Identification of host genes involved in hepatitis C virus replication by small interfering RNA technology

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Hepatitis C virus (HCV) replication is highly dependent on host cell factors. Identification of these host factors not only facilitates understanding of the biology of HCV infection but also enables the discovery of novel targets for anti-HCV therapy. To identify host genes important for HCV RNA replication, we screened a library of small interfering RNA (siRNA) that targets approximately 4,000 human genes in Huh7-derived EN5-3 cells harboring an HCV subgenomic replicon with the nonstructural region NS3-NS5B from the 1b-N strain. Nine cellular genes that potentially regulate HCV replication were identified in this screen. Silencing of these genes resulted in inhibition of HCV replication by more than 60% and exhibited minimal toxicity. Knockdown of host gene expression by these siRNAs was confirmed at the RNA level and, in some instances, at the protein level. The level of siRNA silencing of these host genes correlated well with inhibition of HCV. These genes included those that encoded a G-protein coupled receptor (TBXA2R), a membrane protein (LTβ), an adapter protein (TRAF2), 2 transcription factors (RelA and NFκB2), 2 protein kinases (MKK7 and SNARK), and 2 closely related transporter proteins (SLC12A4 and SLC12A5). Of interest, some of these genes are members of the tumor necrosis factor/lymphotoxin signaling pathway. Conclusion: Findings of this study may provide important information for understanding HCV replication. In addition, these cellular genes may constitute a novel set of targets for HCV antiviral therapy. (HEPATOLOGY 2007.)

Hepatitis C virus (HCV), a member of the Flaviridae family, infects an estimated 170 million patients worldwide.1 It is a leading cause of cirrhosis and hepatocellular carcinoma.2 HCV is an enveloped virus containing a positive-strand RNA genome that encodes structural and nonstructural proteins. Its replication, which occurs in the cytoplasm, is highly dependent on the function of its nonstructural proteins together with host cellular factors. However, the precise roles that cellular factors play in HCV replication have not been well characterized. In addition, structural and nonstructural HCV proteins have been implicated as being involved in various functions within host cells including cell signaling, transcriptional modulation, transformation, and apoptosis.3–8

Current standard therapy for chronic HCV infection involves extended dosing with pegylated interferon (PEG-IFN) and ribavirin (RBV). This regimen provides a sustained virological response in about 50% of patients infected by genotype 1 HCV, which is the prevalent HCV genotype in the United States, Europe, and Japan.9, 10 Unlike how patients infected with genotype 1 HCV respond to the treatment, patients infected with HCV genotypes 2 or 3 have an 80% sustained virological response. PEG-IFN plus RBV therapy is commonly associated with severe side effects. The severe consequences of chronic HCV infection as well as the limitations of the current therapy have created a great demand for new treatments to be developed.

Currently, the major targets for HCV drug discovery are 2 viral enzymes, NS3 protease and NS5B RNA-dependent RNA polymerase. Several protease and polymerase inhibitors have demonstrated inhibitory effects on the HCV replicon system and antiviral activity in humans as well.11–16 Because of the high rate of HCV replication and the error-prone nature of HCV polymerase, it is very likely that drug-resistant HCV variants will emerge in patients treated with HCV polymerase or protease inhibitors. In fact, several studies have reported the selection of HCV mutant replicons resistant to polymerase or protease inhibitors.15, 17–23 Characterization of these mutant replicons showed that single amino acid mutations in the NS3 or NS5B gene could confer significant resistance to these inhibitors. Most inhibitors specific for HCV targets described to date are significantly less effective for treating patients infected with HCV genotypes other than genotype 1. This is because of the high sequence variation among HCV genotypes. In contrast, inhibitors targeting cellular genes may overcome this genotype-specific limitation. Therefore, targeting host proteins as antiviral targets has several advantages. First, an inhibitor that targets a host protein may be active across a broader spectrum of HCV genotypes so as to overcome their great genetic heterogeneity. Second, during the course of treatment, it can potentially minimize the selection of drug-resistant mutants because of the rate of mutation of host genes is lower than that of HCV genes.

