Keratins (KRTs) are predominantly known for their characteristic of forming intermediate filaments within epithelial cells, providing tissues with mechanical support. Indeed, several KRT-linked diseases lead to tissue fragility [1
]. However, KRTs are not exclusively associated with epithelia- and tissue-related diseases. Genetic mouse models highlighted the function of KRTs in protecting hepatocytes from apoptosis and necrosis, and mutations within the KRT8 and KRT18 genes are linked to the progression of liver disease of multiple etiologies [3
]. The expression of a variety of KRTs is triggered by inflammatory cytokines, indicating an important role for these proteins during the cellular stress response [4
]. In particular, KRT18 release into the extracellular space is mediated by hepatocellular damage and is therefore a commonly-used, non-invasive marker of liver diseases [5
]. Additionally, KRT23, strongly upregulated in several human cancers, was suggested to be a ductular reaction marker, since its levels correlate with liver disease severity [6
]. Furthermore, viral infections have been associated with the modulation of KRTs. In particular, the progression of chronic hepatitis B virus infection is considered to associate with the phosphorylation of KRT18 [9
], whereas the cleavage of KRT18 was shown to correlate with the stress response in livers of chronic hepatitis C patients [10
Hepatitis C virus (HCV) is an enveloped positive strand RNA virus belonging to the family of Flaviviridae
. HCV strains are classified into seven genotypes (GT1–7) differing up to 30% at the nucleotide level [11
]. Its 9.6-kb genome consists of one open reading frame, flanked by a 5′ and a 3′ untranslated region. An internal ribosomal entry site enables the expression of the polyprotein, which is subsequently processed into seven non-structural and three structural proteins [12
]. Infection with HCV causes acute hepatitis and progresses to a chronic infection in most cases. Chronic hepatitis C (CHC) patients represent a patient population at high risk for development of serious liver diseases, including steatosis, cirrhosis, and hepatocellular carcinoma [13
]. Although direct-acting antivirals were introduced in 2014 and have replaced PEGylated interferon-α (pegIFN-α) and ribavirin (RBV) as the first choice of anti-HCV treatment, this hepatotropic pathogen is still a global health burden with 71 million infected people, especially since the majority of HCV-infected patients are unaware of their infection status [13
In this study, we analyzed the expression of different KRTs, including KRT23, in vivo and ex vivo upon HCV infection and pegIFN-α therapy. In addition, we analyzed the KRT23 levels in direct acting antiviral (DAA)-treated HCV-infected patients before and after viral clearance. Furthermore, we generated KRT23 expressing and KRT23 knockout cells to determine the influence of KRT23 on the HCV life cycle. Taken together, our data indicate that KRT23 is an HCV host factor, whose expression and secretion correlates with the abundance and clearance of the viral infection.
2. Materials and Methods
2.1. Cell Culture
The Huh-7.5 cell line and HEK 293T cells were cultured in Dulbecco’s Modified Eagle Medium (1 g/L glucose, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (FCS, capricorn scientific, Ebsdorfergrund, Germany), 2 mM l
-glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM of each non-essential amino acid (Invitrogen), 10 U/mL penicillin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 10 μg/mL streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C. In addition, 5 μg/mL blasticidin S (InvivoGen, San Diego, CA, USA) or 2.5 µg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) were added to cells carrying an integrated lentiviral vector for ectopic expression or CRISPR KO, encoding a blasticidin resistance gene or a puromycin resistance gene, respectively. Primary human hepatocytes (PHHs) were isolated from liver specimens, plated at a density of 1.3 × 106
on collagen-coated 6-well dishes, and kept in hepatocyte culture medium (Lonza, Basel, Switzerland) as described [14
2.2. Compounds and Reagents
2-C-methyladenosine (2-CMA) was kindly provided by Timothy Tellinghuisen (The Scripps, Jupiter, FL, USA). Human IFN-α was purchased from SP Europe/Essex Pharma (IntronA) or from Sigma-Aldrich, St. Louis, MO, USA). His-tagged IFN-λ3 and -λ4 were kindly provided by Rune Hartmann (Aarhus, Denmark) and were used as described previously [15
pFK-Jc1, pFK_i389-LucEI/NS3-3′-JFH1_dg, pFK-i341PI-Luc/NS3-3/Con1/ET (replicon with E1202T, I1280T, and K1846T mutations), and bicistronic Renilla
luciferase reporter (RLuc) chimeric HCVcc genomes designated J6/2a/R2a have been described previously [16
]. For generating CRISPR/Cas9 knock out cell lines, the lentiviral plasmid pLenti CRISPR v2 ccdB was used as described previously [21
]. pWPI-empty-BLR and pWPI-3xFLAG-KRT23-BLR (NM_015515.4) were generated by molecular cloning using synthesized gene fragments (gBlocks, IDT).
