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Hepatitis C virus (HCV) is a leading cause of chronic liver disease. The identification and characterization of key host cellular factors that play a role in the HCV replication cycle are important for the understanding of disease pathogenesis and the identification of novel antiviral therapeutic targets. Gene expression profiling of JFH-1-infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defense mechanisms (apoptosis, proliferation, and antioxidant responses), cellular metabolism (lipid and protein metabolism), and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV-associated pathogenesis. These include an increase in proinflammatory and proapoptotic signaling and a decrease in the antioxidant response pathways of the infected cell. To investigate whether any of the host genes regulated by infection were required by HCV during replication, small interfering RNA (siRNA) silencing of host gene expression in HCV-infected cells was performed. Decreasing the expression of host genes involved in lipid metabolism (TXNIP and CYP1A1 genes) and intracellular transport (RAB33b and ABLIM3 genes) reduced the replication and secretion of HCV, indicating that they may be important factors for the virus replication cycle. These results show that major changes in the expression of many different genes in target cells may be crucial in determining the outcome of HCV infection.
Hepatitis C virus (HCV) is a leading cause of chronic liver disease, which affects around 170 million people worldwide. Infections are initially acute, and in many cases the symptoms are mild. However, around 80% of patients eventually develop a persistent chronic infection which can result in steatosis, fibrosis, cirrhosis, liver failure, and, in some cases, hepatocellular carcinoma. The main treatments currently available for chronically infected patients use a combination of pegylated alpha interferon and ribavirin, but these still result in a sustained antiviral response in only about 50% of genotype 1 infections (4). Consequently, more effective antivirals that target either the virus proteins directly or the host cell proteins required during HCV replication are currently being developed. In order to ensure that successful antivirals are generated, it is important that all aspects of the HCV life cycle and HCV-associated pathology are well understood.
One way in which the different host processes that are an essential part of the HCV replication cycle can be studied is to investigate the effect that HCV infection has on cellular gene expression. RNA microarray hybridization is routinely used to investigate host gene expression and allows the entire transcriptomic profile of the cell to be characterized. Microarray analysis of HCV-infected cells can provide an insight into the genes involved in host cell antiviral responses, genes that are essential for the HCV replication cycle, and genes that contribute to HCV-associated liver pathology. Microarray expression profiling has already been used to study host gene expression in cells transfected with RNA encoding either individual HCV genes, HCV subgenomic replicons, or the full-length HCV genome. These studies have demonstrated that the replication of the HCV genome results in the regulation of a small number of host genes involved in lipid metabolism, cellular immunity, proliferation, apoptosis, and molecular transport (2, 5, 15, 32). These studies have provided interesting insights into the HCV replication cycle. However, the biological significance of gene expression patterns identified is less clear, since the full virus replication cycle, including the processes of viral entry, assembly, and exit, does not take place.
The recent discovery of JFH-1, a genotype 2a HCV clone that can undergo a complete infection cycle in cell culture, provides the opportunity to characterize the true effect of HCV infection on host gene expression (51). A recent study investigated the effects that a J6/JFH-1 chimera had on the gene expression profile of Huh7.5 cells during a time course infection with time points of 24, 48, 72, 96, and 120 h (53). The number of host genes regulated during infection was much higher than that previously observed for cells permitting only genome replication, indicating that the full replication cycle has additional effects on host gene expression.
In this study, we present the results from an investigation into the effect that JFH-1 infection has on host gene expression during the early stages of HCV infection, at 6, 12, 18, 24, and 48 h postinfection. Huh7 cells were used for the study to allow investigations to be performed in the most biologically representative host cell system currently available. Previous expression studies have generally been performed using the adapted Huh7.5 cell line, which has a mutation in the RIG-I gene and a defective interferon response that allows higher levels of HCV replication (47). Despite the differences in virus titer, the kinetics of virus replication are thought to be relatively similar in the two cell types (44). Therefore, this study enables a comparison of the host cell responses to HCV infection for the Huh7 and Huh7.5 cell lines. In this study, infection of Huh7 cells with JFH-1 viral particles resulted in the altered expression of host genes involved in innate immune signaling, cellular growth and apoptosis, oxidative stress responses, lipid and protein metabolism, and intracellular transport. Further investigation into several of the genes that had increased levels of expression after HCV infection by small interfering RNA (siRNA) silencing revealed a number of genes which are required for effective virus replication and secretion, including those for TXNIP, RAB27A/B, RAB33B, RAB40B, CYP1A1, and ABLIM3. This work demonstrates the importance of these proteins during HCV infection and opens the way for further analysis of their involvement in the different stages of the virus replication cycle.