In this study, we screened a small interfering RNA (siRNA) library targeting about 4,000 human genes in Huh7 cells containing an actively replicating HCV subgenomic replicon in order to identify host genes important for HCV replication. This library is composed of siRNAs targeting a broad variety of gene families that are potential targets for drug discovery. We have identified several host genes that regulate HCV RNA replication. These genes may provide important information for HCV replication and may serve as potential targets for HCV drug discovery.

Abbreviations

LT, lymphotoxin; NIK, NFκB-inducing kinase; NTR, nontranslated region; SEAP, secreted alkaline phosphatase; ROS, reactive oxygen species; siRNA, small interfering RNA; PKR, interferon-inducible protein kinase; TBXA2R, thromboxane A2 receptor; TNF, tumor necrosis factor.

Materials and Methods

HCV Replicons.

HCV genotype 1b N strain subgenomic replicon cells expressing HCV NS3-NS5B and the secreted alkaline phosphatase (SEAP) reporter protein in EN5-3 cells were licensed from the University of Texas Medical Branch (Fig. 1).24 EN5-3 cells were derived from Huh7 cells by stable transfection with a SEAP reporter plasmid and selection with blasticidin.24 This vector expresses SEAP under the control of a transcriptional regulator, human immunodeficiency virus (HIV) LTR. The HCV genotype 1a subgenomic replicon was constructed in-house (Fig. 1). In this replicon, the N-terminal 73 amino acids of NS3 were derived from the genotype 1b strain con1 sequence, and the remaining NS3-NS5B sequence, as well as the 5′- and 3′ nontranslated regions (NTRs), were derived from genotype 1a strain H77. Amino acid changes at E1202G, S1222T, P1496L, and S2204I (numbered relative to the amino acid position in the viral open-reading frame) were introduced to enhance replication.25, 26 The hepatitis delta virus ribozyme was inserted after the 3′ NTR to generate the exact 3′ end of the HCV sequence after ribozyme cleavage.27 To obtain 1a replicon cells expressing SEAP reporter, EN5-3 cells were transfected with the 1a replicon construct and selected with media containing 200 μg/mL G418 (Invitrogen) and 2 μg/mL blasticidin (Invitrogen).

Figure 1.

Organization of subgenomic HCV replicons. Open-reading frames are shown as boxes and nontranslated regions as bars. Replicons contain HCV nonstructural proteins from either genotype 1b (strain N) or genotype 1a (strain H77), as well as HIV tat-FMDV 2a-neomycin cassette (tat-2A-neo), which drives SEAP expression in replicon cells. Positions of amino acid changes introduced to enhance HCV replication are marked (FMDV, foot-and-mouth disease virus; HDV, hepatitis delta virus; EMCV IRES, encephalomyocarditis virus internal ribosome entry site).

siRNA.

A library of siRNAs that target approximately 4,000 human genes was acquired from the SMARTpool siRNA library designed by Dharmacon. This library consists of siRNAs targeting genes from different families of druggable targets such as protein kinases, G-protein-coupled receptors, and ion channels. Each SMARTpool siRNA consists of 4 siRNA duplexes designed to target different regions of the same target mRNA sequence. It is guaranteed by Dharmacon to silence at least 75% of the expression of a target gene at the mRNA level when a SMARTpool siRNA is transfected at a concentration of 100 nM. HCV NS5B siRNA (5′-aaggucaccuuugacagacug)28 and 5′ NTR siRNA (5′-aaguacugccugauagggugc)29 were used as positive controls. Universal negative control siRNA and individual siRNAs that constituted the SMARTpool siRNAs were purchased from Dharmacon.

Inhibition of HCV Replication.