2.4. Production of Viruses and Pseudoparticles
For production of cell-culture-derived HCV (HCVcc), in vitro transcribed RNA of HCV full-length Jc1 WT and JCR2a were transfected in Huh-7.5 cells. Supernatants, containing HCVcc, were harvested at 48 and 72 h post-electroporation and filtered through a 0.45-μm pore size membrane. Afterwards, HCVcc were concentrated using 100-kDa cutoff Amicon Ultra centrifugal filters (Merck, Darmstadt, Germany).
For production of lentiviral pseudo-particles, HEK 293T cells were transfected with pcz-VSV-G, pCMV-dR8.74, and the respective lentiviral plasmid, by using the PEI method (Carl Roth, Karlsruhe, Germany) or Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Lentiviral pseudoparticles were harvested 48 and 72 h post-transfection and used for transduction of target cells.
2.5. Western Blotting
For Western blot analysis, cells were lysed in RIPA buffer and heated at 95 °C for 5 min with SDS sample buffer. Afterwards, proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes by semi-dry electroblotting. Five percent milk in PBS containing 0.05% Tween (PBS-T) was used to block the membranes. Subsequently, membranes were probed with primary antibodies α-FLAG (1:1000, Sigma-Aldrich, Catalogue Number F3165), α-KRT23 (1:2000, Thermo Fisher, Catalogue Number PA5-50198), α-KRT23 (1:1000, Abcam, Catalogue Number ab156569) α-HCV-NS3 #337 mAb (1:1000), α-GAPDH (1:1000, Sigma-Aldrich, Catalogue Number G9545), and α-β-actin (1:20000, Sigma-Aldrich, Catalogue Number A3854) over night at 4 °C, followed by incubation with secondary horseradish peroxidase conjugated antibodies (Sigma-Aldrich) for 1 h at room temperature. For analysis, membranes were incubated with the ECL Plus detection system (GE Healthcare), SuperSignal Femto Substrate (Thermo Fisher), and Pierce™ ECL Plus Western Blotting Substrate (Thermo Fisher).
2.6. Dot Blot
For dot blot analysis, 3 µL of patient plasma were spotted on polyvinylidene difluoride membranes and air-dried for 1 h at room temperature. Membranes were blocked with 5% milk in PBS containing 0.05% Tween (PBS-T) for 1 h at room temperature and subsequently probed with primary antibodies (α-KRT23, 1:1000, kindly provided by Pavel Strnad [8
]) over night at 4 °C, followed by incubation with secondary horseradish peroxidase conjugated antibodies (Sigma-Aldrich) for 1 h at room temperature. For analysis, membranes were incubated with the ECL Plus detection system (GE Healthcare) and SuperSignal Femto Substrate (Thermo Fisher). Fiji was used to calculate signal intensities of KRT23 on the different dot blots. Therefore, regions of interest with the same size were selected in all samples, and mean grey values were quantified.
2.7. Immunofluorescence Analysis
For immunofluorescence analysis, cells were cultured on cover slips in 24-well plates. After fixation with 3% paraformaldehyde for 10 min, cells were permeabilized by incubation with 0.5% Triton X-100 for 5 min. Subsequently, samples were blocked with 5% FCS in PBS for 1 h at room temperature. For detection of KRT23, samples were incubated with primary antibodies α-FLAG M2 mAb (1:1000, Sigma-Aldrich) over night at 4 °C. Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1:1000, Sigma-Aldrich) by incubation for 1 h at room temperature. Nuclear DNA was stained using DAPI (dilution of 1:10,000).