Huh7.5 and Huh7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37°C with 5% CO2. Huh7 cells were a gift from John Monjardino (24).
Full-length JFH-1 virus was produced using Huh7.5 cells transfected with RNA transcribed from the pJFH-1 plasmid (51). Transfected cells were maintained for 21 days, and growth medium containing infectious JFH-1 was serially passaged onto naive Huh7.5 cells every 4 days. Culture medium containing virus was harvested at each passage, clarified by centrifugation and 0.45-μm filtration, and concentrated using Amicon Ultra-15 centrifugal filter units (Millipore).
To produce infectious HCV Jc1p7FLuc2A, 1 × 107 Huh7.5 cells were electroporated with 10 μg Jc1p7FLuc2A RNA and seeded in a 225-cm2 flask. On the second day postelectroporation, the cells were passaged 1 in 2, and medium containing virus was collected for 5 days and pooled. HCV Jc1p7FLuc2A was constructed from Jc1p7RLuc2A by replacing the Renilla luciferase (Gluc) reporter gene with a firefly luciferase (Fluc) reporter gene. The Flag tag sequence was also removed (28).
A total of 5 × 105 Huh7 cells were seeded in 25-cm2 culture flasks and infected in triplicate with JFH-1 at a multiplicity of infection (MOI) of 3 or mock infected with an equal volume of concentrated conditioned growth medium. At 6, 12, 18, 24, and 48 h postinfection, cellular RNA was extracted using TRIzol reagent (Invitrogen). TRIzol lysates were shipped to Expression Analysis (NC), where RNA was purified, quality tested using an Agilent bioanalyzer, and hybridized onto Human U133 Plus 2.0 Affymetrix microarray chips for fluorescence data acquisition. Raw microarray expression data were processed using Array Studio software (Omicsoft) to generate values representing fold changes in gene expression. An average of the triplicate values was used to calculate fold change, and each value was assessed for its statistical significance, using analysis of variance (ANOVA). Host genes demonstrating at least a 2-fold change in expression and a >95% probability of being expressed differentially (P ≤ 0.05) were selected for further investigation.
The infectivity of JFH-1 for Huh7 cells was tested prior to the microarray experiment. An MOI of 3 was shown to be appropriate for complete infection of Huh7 cells (see Table S1 in the supplemental material).
Gene ontology programs used to identify expression patterns in the data included Ingenuity pathway analysis (Ingenuity Systems) and PANTHER classification (49) software.
Heat maps representing the microarray data were generated using Multi Experiment Viewing (MEV) software, available within the TM4 microarray software suite. Hierarchical clustering by the Pearson correlation was used to cluster the genes in each heat map (43). The log2 expression data represented in the heat maps can be found in Table S2 in the supplemental material.
Total RNA from a replica microarray infection experiment was extracted using TRIzol reagent and treated with DNase (Ambion Turbo DNase). cDNA was synthesized from 2 μg of RNA, using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) and oligo(dT) primers (Invitrogen). Quantitative reverse transcription-PCR (qRT-PCR) was performed using a Quantitect SYBR green PCR kit (Qiagen). Ten-microliter reaction mixtures were set up using 900 ng of cDNA according to the manufacturer's instructions. The comparative threshold cycle (CT) method was used to calculate the fold change in gene expression, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and large ribosomal protein (RPLPO) as the normalizing control genes (38). PCR primers were designed using Primer3 software (sequences are available in Table S3 in the supplemental material).
A total of 1 × 105 Huh7 cells were seeded in 24-well plates and transfected with a 50 nM concentration (total) of two siRNA molecules for each target (25 nM siRNA 1 plus 25 nM siRNA 2), using Lipofectamine 2000 (Invitrogen) (siRNA sequences are available in Table S4 in the supplemental material). After an 8-h incubation period, the transfection medium was replaced with growth medium. The transfected cells were incubated for 48 h and infected with JFH-1 at an MOI of 3. Four hours after infection, the cells were washed and incubated with growth medium for another 48 h. Intracellular RNA was extracted using TRIzol reagent, treated with DNase, and used directly for qRT-PCR for quantification of target mRNA and JFH-1 RNA. qRT-PCR was performed using a One-Step Quantitect SYBR green RT-PCR kit (Qiagen). Ten-microliter reaction mixtures were set up using 250 ng of RNA according to the manufacturer's instructions. The comparative CT method was used to calculate the fold change in JFH-1 RNA and target mRNA abundance, with GAPDH as the normalizing control gene.