Two methods were used to measure the inhibition of HCV replication after transfection of replicon cells with siRNA. First was the SEAP reporter assay, in which HCV subgenomic replicon cells (5,000 cells per well) were seeded onto 96-well plates in DMEM containing 10% fetal bovine serum (FBS). The next day, siRNA was transfected into replicon cells at 100 nM by oligofectamine (Invitrogen) in a volume of 100 μL of Opti-MEM (Invitrogen) according to the manufacturer's protocol. After 4 hours of incubation at 37°C, 100 μL of DMEM containing 20% FBS was added without removing the transfection mixture. Four days after transfection, the SEAP activity in the supernatant of the transfected replicon cells was determined using Phospha-Light SEAP reporter gene assay reagents (Applied Biosystems). Toxicity in the remaining cells was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) colorimetric assay.30 The second method was the RNA quantification assay, in which total RNA was extracted from replicon cells 4 days after siRNA transfection using an RNeasy 96 kit (Qiagen). After extraction, RNA was eluted in 300 μL of nuclease-free water, and 5 μL was analyzed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR; Applied Biosystems) using primers specific to HCV 5′ NTR,15 β-actin (TaqMan β-actin control reagents, Applied Biosystems) or host genes (Assays-on-Demand Gene Expression products, Applied Biosystems) in order to quantitate the amount of viral or host-specific RNA in the siRNA-transfected cells.

Western Blot.

Cells were lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 100 μg/mL PMSF, 1% NP-40, and 0.5% sodium deoxycholate) in the presence of a protease inhibitor cocktail (Roche). Cell lysates were sonicated for 30 seconds on ice and then centrifuged at 11,000g at 4°C for 10 minutes. Supernatants were collected, and equal amounts of protein were subjected to electrophoresis in 4%–20% gradient gels (Bio-Rad). After electrophoresis, protein was transferred onto polyvinylidene difluoride membranes (Bio-Rad), blocked, and incubated with the indicated antibodies at 4°C overnight. Antibodies for thromboxane A2 receptor (TBXA2R; Cayman), RelA (Invitrogen), or β-actin (Sigma) were used at the dilution recommended by the manufacturers. After incubation with the appropriate secondary antibody, the Western blot signal was detected using the 4CN Peroxidase Substrate System (KPL) or Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).

Results

Development of a High-Throughput Assay to Screen siRNA in HCV Replicon Cells.

To identify host genes important for HCV replication, a high-throughput assay was set up to screen a library of siRNAs in cells containing an HCV (genotype 1b) subgenomic replicon that expresses the SEAP reporter (Fig. 1).24 Activity of the SEAP reporter of this replicon has been shown to be proportional to the replication of HCV RNA31 and was therefore used to measure the replication of the HCV replicon in this assay. Three transfection reagents (TransIT-TKO, lipofectamine 2000, and oligofectamine) were compared, and oligofectamine was found to be the most effective and least toxic in introducing siRNA into HCV replicon cells (data not shown). The assay was established using 2 siRNAs that had been shown to be very effective in inhibiting HCV replication. One siRNA targets HCV NS5B polymerase, whereas the other targets the 5′ NTR region.28, 29 To adapt the siRNA transfection assay to 96-well format for high-throughput screening, the number of replicon cells transfected and the culture time after transfection were optimized. The best detection window was achieved when 5,000 replicon cells were plated per well for transfection and harvested 4 days after transfection (data not shown). Under these optimized conditions, both control siRNAs inhibited about 85% of SEAP reporter activity and about 95% of HCV RNA replication (Table 1). The lowered inhibition in the SEAP reporter assay was probably a result of the background SEAP signal caused by the long half-life of this enzyme. In addition, very little toxicity was detected by either the MTT or β-actin RNA assay. No apparent reduction in the signal was observed when cells were transfected with a universal negative control siRNA that has no homology to any known gene (Table 1).

Table 1. Activity of HCV siRNA in HCV Genotype 1b Replicon Cells in High-Throughput Screening Assay
siRNAInhibition (%)*
SEAPHCV RNAMTTβ-Actin RNA
  • *

    Inhibition relative to mock-transfected cells.

  • 100 nM siRNA tested.

HCV NS5B87 ± 898 ± 118 ± 210 ± 5
HCV 5′NTR85 ± 693 ± 137 ± 36 ± 6
Universal negative8 ± 29 ± 45 ± 29 ± 5

siRNA Screening Strategy.

