JFH-1 infection regulates Huh7 cell gene expression.
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 . 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
). 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).
Genes significantly regulated during JFH-1 infection
Gene ontology analysis.
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.
JFH-1 infection alters the expression of host genes involved in cellular defense mechanisms.
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. 1. Host genes regulated by JFH-1 infection. The expression profiles of host genes significantly regulated (≥2-fold; P ≤ 0.05) following infection of Huh7 cells with JFH-1 were characterized using microarray analysis. Fold changes in gene (more ...) (i) JFH-1 infection stimulates the expression of proinflammatory antiviral response genes.
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).
(ii) JFH-1 infection regulates the expression of cellular antioxidant response 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).
(iii) JFH-1 infection regulates the expression of genes controlling cellular survival.
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.
JFH-1 infection alters the expression of genes involved in metabolism and transport.
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. .
(i) JFH-1 infection regulates host genes controlling intracellular lipid metabolism.
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).
(ii) JFH-1 infection regulates the expression of genes involved in host protein synthesis, degradation, and posttranslational modification.
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).
(iii) JFH-1 infection regulates genes controlling intracellular protein transport and vesicle trafficking.
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).
(iv) JFH-1 infection regulates the expression of genes encoding actin and microtubule binding proteins.
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.
qRT-PCR investigation of host gene expression.
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. . 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. . 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. ). 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. ). 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. ). 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. 2. Quantitative PCR investigation into host gene expression. qRT-PCR was used to investigate the effect of JFH-1 infection on Huh7 cell gene expression. Fold changes in host gene expression were calculated by comparing gene expression in Huh7 cells infected (more ...)
FIG. 3. Temporal regulation of host gene expression. qRT-PCR quantification of host gene expression at 6, 12, 18, 24, and 48 h post-JFH-1 infection was performed for TXNIP, ABLIM3, RAB27A, the metallothionein gene family (qRT-PCR data) (left), and the metallothionein (more ...) siRNA silencing identifies host genes with a key role in the HCV replication cycle.
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. (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%.
FIG. 4. (Top two panels) Effect of host gene silencing on JFH-1 replication and secretion. siRNA silencing of host gene expression was used to investigate the role of TXNIP, CYP1A1, ABLIM3, RAB33B, RAB27A, RAB27B, and RAB40B during JFH-1 replication. Huh7 cells (more ...)
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. , 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. . 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.
FIG. 5. Effect of host gene silencing on Jc1 replication. siRNA silencing of host gene expression was used to investigate the role of TXNIP, CYP1A1, and ABLIM3 during Jc1 replication. Huh7.5 cells treated for 48 h with siRNA molecules targeting the expression (more ...)