2.8. Real-Time Quantitative PCR
To extract total RNA from cell cultures, the Nucleospin RNA II kit (Machery Nagel) was used, according to the manufacturer’s instructions. For synthesis of cDNA, the PrimeScript First Strand cDNA synthesis kit (TaKaRa) was used, following the manufacturer’s instructions. Quantitative PCR was performed with SYBR Premix Ex Taq (Takara), the LightCycler480 system (Roche), and the respective primers. The primer sequences for amplification of the HCV genome and of each gene product (KRT23 and GAPDH) have been described previously [22
2.9. In Vitro Transcription and Electroporation of Huh-7.5 Cells
transcripts were prepared and used for electroporation-based transfection as described recently [25
]. Subsequently, cells were seeded in 96-well plates and subjected to further analysis.
2.10. HCV Infection Assays
The day before inoculation, Huh-7.5 cells and derivatives were seeded at a density of 1 × 104/well in 96-well plates. Cells were inoculated with the respective virus particles at 37 °C for 4 h. Afterwards, the viral inoculum was replaced by fresh culture medium.
2.11. Luciferase Assays
To quantify the HCV Renilla-reporter virus, cells were washed once with PBS and lysed in H2O. After storage at −80 °C for at least 15 min, cell lysates were subjected to luciferase activity measurement. Therefore, samples were incubated with luciferase substrate (1 μmol/L of coelenterazine in PBS, PJK GmbH, Kleinblittersdorf, Germany), and Renilla luciferase activity was measured in a luminometer (Lumat LB9507, Berthold). For quantification of the replication activity of firefly reporter subgenomic constructs, cells were washed once with PBS and lysed with lysis buffer (containing 0.1% Triton-X100, 25 mmol/L glycylglycine, 15 mmol/L MgSO4, 4 mmol/L EGTA tetrasodium, and 1 mmol/L dithiothreitol, pH 7.8). After storage at −20 °C for at least 30 min, cell lysates were incubated with luciferase substrate (200 µmol/L luciferin, 25 mmol/L glycylglycine, pH 8), and luciferase activity was measured with a luminometer (Lumat LB9507, Berthold).
2.12. Generation of CRISPR/Cas9 Knockout Cells
CRISPR/Cas9 knockout cells were generated as described previously [26
]. The web tool CHOPCHOP was used to identify two single-guide RNA (sgRNA) sequences [27
]. The two sgRNAs, as well as a non-targeting control were cloned into pLenti CRISPR v2 ccdB. For the production of lentiviral pseudo-particles, HEK 293T cells were transfected with pcz-VSV-G, pCMV-dR8.74, and the respective lentiviral plasmid, by using the Lipofectamine 2000 (Thermo Fisher). Lentiviral pseudoparticles were harvested 24 and 48 h post-transfection and used for transduction of target cells. Target cells were inoculated with lentiviruses for 4 h and selected with Puromycin 48 h post-infection. For validation, the sgRNA target sequence in the genomic DNA was analyzed by Sanger sequencing. sgRNAs and corresponding primers for amplification of the genomic DNA are listed in Appendix Table A1
2.13. Statistical Methods
For data analysis, GraphPad Prism 8 software was used. Two-tailed Student’s t-test and one-way analysis of variance (ANOVA) adjusted with Dunnett’s multiple comparison test were performed to evaluate statistical significance. Values <0.05 (*), <0.01 (**), and <0.001 (***) were considered statistically significant.
In the last few years, the classical role of KRTs in forming intermediate filaments was expanded by studies demonstrating that KRTs influence a variety of cellular processes including cell signaling, apoptosis, and stress responses [1
]. Recently, the dramatic changes in KRT23 expression levels in the context of liver disease were reported [8
]. In this study, we analyzed its role in the life cycle of the hepatotropic pathogen HCV and consequently discovered that KRT23 is an HCV-induced pro-viral factor.