Using the high-throughput screening assay, we screened a library of siRNAs that target approximately 4,000 human genes from different families of druggable targets such as protein kinases, G-protein-coupled receptors, and ion channels to identify host factors important for HCV replication. In the primary assay, SMARTpool siRNA consisting of 4 individual siRNAs was transfected into HCV replicon cells at a final concentration of 100 nM (Fig. 2). Inhibition of HCV replication in the siRNA-transfected cells was detected by reduction of the SEAP reporter activity. The cytotoxicity of the transfected siRNA was monitored by the MTT assay. On the basis of the degree of inhibition of SEAP reporter activity (at least 30% higher than the median SEAP reading on the same plate) and the degree of toxicity (at most 25% higher than the median MTT reading on the same plate), more than 200 siRNAs were selected for retest in secondary assays. In the secondary assays, siRNA was transfected at 50 nM into replicon cells. RNA copy numbers of both HCV and β-actin, a housekeeping gene, in the siRNA-transfected cells were determined by quantitative real-time RT-PCR to confirm the inhibition of HCV RNA replication and to monitor cellular cytotoxicity, respectively. After evaluation by the secondary assays, hits were chosen on the basis of the degree of inhibition of HCV replication (>60%) and toxicity (<30%) compared with the effect of a universal negative control siRNA transfected into replicon cells. The siRNA hits selected in the secondary assays were further evaluated by individual siRNAs that made up the SMARTpool as described in the Evaluation of Host siRNA Hits section.

Figure 2.

The siRNA high-throughput screen in HCV 1b replicon cells. In the primary screen, inhibition of HCV replication and cytotoxicity mediated by the SMARTpool host siRNA were measured by the SEAP reporter assay and the MTT assay, respectively. In the secondary assays, inhibition of HCV replication and cytotoxicity were confirmed by real-time RT-PCR measuring HCV RNA and β-actin RNA, respectively. Further evaluation of the siRNA hits was carried out using the individual siRNAs that made up the SMARTpool.

Identification of Host siRNA Hits.

Table 2 lists 9 host genes identified from the more than 200 initial hits by siRNA screening as being involved in HCV replication. Among the host genes identified were those that encoded a G-protein coupled receptor (TBXA2R), transcription factors (RelA and NFκB2), protein kinases (MKK7 and SNARK), and transporter proteins (SLC12A4 and SLC12A5). Some of these hits, for example, TBXA2R, SLC12A4, and SLC12A5, inhibited more than 90% of HCV replication (Table 2). The silencing of host gene expression was examined for only 7 of the 9 siRNA hits because of the limited availability of detection reagents. All demonstrated more than 60% knockdown of the host RNA. In addition, all induced little cellular toxicity, with most inhibiting less than 25% of the β-actin RNA. To determine whether these hits could inhibit replication of HCV genotypes other than genotype 1b, the siRNA hits were tested in cells harboring an HCV genotype 1a subgenomic replicon (Fig. 1). All the tested siRNAs had an inhibitory effect on 1a HCV RNA replication, indicating that these siRNAs were effective against replication of both 1a and 1b HCV (Table 2). In addition to the subgenomic HCV region, all replicons described so far are bicistronic replicons that include the coding regions of HIV tat, foot-and-mouth disease virus 2A proteinase, neomycin resistance marker, and encephalomyocarditis virus internal ribosome entry site (IRES; Fig. 1). To exclude the possibility that the inhibition by the siRNA hits was actually a result of their effect on these non-HCV regions, the inhibitory effect of the siRNA hits was confirmed in cells harboring an HCV monocistronic subgenomic replicon by real-time RT-PCR (Table 2). This replicon contained an HCV IRES driving the expression of a hygromycin resistance marker coupled to the HCV (1b-N strain) NS3-NS5B region but none of the non-HCV regions mentioned above (unpublished data). The degree that HCV RNA replication was inhibited by the siRNA hits using this monocistronic replicon was very similar to that seen with the bicistronic replicons, indicating the siRNAs did not affect these non-HCV regions (Table 2).

