Gene expression profiling indicates the roles of host oxidative stress, apoptosis, lipid metabolism, and intracellular transport genes in the replication of hepatitis C virus

Abstract

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.

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 expression were calculated by comparing gene expression in JFH-1-infected Huh7 cells to that in mock-infected Huh7 cells at 6, 12, 18, 24, and 48 h postinfection. Genes regulated by JFH-1 infection were involved in either host defense mechanisms (top) or cellular metabolism and transport (bottom). Genes shown in red were upregulated, genes shown in green were downregulated, and genes in black showed no change in expression in JFH-1-infected cells compared to mock-infected cells. Expression profiles are represented on a 2-fold-change scale. Fold change values were calculated from the averages of the expression values collected for triplicate samples at each time point.

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 with JFH-1 to that in mock-infected cells. Fold changes in gene expression were calculated for genes involved in host defense mechanisms (A) and in cellular metabolism, vesicle trafficking, and transport (B). For the majority of the genes shown, expression was quantified in cells infected for a total of 48 h. The only exception was the ABLIM3 gene, which was quantified at 24 h postinfection. The qRT-PCR data shown represent the means ± standard deviations (SD) for triplicate infections (white bars). The corresponding microarray gene expression data are also shown (gray bars). The microarray data for some genes relied on single probe sets, and therefore standard deviations are not displayed. The P value for each qRT-PCR data point was calculated to be <0.05 by an unpaired t test.

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 gene family (microarray data) (right). Fold changes in gene expression are shown for the qRT-PCR data (open symbols) and the corresponding microarray data (closed symbols). Metallothionein genes are represented as follows: triangles, MT2A gene; diamonds, MT1M gene; squares, MT1F gene; and circles, MT1E gene. The means for triplicate infections are shown for the qRT-PCR and microarray data.

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 treated for 48 h with siRNA molecules targeting the expression of selected host genes were infected with JFH-1. A pool of two siRNA molecules was used for each target. At 48 h postinfection, total RNA was extracted and used in qRT-PCR for quantification of target mRNA (top) and JFH-1 RNA (middle). GAPDH expression was used to normalize the gene expression data. The expression data collected for the siRNA-treated samples were compared to those for cells treated with a nontargeting siRNA control. An siRNA molecule targeting the HCV IRES sequence was used as a positive control for the experiment. Values shown represent the means ± SD for duplicate samples in three separate experiments. P values for the qRT-PCR data were calculated to be <0.05 by a two-tailed unpaired t test. (Bottom) Medium collected from the siRNA transfection/JFH-1 infection experiments was used to infect a monolayer of Huh7 cells grown in 96-well plates. At 48 h postinfection, the cells were stained for HCV core protein and visualized by fluorescence microscopy. Focus-forming units were counted for each well, and a comparison was made between the wells infected with medium from target-specific siRNA transfections and those with medium from the nontargeting siRNA transfections. Values shown represent the means ± SD for duplicate wells for all samples collected in the three siRNA experiments. P values for the core staining data were calculated to be <0.05 by an unpaired t test.

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 of selected host genes were infected with luciferase-tagged Jc1. At 48 h postinfection, luciferase activity was determined. Luciferase data collected for the siRNA-treated samples were compared to those for cells treated with a nontargeting siRNA control. An siRNA pool targeting a host factor, DDX3X, was used as a positive control for the experiment. Values shown represent the means ± SD for four replicate samples in three separate experiments. P values for the luciferase data were calculated to be <0.05 by an unpaired t test.