Medium from each sample in the siRNA transfection/JFH-1 infection experiments was collected and stored at −20°C. A total of 1 × 104 Huh7 cells were seeded in 96-well plates and infected with the transfection sample medium, which had been diluted 1 in 100 in DMEM. At 48 h postinfection, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and incubated for 1 h at room temperature with an anti-HCV core antibody (mouse monoclonal antibody C7-50 [Abcam]) diluted 1 in 400 in 1% bovine serum albumin (BSA)-phosphate-buffered saline (PBS). The cells were then washed in PBS three times for 5 min each and incubated for 1 h at room temperature with Alexa Fluor 594-conjugated donkey anti-mouse polyclonal antibody (Invitrogen) diluted 1 in 1,000 in 1% BSA-PBS. The cells were washed in PBS three times for 5 min each and mounted using Mowiol mounting medium. Core staining was visualized using a Nikon Eclipse TE2000S fluorescence microscope. The number of core-positive foci was counted for each sample. The focus numbers counted for wells infected with medium from target-specific siRNA transfections were compared to the focus numbers counted for wells infected with medium from the nontargeting siRNA transfections. Each medium sample was analyzed in duplicate, and all samples from the three transfection experiments were investigated.
A total of 5 × 103 Huh7.5 cells were transfected with 25 nM siRNA molecules and Lipofectamine 2000 in a 96-well plate (siRNA sequences are available in Table S4 in the supplemental material). Cells were infected at 48 h posttransfection with full-length infectious HCV Jc1p7FLuc2A at an MOI of 0.1. At 48 h postinfection, cell culture medium was removed and the cells were incubated with 25 μl of Steady-Glo luciferase assay reagent (Promega). After 10 min of incubation, the luciferase signal was read using an Envision luminometer (Perkin-Elmer).
Calculations of statistical significance for qRT-PCR and luciferase data were performed using a two-tailed unpaired t test in GraphPad Prism software. Data points with a P value of <0.05 were considered statistically significant.
The GEO accession number for the data associated with this paper is GSE20948.
The effect of HCV infection on host gene expression was investigated using Huh7 cells infected with JFH-1 for 6, 12, 18, 24, and 48 h. Total RNA extracted from cells was hybridized to Affymetrix Human U133 Plus 2.0 GeneChips for gene expression analysis. The total number of genes significantly regulated at each time point is shown in Table Table1.1. Genes showing at least a 2-fold change in expression and a >95% probability of being differentially expressed (P ≤ 0.05) were considered to be significantly regulated by infection. These selection criteria were chosen to enable a comparison of the results in this study to those observed in previously published HCV microarray studies which used the same selection cutoff values (15, 32, 53). The total number of genes regulated by infection increased over time. JFH-1 RNA also increased in abundance during the course of infection (see Fig. S1 in the supplemental material).
Gene ontology analysis of the microarray expression data was performed using the PANTHER classification program and Ingenuity pathway analysis software. These programs indicated that the host genes with altered expression levels during JFH-1 infection were involved in a range of biological processes. In order to identify host gene expression patterns for further investigation, a focus was placed upon the processes that were overrepresented in the microarray data set and upon those with prior evidence for a role in the replication cycle of other RNA viruses. The genes chosen for further investigation included those involved in pathways regulating host defense mechanisms (e.g., inflammation, oxidative stress, and apoptosis), host metabolism (e.g., lipid and protein metabolism), and intracellular transport (e.g., vesicle trafficking and cytoskeleton). Many of the host genes significantly regulated by infection were also involved in processes such as vision, taste, and embryogenesis. These processes were not considered relevant to the study and were therefore excluded from the analysis.
Many of the genes regulated following infection were involved in host defense mechanisms that protect the cell against infection and oxidative stress and in turn determine the fate of cellular survival. The expression profiles for the cellular defense genes regulated by JFH-1 infection are represented by heat map diagrams in Fig. Fig.11.
Genes involved in innate immune signaling increased in expression following JFH-1 infection, including those involved in type I and II interferon responses (e.g., IRF1, IRF9, and MX1 genes), the complement cascade (e.g., MBL2 and MASP1 genes), and the production of proinflammatory chemokines and cytokines (e.g., interleukin-8 [IL-8] and CXCL1, -2,-3, -5, -6, and -16 genes). Increased expression of genes encoding negative regulators of the interferon response was also observed, including that of several members of the SOCS gene family (e.g., SOCS2 and -3 genes).