Table 2. Activity of SMARTpool Host siRNA in HCV Replicon Cells*
Host siRNAGene function1b HCV RNA inhibition (%)1a HCV RNA inhibition (%)MonoHCV RNA inhibition (%)Host gene RNA silencing (%)β-actin RNA inhibition (%)IFN gene induction
  • Abbreviation: ND, not determined; NA, not applicable.

  • *

    Determined by quantitative real-time RT-PCR. Activity is calculated relative to the effect of the negative control siRNA.

  • 50 nM siRNA tested.

  • Monocistronic 1b subgenomic replicon.

TBXA2RG-protein coupled receptor through which TBXA2 mediates intracellular calcium mobilization91 ± 1883 ± 1385 ± 989 ± 725 ± 7Negative
RelADimerizes with NFκB1/NFκB2 to form NFκB complex, a transcription factor in TNF signaling pathway80 ± 1180 ± 1177 ± 688 ± 1011 ± 8Negative
NFκB2Dimerizes with RelA/RelB to form NFκB complex, a transcription factor in TNF signaling pathway82 ± 1586 ± 776 ± 486 ± 625 ± 5Negative
LTβMembrane protein that complexes and anchors lymphotoxin-alpha to cell surface81 ± 1683 ± 1275 ± 298 ± 1118 ± 10Negative
MKK7Protein kinase in MAP signaling pathway80 ± 985 ± 782 ± 593 ± 822 ± 8Negative
TRAF2Adaptor protein in TNF signaling pathway65 ± 10ND70 ± 770 ± 1023 ± 12ND
SNARKProtein kinase activated by AMP-dependent kinase61 ± 12NDND85 ± 101 ± 2ND
SLC12A4Cation-coupled chloride transporter98 ± 1391 ± 10NDND25 ± 5ND
SLC12A5Cation-coupled chloride transporter94 ± 1490 ± 12NDND29 ± 9ND
5′NTRPositive control91 ± 1186 ± 985 ± 8NA12 ± 5ND

Evaluation of Host siRNA Hits.

Host siRNA hits were evaluated in additional assays to determine the specificity of their inhibition of HCV replication. In figure 3, thromboxane A2 receptor (TBXA2R) is used as an example to illustrate this evaluation. All the siRNA SMARTpools tested in the primary and secondary assays were composed of 4 individual siRNAs targeting different regions of the same gene. These siRNAs might vary in the efficiency with which they could silence target gene expression and induce cellular toxicity from possible nonspecific targeting. To identify which of the 4 TBXA2R siRNAs was most active in gene silencing and least cytotoxic, individual siRNA duplexes that constituted the SMARTpool siRNA were tested in 1b HCV replicon cells. Of the 4 siRNA duplexes tested, numbers 1 and 2 inhibited approximately 90% of HCV replication, whereas the other 2 siRNA duplexes inhibited 60%–70% of replication (Fig. 3A). The HCV inhibition mediated by the 4 siRNAs correlated well with the TBXA2R silencing ability of the 4 siRNAs (Fig. 3B). All the individual siRNAs had minimal toxicity (<15%), except for siRNA number 2, which inhibited approximately 40% of β-actin RNA (data not shown). Next, the most potent TBXA2R siRNA without significant toxicity (number 1) was tested in a dose–response assay to determine its specificity in inhibiting HCV replication. This siRNA displayed excellent dose–response inhibition of HCV, and it maintained half its inhibitory effect at concentrations as low as 1 nM (Fig. 3C). More important, there was good correlation of the dose response between the silencing of TBXA2R and HCV RNA replication. Similar experiments were carried out on most other siRNA hits, all of which showed good dose responses (Fig. 4). Next, we tested whether the silencing of TBXA2R RNA correlated with the knockdown of its protein expression. As shown in Fig. 3D, TBXA2R siRNA number 1 was able to substantially reduce expression of TBXA2R protein, as detected by Western blot, whereas neither the NS5B positive control siRNA nor the universal negative control siRNA had any effect on TBXA2R protein expression. The knockdown of expression of RelA protein was also confirmed by Western blot (data not shown). Because specific antibodies to other host proteins were not available, it was not possible to detect their expression knockdown.

