stat1 knockout MEFs (iMEFs) expressing the human HCV entry factors, miR-122 and mApoE (Vogt et al., 2013).

Unlike these previous efforts, we wanted to focus on the replication of a non-selectable, full-length genome during infection without exogenous disruption of cellular immune responses as this is most immediately relevant and critical for generating a successful HCV animal model. Towards this aim, we engineered murine Hep56.1D hepatoma cells to express a variety of factors to overcome the various blocks at the level of entry and replication. These so-called Clone 8 cells demonstrated sustained de novo replication compared to the parental Hep56.1D cells, regardless of whether mApoE was present. Addition of murine CypA resulted in a further increase in replication that was even greater for human CypA. The phenotypes of the triple mutants in this murine context were the reverse of those observed in Huh7.5-shRNA CypA cells, suggesting that the mechanism by which these mutants function could depend to some extent on the species specificity of the cellular environment, which will be an interesting area for future study.

In spite of the elevated replication and presence of mApoE in our Clone 8 cells, infectious particle production was not even a log greater than the parental Hep56.1D cells. This is similar to previous observations that when infecting humanized, MAVS^-/- MLTs with full-length genomes, infectious particle release is negligible and viral replication lower compared to transfecting in the genome (Frentzen et al., 2014). This raises the question of whether viral replication must be enhanced further in order to achieve greater viral release and/or packaging or whether factors directly important to these final steps of the viral life cycle are still missing. The evidence that infection with selectable HCVcc, so that the population is made up of cells containing HCV genome (Vogt et al., 2013), results in efficient particle release as does transfecting in RNA to ‘hit’ more cells (Frentzen et al., 2014) suggests the former. However, in both of these cases, it is unclear how much blunting the innate immune response additionally contributed to these observations. Furthermore, if there is (a) murine factor(s) that is having an inhibitory effect on particle release, as observed for murine tetherin’s block of HIV-1 particle release due to its resistance to Vpu-mediated degradation (McNatt et al., 2009), more viral replication may be required to overcome this. The luciferase assay we used is extremely sensitive, capturing even low levels of replication that may simply be insufficient for robust infectious particle production. To enhance replication, other (co-)factors/adaptors may be necessary to facilitate the interactions of CypA and are simply missing in murine cells or are incompatible orthologs of such factors. As observed for PI4KA, it is also possible that the effects of CypA we observe in the hepatoma lines tested may differ in a primary hepatocyte context and/or with the use of patient HCV isolates (Harak et al., 2017).

Understanding the mechanistic basis for the differences we observed in the respective rescue abilities of the CypA orthologs and mutants we tested remains an important area for future research. Indeed, these mutants may prove useful for delineating both the direct and indirect interactions necessary for robust HCV replication. Extensive study of human CypA has determined interactions with multiple HCV proteins. A connection between CypA and the HCV RdRp is evidenced by mutations in NS5B linked to CsA resistance (Fernandes et al., 2007; Robida et al., 2007; Yang et al., 2008a) and indications that NS5B can bind at the CypA active site (Chatterji et al., 2009; Yang et al., 2008a), which might enhance the viral protein's RNA-binding capcacity as a result (Nag et al., 2012).

The interaction of CypA with the HCV phosphoprotein NS5A is far better characterized. Mutations specifically in domain 2 of NS5A were linked to CsA treatment resistance among patients (Goto et al., 2009). CypA stably binds NS5A and this interaction is dependent on the protein’s PPIase activity (Chatterji et al., 2010b), which promotes the isomerization of multiple conserved proline residues in domains II and III of NS5A, contributing to the protein’s proper folding (Coelmont et al., 2009; Foster et al., 2011; Grisé et al., 2012; Hanoulle et al., 2009; Verdegem et al., 2011). As with NS5B, CypA is also believed to promote the RNA-binding capacity of NS5A (Nag et al., 2012). Upon inhibition of CypA activity, lipid and protein trafficking patterns associated with the formation of lipid droplets and the VLDL synthesis pathway are altered (Anderson et al., 2011). This has led to the hypothesis that CypA might facilitate the trafficking and assembly function of NS5A, which is known to localize to lipid droplets during the assembly of progeny virions (Nag et al., 2012). Furthermore, whether CypA expression or just its PPIase activity is inhibited, formation of double membrane vesicles (DMVs) – a prevalent component of the membranous web derived from the host endoplasmic reticulum (ER) which serves as scaffolding for viral genome replication (Paul et al., 2013; Romero-Brey et al., 2012) – is abrogated (Chatterji et al., 2015). A portion of host CypA is already localized to ER compartments, which could be advantageous for later HCV replication complex formation in these same regions (Chatterji et al., 2010a). Within these DMVs, HCV replication complexes can then more safely assemble outside of the purview of host nucleic acid sensors and other defenses. Indeed, treatment of infected cells with cyclophilin inhibitors leads to changes in the ER that temporarily prevent re-infection (Chatterji et al., 2016). Finally, an interaction between CypA and the viral assembly factor and protease NS2 has also been suggested, perhaps impacting the cleavage of NS2 from NS3 by the NS2-NS3 protease (Ciesek et al., 2009) and/or the overall proteolytic processing of the HCV polyprotein (Kaul et al., 2009). It will be important to determine if the mutants and orthologs we examined here maintain or disrupt these known interactions or instead participate in novel, undocumented interactions.

In this study, we focused on the contribution of CypA to the host restriction of HCV, but undertaking these same experiments with the other well-described HCV replication factor PI4KA is also of great interest. The percent amino acid similarity between human PI4KA and its orthologs in the species we examined in this study is lower compared to CypA, ranging from 83.8% (chimpanzee) to 99.5% (rhesus macaque) for PI4KA (Supplementary file 2) versus 97–100% for CypA. However, assessing the PI4KA orthologs for all of these species is challenging, as a strong candidate for a PI4KA ortholog is either absent for a given species (i.e. tree shrew) or is a possible ortholog by our own sequence homology search but not formally annotated (i.e. gorilla and bonobo). Some annotations for a predicted PI4KA ortholog are also more than 100 amino acids shorter than that of human, which is 2102 amino acids. Thus, determining the ‘true’ PI4KA ortholog for a given species requires additional corroboration.

The engineered murine hepatoma line we have generated will serve as a ready platform for testing the potential of additional human factors or humanized murine factors to increase the replication capacity of HCV in these cells. If augmentation of viral replication is seen in vitro, genetic engineering approaches could be utilized to generate mice expressing these human orthologs or ‘humanized’ alleles . Such genetic humanization approaches would finally create the possibility to study HCV infection and pathogenesis in an immunocompetent small animal model, informing future vaccine development and testing.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Jenna M Gaska

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1706-9701

2. Metodi Balev

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

3. Qiang Ding

   Department of Molecular Biology, Princeton University, Princeton, United States

   Present address

   School of Medicine, Tsinghua University, Beijing, China

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Brigitte Heller

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Alexander Ploss

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   aploss@princeton.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9322-7252

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