The expression of genes involved in cellular antioxidant responses decreased following infection, including that of genes transcribed following NRF2 activation (e.g., CAT and EPHX1 genes), members of the metallothionein gene family, genes for the glutathione S-transferase enzymes (e.g., GSTM1 to -4 genes), and genes for other key antioxidants (e.g., ATOX1 and GLRX genes). Expression patterns promoting an increase in intracellular reactive oxygen species were also observed, as genes involved in the inhibition of antioxidant proteins (e.g., TXNIP gene) and the production of reactive oxygen species (e.g., DDAH1 and AOX1 genes) significantly increased in expression. Despite the decreased expression of genes involved in cellular antioxidant pathways, genes involved in cellular detoxification actually increased in expression, including the aryl hydrocarbon receptor (AHR) pathway genes (e.g., AHR, CYP1A1, and CYP1B1 genes) and genes for other important detoxification enzymes (e.g., ESD, NNMT, and UGT2B4 genes).
The expression of genes controlling apoptosis and cell cycle arrest was significantly regulated by infection. Transcriptional changes that promoted apoptosis and cell cycle arrest were observed. These included an increase in the expression of genes that induce apoptosis, such as those involved in p53 and transforming growth factor beta (TGFβ) signaling (e.g., PMAIP and TP53BP2 genes), an increase in the expression of proapoptotic transcription factor genes (e.g., FOXO3 and ATF4 genes), and an increase in the expression of genes promoting cell cycle arrest (e.g., INHBE and STC2 genes). Decreases in the expression of genes required for cell cycle progression (e.g., CCNB1, CCNE1, and SKP2 genes), cytokinesis (e.g., CP110 and SPC24 genes), and S-phase DNA synthesis (e.g., RFC3 and RFC5 genes) were also observed. At the same time, genes promoting apoptosis and inhibition of the cell cycle also decreased in expression (e.g., BCL2L11, DFFB, CDKN3, and CDKN2C genes), and genes promoting cellular survival, cell cycle progression, and cytokinesis increased in expression (e.g., BNIP1, PRNP, CDK6, and MIS12 genes), demonstrating that conflicting transcriptional changes occur following infection.
A second class of host genes significantly regulated by JFH-1 infection were those involved in more general housekeeping functions of the cell, including cellular metabolism and intracellular transport. The expression profiles of the genes involved in these processes are represented by heat map diagrams in Fig. Fig.11.
Host genes involved in the biosynthesis, degradation, and transport of intracellular lipids were significantly regulated following infection. Genes involved in the cholesterol biosynthesis pathway decreased in expression following infection (e.g., SQLE and HMGCR genes). However, genes regulating the transfer of geranyl moieties from the mevalonate cholesterol pathway to cellular proteins actually increased in expression (e.g., GGPS1 and PGGT1B genes), indicating that the different branches of the cholesterol synthesis pathway are differentially regulated during infection. Genes involved in the synthesis and transport of cellular sphingolipids and phospholipids increased in expression following infection (e.g., SGPP1, SPTLC1, CHKA, and ACSL3 genes). The expression of genes involved in fatty acid metabolism was also significantly regulated following infection, including a decrease in the expression of genes involved in the degradation and oxidation of fatty acids (e.g., ACAT2 and ACADSB genes) and an increase in the expression of genes involved in the synthesis and transport of fatty acids (e.g., ELOVL4, ACSL3, VLDLR, and FABP3 genes) and the transcription of genes controlling fatty acid metabolism (e.g., PPARGC1A and TXNIP genes).
The expression of genes involved in host protein synthesis and protein processing increased following infection. Increased expression was observed for the genes that control amino acid metabolism (e.g., PSAT1 and BCAT1 genes), tRNA synthesis (e.g., AARS, SARS, GARS, and WARS genes), production of ribosomes (e.g., RPL13 and RPL37 genes), pre-mRNA processing and splicing (e.g., HNRNPH3 and QKI genes), protein translation (e.g., eIF-4E and eIF4EBP1 genes), and posttranslational modification of proteins by glycosylation (e.g., MGAM gene) and sulfation (e.g., SULT2A1 gene). In addition to promoting increased rates of protein synthesis, there may also be a decrease in the rate of protein degradation, as host genes involved in the polyubiquitination of proteins and the function of the proteasome and lysosome decreased in expression (e.g., UBE3B, PSMAI, and CTSC genes) and genes promoting the disassembly of polyubiquitin increased in expression (e.g., USP15 and OTUD1 genes).