Figure 3.

Activity of the individual TBXA2R siRNAs that constituted the SMARTpool TBXA2R siRNA in HCV 1b replicon cells. (A) Results of real-time RT-PCR assay showing inhibition of HCV RNA replication by 50 nM of 4 individual TBXA2R siRNAs (HCV NS5B siRNA was used as a control). (B) Results of real-time RT-PCR assay showing silencing of expression of TBXA2R RNA by 50 nM of 4 individual TBXA2R siRNAs (HCV NS5B siRNA was used as a control). (C) Results of real-time RT-PCR assay showing dose response of individual TBXA2R siRNA (number 1) in inhibiting HCV RNA replication, silencing of TBXA2R RNA expression, and inhibiting β-actin RNA expression. (D) Inhibition of TBXA2R protein expression by 50 nM of individual TBXA2R siRNA (number 1) as detected by Western blot.

Figure 4.

Dose–response of individual siRNA hits in HCV 1b replicon cells. The most potent siRNA duplexes from the respective siRNA SMARTpools were transfected into replicon cells at a range of concentrations. Silencing of host RNA expression, inhibiting of HCV RNA replication, and inhibiting of β-actin RNA expression were detected by real-time RT-PCR. (A) LTβ siRNA, (B) RelA siRNA, (C) NFκB2 siRNA, (D) MKK7 siRNA, and (E) TRAF2 siRNA.

Effects of siRNA Hits on Interferon-Inducible Genes.

Some siRNAs, despite their short length, can activate IFN-inducible protein kinase (PKR) and global up-regulation of IFN-stimulated genes.32 Although part of the IFN signaling pathway is inhibited by HCV NS3/4A in HCV replicon cells,33–35 it was crucial to confirm that the siRNA hits did not exert their inhibitory effect on HCV through nonspecific activation of PKR and other IFN-inducible genes. To address this, siRNA was transfected into replicon cells, and its effects on PKR and 2 other well-known IFN-inducible genes (oligoadenylate synthetase [OAS] and myxovirus-resistance protein [Mx]) were compared with those of IFN-α incubated with replicon cells. Both TBXA2R siRNA (number 1) and IFN-α inhibited approximately 90% of HCV replication (Fig. 5A). In addition, IFN-α substantially activated expression of PKR, OAS, and Mx genes (Fig. 5B-D). In contrast, the TBXA2R siRNA did not induce the expression of any of these genes, confirming that it did not mediate its inhibition of HCV replication by nonspecific activation of PKR and, more important, by the IFN signaling pathway. The same evaluations were done for 4 other siRNA hits, none of which were found to activate expression of IFN-inducible genes (Table 2).

Figure 5.

Lack of activation of IFN-inducible genes by TBXA2R siRNA. HCV 1b replicon cells were treated with 10 IU/ml of interferon-α (IFN), transfected with 50 nM universal negative control siRNA (Neg), or transfected with individual TBXA2R siRNA (number 1). Three days after transfection, HCV, PKR, OAS, and Mx RNA was detected by real-time RT-PCR. (A) Inhibition of HCV RNA replication, (B) activation of PKR RNA expression, (C) activation of OAS RNA expression, and (D) activation of Mx RNA expression were calculated relative to the effect of the universal negative control siRNA.

Involvement of Tumor Necrosis Factor/Lymphotoxin Signaling Pathway in HCV Replication.

Of the 9 siRNA hits identified in this study, 4 are members of the tumor necrosis factor/lymphotoxin (TNF/LT) signaling pathway: LTβ, TRAF2 (TNF receptor-associated factor 2), RelA, and NFκB2 (nuclear factor κB2)/p52 (Fig. 6). TNF and LT are members of the TNF superfamily, a diversified family of ligands and receptors that control signaling pathways leading to cell death, survival, and cellular differentiation. In this pathway, many factors can induce activation of the downstream NFκB complex through different intermediates (Fig. 6).36 For example, the LTα1β2 ligand, formed by the complex of LTβ with LTα, signals through the lymphotoxin beta receptor (LTβR). This signaling results in activation of the inhibitor of NFκB kinase (IKK), which phosphorylates the inhibitor of NFκB (IκB) and leads to its degradation. As a result, the NFκB complex translocates into the nucleus to regulate the expression of its effector genes. The NFκB complex is formed by NFκB1 (p50) or NFκB2 (p52) binding to Rel, RelA, or RelB. The transcription of NFκB2 (p52) is dependent on RelA/NFκB1 linking these pathways together (Fig. 6).