In our initial experiments, in which we analyzed the expression of KRTs in PHHs, we observed the highest expression levels for the hepato-characteristic KRT8 and KRT18 and robust levels for hepatocyte progenitor characteristic KRT7 and KRT19 (Figure 1
A). Since the expression of distinct KRTs is restricted to specific tissues and the substitution of primary KRTs by others occurs in a tissue-specific manner, we observed an extreme variable expression of KRT23 in hepatocytes (Figure 1
A). Further analysis of KRT expression in a CHC patient cohort revealed an upregulation of KRTs, including KRT23, in CHC patients compared with uninfected patients (Figure 1
B). This upregulation might indicate a stress-induced upregulation of KRTs and/or a further substitution of the primary KRTs by other ones in a disease-dependent manner. However, transcriptomics data from liver biopsies do not take into account that the majority of the cells in the sample are HCV negative, and rather indicate a global alteration caused by HCV infection. Furthermore, Guldiken and colleagues observed that KRT23 upregulation is independent of disease etiology, but dependent on disease progression [8
]. However, subsequent analysis of PHHs and hepatoma cells supported the hypothesis that KRT23 is upregulated in an HCV-dependent manner on a single cell level (Figure 2
C,E). Notably, elevated KRT23 mRNA were also detected in 2´CMA-treated and HCV-infected cells (Figure 2
C), pointing to a potential replication-independent induction of KRT23. By analyzing the KRT23 protein levels in hepatoma cells, we detected the disappearance of the designated 33-kDa KRT23 isoform, but increased signal intensities of the 48 kDa KRT23 isoform in the HCV challenged cells compared with the mock challenged cells. These results suggest an HCV-mediated shift of KRT23 isoform abundance and a preferential expression of the 48-kDa KRT23 isoform. KRT23 is reported to be induced in a peroxisome proliferator-activated receptor alpha (PPARα)-dependent and MYC amplified fashion, highlighted by several PPARα and MYC binding sites, as well a PPARα-deficient mouse model [35
]. Interestingly, several studies reported an HCV-core induced activation of and interference with PPARα [36
], as well as MYC [39
], which subsequently might explain the induction of KRT23 upon HCV challenge.
Following experiments and analysis of publically-available transcriptomics data revealed that expression levels of most KRTs are not modulated by pegIFN-α therapy (Figure 3
A). In contrast, we detected decreased KRT23 expression 48 h post-IFN injection (Figure 3
A), which is associated with the early phase of antiviral efficiency of IFN treatment [40
], indicating a correlation between KRT23 mRNA level decrease and the drop of the viral load. Importantly, IFN-α treatment of PHHs in the absence of HCV did not alter the KRT23 mRNA levels (Figure 3
B), indicating that IFN-α has no pronounced direct effect on KRT23 expression. By analyzing the KRT23 levels of HCV-positive patients, we observed a decrease of KRT23 sera levels caused by DAA treatment concomitant with viral clearance (Figure 3
C). Taking the findings of Guldiken and colleagues that KRT23 is a stress-induced marker into account, the decline of KRT23 levels highlights the regenerative potential of the liver and the potential of DAA to revert the progression of liver diseases.
Experimental settings with both viral particles and subgenomic replicons of HCV indicated that forced expression of KRT23 positively affects the replication of HCV (Figure 4
C,D). The non-affected replication capacity in KRT23 KO cells may be based on phenotypic compensation by counter-regulation of other KRTs or could also indicate that KRT23 is not essential for the HCV life cycle progression. Regarding a potential interplay between HCV and KRT23, Liffers and colleagues observed direct interaction of KRT23 with KRT8, KRT18, plectin1, 14-3-3ε, heat shock protein (HSP) 60, and HSP70 [41
]. Intriguingly, the three latter proteins are reported to directly interfere with HCV proteins [42
]. For example, Gonzales and colleagues reported that HSP70 interacts with NS5A and treatment with Quercetin, an HSP expression inhibitor, led to decreased virus production [45
], pointing to a role of HSPs in the HCV life cycle. 14-3-3ε, which is known to interfere with several cell signaling components, does not only interact with KRT23, but was also shown to interact with the HCV entry receptor CD81 and HCV core [46
]. Taken together, the interference of KRT23 interactors with the HCV life cycle supports our hypothesis of a potential pro-viral role for KRT23.
In summary, our data indicate that KRT23 is an HCV-induced pro-viral host factor. Further studies will help to investigate the HCV-mediated regulation of KRT23, giving insight into the molecular mechanisms of how KRT23 supports the HCV life cycle. Of note, the decrease in KRT23 plasma levels concomitant with HCV RNA clearance highlights the regenerative potential of the liver after HCV infection. Thus, the idea to use KRT23 as a serum marker to monitor hepatic disease progression may be extended by the usage of KRT23 to monitor liver regeneration.