The expression of genes involved in protein sorting and vesicle trafficking increased following infection. This included the genes responsible for regulating endosomal vesicle trafficking (e.g., SNX12, SNX16, and PICALM genes), trafficking between the endoplasmic reticulum (ER) and the Golgi body (e.g., RAB6B, RAB7L1, RAB33B, RAB40B, and ARF-GEF1 genes), and trafficking of secretory vesicles to the plasma membrane (e.g., RAB27A and RAB27B genes).
Genes that control the function of actin and microtubule cytoskeleton filaments were also significantly regulated by infection. A large proportion of the genes encoding actin and microtubule binding proteins decreased in expression (e.g., TUBA1A and MYL9 genes). A small number of genes encoding actin binding proteins also increased in expression (e.g., ABLIM3 gene).
Further details of the genes regulated by JFH-1 in the time course infection experiment are shown in Table S5 (A to C) in the supplemental material for the host defense genes and in Table S6 (A to D) in the supplemental material for the metabolism and transport genes.
Total RNA extracted from a replicate microarray infection experiment was used for qRT-PCR quantification of gene expression to validate the data obtained through microarray analysis. The expression of genes involved in antiviral signaling, oxidative stress responses, apoptosis, lipid metabolism, and intracellular transport was investigated in the RNA extracted at 48 h postinfection. Fold changes in gene expression calculated by qRT-PCR and microarray analysis are shown in Fig. Fig.2.2. The microarray and qRT-PCR experiments demonstrated reproducible gene expression patterns, although larger fold change values were obtained with qRT-PCR for the majority of the genes tested. For several of the genes identified, qRT-PCR was also used to investigate the level of expression in the RNAs extracted at 6, 12, 18, and 24 h postinfection. Microarray and qRT-PCR data for the temporal regulation of expression of the TXNIP, RAB27A, ABLIM3, and metallothionein genes are shown in Fig. Fig.3.3. Different patterns were observed in the transcription of host genes following JFH-1 infection. For example, the TXNIP gene was consistently transcribed at a high level from 12 to 48 h postinfection (Fig. (Fig.3).3). In contrast, the ABLIM3 gene was expressed at increasing levels between 6 and 18 h postinfection. However, at 18 h postinfection, the transcription of the ABLIM3 gene reached a maximum level, and it returned to normal levels of expression by 48 h (Fig. (Fig.3).3). Other genes, such as the RAB27A gene, did not show any change in expression until a later stage of infection, when gene expression increased to reach a maximum level at 48 h postinfection (Fig. (Fig.3).3). Many host genes were also significantly downregulated following infection, including members of the metallothionein gene family, which consistently showed a decrease in expression from 6 to 24 h postinfection (Fig. (Fig.33).
To investigate whether the host genes regulated by infection are important components of the HCV replication cycle, siRNA silencing of host gene expression was performed for selected targets. The effect of host gene silencing on JFH-1 replication was investigated by qRT-PCR quantification of JFH-1 RNAs in the siRNA-treated cells. siRNA molecules which reduced intracellular JFH-1 RNA levels included those targeting the expression of the TXNIP, CYP1A1, ABLIM3, RAB27A, RAB27B, RAB33B, and RAB40B genes. qRT-PCR quantification of target mRNAs and JFH-1 RNAs following siRNA silencing is shown in Fig. Fig.44 (top two panels). An siRNA directly targeting the HCV internal ribosome entry site (IRES) sequence was included in the assay as a positive control for JFH-1 RNA knockdown. In comparison to a nontargeting siRNA, siRNAs targeting TXNIP gene expression had the greatest effect on JFH-1 replication, reducing JFH-1 RNA levels by 85%. The knockdown of other host genes also had significant effects, with CYP1A1, RAB33B, and RAB40B gene knockdown reducing JFH-1 RNA levels by 60%, ABLIM3 and RAB27B gene knockdown reducing JFH-1 RNA levels by 50%, and RAB27A gene knockdown reducing JFH-1 RNA levels by 40%.