Figure 6.

TNF/LT signaling pathway. Various ligands (TNF, LTα3, TNFSF14, and LTα1β2) could bind to their cognate receptors (TNFR1, LTβR, TNFR2, and HVEM) to induce the downstream signaling pathway. This signaling could result in (1) induction of apoptosis through TRADD; (2) activation of the classical NFκB pathway, leading to the activation of the NFκB (RelA/p50[NFκB1]) complex; or (3) activation of the nonclassical NFκB pathway through LTβR, resulting in activation of the NFκB (RelB/p52[NFκB2]) complex (*siRNA hits identified by high-throughput screening; #siRNA hits identified by further experiments; TNFSF14, tumor necrosis factor ligand superfamily, member 14 [also known as LIGHT]; TNFR, tumor necrosis factor receptor; HVEM, herpes simplex virus entry mediator; TRADD, TNFR1-associated death domain; FADD, Fas-associated death domain). This figure is adapted from Schneider et al.36

To determine if the proteins encoded by these 4 siRNA hits crosstalk with each other to regulate the replication of HCV, siRNAs targeting 2 of the genes (inhibitor of NFκB kinase beta [IKKβ] and NFκB-inducing kinase [NIK]) that propagate the signaling of the siRNA hits in the TNF/LT cascade were tested in HCV replicon cells. IKKβ was chosen because it is a major component of the IKK complex, which regulates activation of the NFκB [RelA/p50(NFκB1)] complex. NIK was tested because it is required for LTβ-mediated activation of a different form of the NFκB complex [RelB/p52(NFκB2)]. As shown in Table 3, the siRNA that silenced IKKβ gene expression also inhibited HCV RNA replication. The NIK siRNA also had some inhibitory effect on RNA replication of HCV. However, the role of NIK in HCV replication requires further investigation because the expression of NIK RNA in replicon cells was quite low (data not shown).

Table 3. Activity of siRNA Targeting Members of TNF/LT Signaling Pathway in HCV Genotype 1b Replicon Cells*
siRNA1b HCV RNA inhibition (%)Host gene RNA silencing (%)β-Actin RNA inhibition (%)
  • *

    Determined by quantitative real-time RT-PCR. Activity was calculated relative to the effect of the negative control siRNA.

  • 50 nM siRNA tested.

IKKβ72 ± 992 ± 622 ± 5
NIK69 ± 1164 ± 156 ± 10

Discussion

In the present study, we identified several host genes, the silencing of which by siRNA resulted in inhibition of HCV replication in replicon cells. Inhibition of HCV RNA replication was initially detected by a SEAP reporter assay and later confirmed by an RNA quantitation assay. These siRNA hits inhibited RNA replication of both HCV 1a and 1b replicons. The ability of the siRNA hits to inhibit HCV replication correlated well with their ability to silence host gene expression. Furthermore, we confirmed that inhibition of HCV replication mediated by these siRNA hits was not a result of nonspecific activation of IFN-inducible genes. Taken together, these results strongly support the involvement of these host factors in HCV replication.

It is notable that among the inhibitory siRNA hits were a group of genes belonging to the TNF/LT signaling pathway. Results from recent studies may shed some light on how the TNF/LT cascade regulates HCV replication. By performing microarray studies with a cell line expressing HCV NS5A, it was shown that many of the genes up-regulated by NS5A contained one or more NFκB binding sites within their promoter regions.37 In addition, NFκB was activated in NS5A-expressing cells.37, 38 These findings suggest that activation of NFκB may be involved in HCV replication, in agreement with our siRNA silencing data, which showed that several factors that constitute the pathway leading to the activation of the NFκB complex could regulate HCV replication. Interestingly, one of the siRNA hits, TRAF2, has been shown to complex with NS5A.39 This interaction may modulate activation of the NFκB complex. HCV may interact with the LT pathway by more than one mechanism because HCV core protein has been shown to interact with the LT pathway as well.40, 41

One of the major proposed roles of NFκB in cells harboring HCV is to regulate the expression of genes essential for protecting cells from apoptosis induced by HCV.42 The principal site of HCV replication is on the membranes of the endoplasmic reticulum (ER). NS5A is associated with the ER membrane and is capable of inducing ER stress.43, 44 As a consequence of ER stress, calcium is released from the ER and taken up by mitochondria, generating reactive oxygen species (ROS). ROS by itself promotes apoptosis and also inhibits HCV replication.45 It is believed that ROS activates dampening pathways such as NFκB,46 which induces expression of antiapoptotic genes to counteract the apoptotic processes, an essential event for the survival of cells harboring HCV.47 In addition, dampening of ROS production by NFκB would allow replication of HCV. Thus, NFκB may support HCV replication through these 2 mechanisms. However, NFκB could also induce an antiviral state in the cells through activation of IFN-dependent genes.48, 49 Therefore, survival of HCV in host cells may depend on a critical balance between ROS dampening and antiviral activity of NFκB.

Another siRNA hit was TBXA2R, the receptor of a potent stimulator of platelet aggregation and constrictor of smooth muscles. TBXA2R, which is predominantly expressed in platelets, is also expressed in replicon cells (Fig. 3), and its RNA could be detected in human liver cells, the primary cells that support HCV replication (data not shown). TBXA2R was up-regulated in NS5A-expressing cells when examined by microarray studies,37 but the significance of this up-regulation in the replication of HCV remains to be established. TBXA2R may be involved in the action of NS5A in altering calcium homeostasis in the ER because signal transduction through TBXA2R results in intracellular calcium mobilization. Alternatively, TBXA2R may be involved in activation of calpain protease, which is initiated by elevation of calcium levels.50 NS5A has been shown to be both an inducer and a substrate of calcium-dependent calpain protease.50 Cleavage of NS5A by calpain may play a role in the modulation of the function of NS5A. For the rest of the siRNA hits (MKK7, SNARK, SLC12A4, and SLC12A5), very little data has been published that links their functions to HCV replication.

In this study, we used siRNA to identify host genes important for HCV replication. Future experiments that could confirm the roles of these host proteins include overexpression of the host genes in HCV replicon cells, which may result in enhancement of HCV RNA replication. Additionally, overexpression of dominant-negative mutants of these host genes could lead to competition of the mutants with wild-type host genes, causing inhibition of HCV replication. Another approach would be to test if known inhibitors of the host proteins could inhibit HCV replication. siRNAs identified in this study inhibit replication of both the 1a and 1b HCV replicons. It would be interesting to test these siRNA hits in replicon cells generated from other HCV genotypes or cellular backgrounds. More important, the effects of these hits should be confirmed in a full-length HCV replicon and in infectious HCV tissue culture systems. Data presented in this report focus on siRNAs that inhibit HCV replication. Preliminary data from our screen also indicated that we had identified some host genes that might suppress HCV replication because siRNA silencing of these genes led to enhancement of HCV RNA replication (data not shown).

A better understanding of the biology of the interaction of HCV and host may enable identification of novel cellular targets for drug discovery. Inhibitors targeting virus–host interaction do not necessarily have a higher level of undesirable side effects than do direct antiviral therapies. Drugs other than anti-infectives are directed against host targets, yet they do not always cause more adverse effects than do standard antivirals. Given the possible advantages of lower incidence of resistance and the broader spectrum of activity for various HCV genotypes, it is not surprising that the search for compounds targeting host genes has become one of the possible approaches to developing treatments for HCV infection.

Acknowledgements

The authors thank Drs. Steve Fesik, Yu Shen, and Dimitri Semizarov (Abbott Laboratories, Cancer Research) for helpful discussions and suggestions.

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