Since the JFH-1 replication cycle results in the production of infectious virus particles, the effect of host gene silencing on the secretion of JFH-1 was also investigated. Medium collected from the siRNA transfection/JFH-1 infection experiments was used to infect naïve Huh7 cells. JFH-1 virus titers were then compared for the medium collected from the target-specific siRNA transfections and the medium collected from the nontargeting siRNA transfections, as shown in Fig. Fig.4,4, bottom panel. JFH-1 secretion was reduced following the silencing of specific host genes. The most significant reduction in particle secretion occurred following the silencing of the TXNIP gene, where a 90% reduction in core-positive foci was observed. Silencing the expression of the CYP1A1 gene reduced JFH-1 secretion by 75%, RAB27B and RAB33B gene silencing reduced JFH-1 secretion by 70%, RAB40B and RAB27A gene silencing reduced levels by 60%, and ABLIM3 gene silencing reduced levels by 50%.
The effect of gene silencing on HCV replication was also investigated using a luciferase-expressing HCV clone, Jc1. Jc1 is a fully infectious chimeric virus constructed from the JFH-1 backbone, with the 5′ end of the genome (core-E1-E2-P7-NS2) replaced with the sequence from the J6 genotype 2a HCV clone (39). The relative luciferase activities of Jc1-infected Huh7.5 cells following siRNA silencing of the DDX3X, TXNIP, CYP1A1, and ABLIM3 genes are shown in Fig. Fig.5.5. DDX3X is a host factor previously shown to play an essential role in the HCV replication cycle (3). siRNAs targeting the expression of DDX3X were used as a positive control for the assay. In support of the qRT-PCR data, the siRNA molecules that reduced the Jc1 luciferase signal by the largest amount were those targeting TXNIP gene expression, which reduced the signal by 80 to 85%. For comparison, silencing of the CYP1A1 gene reduced the luciferase signal by 75 to 80% and silencing of the ABLIM3 gene reduced the luciferase signal by 50 to 60%. The viability of siRNA-transfected Huh7 and Huh7.5 cells was tested using Alamar Blue and MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (see Fig. S2 in the supplemental material). siRNA molecules were shown to have no significant effects on cell viability when tested at 72 h posttransfection.
The results of this study demonstrate that host genes involved in cellular defense, metabolism, and intracellular transport are regulated during the early stages of HCV infection. The total number of genes regulated by infection increased over time, which may correspond to the increasing viral RNA titers observed (see Fig. S1 in the supplemental material). The microarray study findings were confirmed by qRT-PCR, which showed higher fold change expression values for many of the genes tested. This has been observed in other gene expression studies and may result from the greater sensitivity of qRT-PCR (31, 53).
To compare the findings of this study to previous HCV microarray studies, genes showing at least a 2-fold change in expression were selected for further analysis. Many of the genes selected were regulated in a highly coordinated manner (e.g., cholesterol biosynthesis genes, tRNA synthetase genes, and metallothionein genes), indicating that the regulation of these genes may significantly impact cellular function. By comparing the findings of this study to previously published HCV microarray studies, overlapping expression patterns were observed, including the regulation of genes involved in proliferation, apoptosis, cytokine and chemokine production, and lipid metabolism (1, 2, 32, 53). However, many novel host genes regulated by HCV infection were also identified, including those involved in protein synthesis and degradation, posttranslational modification, and vesicular trafficking. By investigating the effect of the full replication cycle on host expression, this study has shown that many more host genes (>2,000 genes) are regulated than those in studies where genome replication alone takes place (<200 genes) (1, 2, 32). A recent study demonstrated that the binding and entry of HCV-like particles into HepG2 cells altered the expression of 565 host genes, indicating that virus entry alone significantly alters host expression (18). Furthermore, in this study, we also observed a larger number of host genes regulated during infection and a more immediate transcriptional response to infection than those in a recent microarray study investigating J6/JFH-1 infection of Huh7.5 cells. In our study, 1,000 genes were regulated at 24 h postinfection; for comparison, fewer than 50 genes were regulated in the study investigating Huh7.5 cell infection (53). Several genes in our study were also significantly regulated between 6 and 24 h postinfection, with less-significant expression changes at 48 h postinfection (e.g., ABLIM3 and metallothionein genes). The identification of genes which are regulated only during the early phases of infection may be important. This is supported by the observation that siRNA silencing of the actin binding protein ABLIM3 in this study reduced the replication of HCV. Differences in the magnitude and timing of the transcriptional responses observed in the two studies may be due to the regulation of host gene expression following activation of the antiviral RIG-I pathway, which is functional in Huh7 cells but not in Huh7.5 cells (47). The previous microarray study also used UV-inactivated virus as the negative-control treatment, which does not permit identification of genes regulated during virus entry.
By performing our study in Huh7 cells, in which RIG-I signaling is intact, the gene expression patterns observed may reflect more accurately those typical of an in vivo HCV infection. Studies of HCV-infected chimpanzee and chimeric SCID-beige/Alb-uPA mouse livers showed a number of host gene transcriptional changes during infection, some of which were also observed in our study but not in the Huh7.5 J6/JFH-1 infection study. This included genes involved in the double-stranded RNA response pathway (e.g., OAS3, MX1, IFIT1, GBP1, and CXCL2 genes) and the synthesis of fatty acids (e.g., SREBP gene) (46, 52).
In this study, a number of the genes regulated by JFH-1 infection were shown to play a role in cellular defense. An increase in the expression of genes that encode proinflammatory cytokines and chemokines was observed, supporting prior evidence that infected hepatocytes may act as a major source of inflammation in the liver (53). In particular, CXCL chemokines (1, 2, 3, 5, 6, and 8), which act via CXCR1 and CXCR2 receptors and are known to stimulate the chemotaxis and respiratory burst activity of neutrophils (12), as well as the induction of hepatocyte proliferation (41), all had elevated mRNA levels. Increased expression was also observed for several SOCS genes, which act as negative feedback regulators of cytokine function (56). This has been observed in previous studies and may contribute to the alpha interferon resistance that can develop in HCV patients (10, 37, 53). An increase in the expression of fibrosis-promoting complement factors, MASP1 and MBL2, was also observed, indicating that they may be responsible in part for the liver fibrosis that frequently occurs in HCV-infected patients (8, 19). HCV infection is known to stimulate the production of reactive oxygen species, which promote oxidative stress-induced damage in patients (19). A significant reduction in the expression of genes that function in multiple antioxidant pathways was observed (e.g., metallothionein genes), indicating that damage caused by oxidative stress may be amplified by the absence of a functional antioxidant response. Decreased expression of antioxidant genes (e.g., catalase gene) has previously been shown to occur following cytokine stimulation (13). The increased expression of cytokines observed in this study may therefore be responsible for the changes observed in antioxidant expression. Genes controlling cellular proliferation and apoptosis were also significantly regulated during JFH-1 infection, supporting evidence from recent studies of an increase in proapoptotic and growth arrest signaling during HCV infection (26, 53). These findings indicate that the hepatocyte cell death frequently observed in patients may result from a virally driven process of apoptosis in addition to the proapoptotic actions of cytotoxic immune cells (19).
By comparing our study to a recent study on J6/JFH-1 infection of Huh7.5 cells (53), we observed differences in the transcriptional regulation of host genes involved in the antioxidant response and the induction of interferon response genes, indicating that RIG-I signaling in Huh7 cells may be important for the regulation of these genes. However, there were fewer differences in the expression patterns of genes that control cytokine and chemokine production and cellular survival, indicating that RIG-I signaling may be less important in controlling these host cell responses to infection.
In this study, host genes involved in cellular metabolism and intracellular transport were also significantly regulated by JFH-1 infection. Although previous studies have shown that HCV can alter lipid metabolism (32), this study demonstrated that genes involved in protein synthesis and degradation, posttranslational modification, vesicle trafficking, and cytoskeleton function are also regulated following infection.
Lipids play an important role in HCV infection: cholesterol, sphingolipids, and phospholipids are key components of the membranous web structure, and fatty acids are required for the formation of lipid droplet virus assembly sites (30, 55). Consistent with this, genes controlling the synthesis and transport of sphingolipids, phospholipids, and fatty acids increased in expression following infection. Other studies of HCV-infected chimpanzee and replicon-transfected cells have previously demonstrated increased expression of fatty acid and sphingolipid synthesis genes (32, 46). Expression of HCV core protein also increased the expression of genes encoding lipogenic enzymes and key transcription factors (e.g., SREBP1 gene) (23). Transcriptional regulation of lipid metabolism may therefore result from the direct effects of individual HCV proteins.
Many of the lipid metabolism genes upregulated in this study are targets of the PPARα transcription factor (40). One of these genes, the TXNIP gene, which functions as a negative feedback inhibitor of PPARα (33), was shown to be essential for HCV replication and secretion by siRNA silencing. A recent study demonstrating that agonists of PPARα suppress the replication of HCV indicated that the inhibition of PPARα by TXNIP may be important for HCV infection (32). Increased expression of TXNIP also occurs when intracellular glucose levels are high (14). A recent siRNA screen for host factors involved in JFH-1 HCV replication demonstrated that silencing MXLIPL, a glucose-responsive transcription factor that induces TXNIP expression, also significantly reduced JFH-1 replication. This further indicates that expression of TXNIP may be important during the HCV replication cycle (27). Another PPARα target gene upregulated during infection was the CYP1A1 gene, also known for its role in the AHR detoxification pathway (22, 40). In this study, siRNA silencing of CYP1A1 gene expression reduced HCV replication and secretion. Activation of CYP1A1 gene expression, either through the AHR pathway or through PPARα, has been shown to control intracellular lipid metabolism (40, 45). The requirements for CYP1A1 expression during HCV infection may therefore be related to its ability to regulate important lipid metabolites required during the HCV replication cycle.
Another lipid metabolic pathway regulated by infection in this study was the synthesis of cholesterol. Genes involved in the geranylgeranylation of host proteins, a process controlled by the mevalonate cholesterol pathway, were upregulated following infection, consistent with the role of geranylgeranylated proteins in HCV replication (54). However, the majority of genes controlling cholesterol synthesis actually decreased in expression following infection. Reduced cholesterol levels have also been observed in HCV patient serum (16), and other RNA viruses, including measles virus, have also been shown to reduce cholesterol synthesis during infection (42). In contrast, a recent study investigating replicon-transfected Huh7.5 cells demonstrated an increase in the expression of cholesterol synthesis genes (32). The disparity in these results may reflect the reduced interferon response in the Huh7.5 cells used in the replicon study. MicroRNA miR122, which positively regulates cholesterol biosynthesis, is downregulated by the beta interferon pathway (17, 36). The absence of a functional interferon signaling pathway in Huh7.5 cells may prevent the downregulation of miR122, enabling expression of cholesterol synthesis genes.
Other metabolic processes transcriptionally regulated during HCV infection in this study included protein synthesis, protein turnover, and posttranslational protein modification. HCV has not previously been shown to regulate these processes, but other viruses, such as HIV, have been shown to boost protein synthesis and to utilize protein sulfation for virus protein modification (6, 9). The glycosylation enzyme MGAM, which was upregulated in this study, is known to be important for HCV structural protein processing (11), indicating that the key enzymes required during infection may be upregulated. The coordinated regulation of functionally related genes, such as the tRNA synthetase genes, also indicates that key transcription factors regulating these pathways may be activated.
Recent studies have demonstrated that host proteins regulating intracellular transport are important for the HCV replication cycle (7, 20, 25). In this study, host genes regulating vesicle trafficking between the ER, Golgi body, and plasma membrane were upregulated, indicating that these genes may be important for viral entry and exit and the construction of virus replication complexes. An increase in the expression of genes encoding components of the endocytic machinery (SNX12 and SNX16 genes) was observed and may be important for the entry of HCV by clathrin-dependent endocytosis. Increased expression of RAB6B and RAB33B, which control retrograde transport from the Golgi body to the ER, may facilitate the accumulation of virus proteins during replication complex formation (34, 50). Increased expression of RAB33B may also be required, as it plays a role in autophagy, which was recently shown to be important for HCV replication and assembly (21, 48). An increase in the expression of the secretory vesicle membrane proteins RAB27A, RAB27B, and RAB37, usually expressed only in cells requiring high levels of secretion (29, 35), indicates that these factors may facilitate viral exit. Evidence to support the role of these genes in viral secretion was obtained in the siRNA silencing experiments performed in this study, in which the knockdown of RAB27A and RAB27B had more significant effects on JFH-1 viral secretion than on JFH-1 genome replication.
In this study, we have demonstrated that JFH-1 infection has widespread effects on the expression of host genes involved in cellular defense mechanisms, metabolism, and intracellular transport. By comparing our findings to those from previous expression profiling studies, we have provided further evidence to support the expression patterns already identified. However, we have also characterized a number of novel host genes transcriptionally regulated during infection that may act as important factors for the HCV replication cycle. The gene expression patterns identified may also provide insight into some of the underlying causes of HCV-associated liver pathology frequently observed in chronically infected patients. Analysis of the expression patterns of host genes affected by HCV at the protein level and investigation of changes to host gene expression in vivo in HCV-infected liver tissue from patients will help to demonstrate the roles of these genes in HCV replication and pathogenesis.
We thank Charles Rice for provision of the Huh7.5 cell line and the Jc1p7Gluc2A plasmid and Takaji Wakita for the pJFH-1 plasmid.
This work was supported by GlaxoSmithKline and the Biotechnology and Biological Sciences Research Council UK.
Published ahead of print on 3 March 2010.
†Supplemental material for this article may be found at http://jvi.asm.org/.