Metabolomic profile of hepatitis C virus-infected hepatocytes

Abstract

Hepatitis C virus (HCV) is capable of disrupting different facets of lipid metabolism and lipids have been shown to play a crucial role in the viral life cycle. The aim of this study was to examine the effect HCV infection has on the hepatocyte metabolome. Huh-7.5 cells were infected using virus produced by the HCV J6/JFH1 cell culture system and cells were harvested 24, 48, and 72-hours following infection. Metabolic profiling was performed using a non-targeted multiple platform methodology combining ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS(2)) and gas chromatography/mass spectrometry (GC/MS). There was a significant increase in a number of metabolites involved in nucleotide synthesis and RNA replication during early HCV infection. NAD levels were also significantly increased along with several amino acids. A number of lipid metabolic pathways were disrupted by HCV infection, resulting in an increase in cholesterol and sphingolipid levels, altered phospholipid metabolism and a possible disruption in mitochondrial fatty acid transport. Fluctuations in 5'-methylthioadenosine levels were also noted, along with alterations in the glutathione synthesis pathway. These results highlight a number of previously unreported metabolic interactions and give a more in depth insight into the effect HCV has on host cell biochemical processes.

Figure 1. Altered lysophospholipid metabolism.

Bar charts show the scaled ion intensity for (A) inositol-based lysophospholipids, and (B) Inositol-based lysophospholipid metabolites in HCV-infected cells (black bars) and mock-infected cells (white bars) 24, 48, and 72 hours post-infection. An asterisk (*) denotes a statistically significant difference with p<0.05. Data are presented as the mean of three separate experiments with error bars representing standard deviation. CM, conditioned media.

Figure 2. Increased cholesterol and sphingoid base production.

Bar charts show the scaled ion intensity for (A) lathosterol, (B) cholesterol, (C) sphingosine, and (D) sphinganine in HCV-infected cells (black bars) and mock-infected cells (white bars) 24, 48, and 72 hours post-infection. An asterisk (*) denotes a statistically significant difference with p<0.05. Data are presented as the mean of three separate experiments with error bars representing standard deviation. CM, conditioned media.

Figure 3. Disruption in fatty acid metabolism during HCV infection.

Fatty acid oxidation pathway and bar charts showing the scaled ion intensity for coenzyme A, pantothenic acid, and carnitine derivatives in HCV-infected cells (black bars) and mock-infected cells (white bars) 24, 48, and 72 hours post-infection. Increased fatty acid concentration in HCV-infected cells 72 hours post-infection is also depicted. An asterisk (*) denotes a statistically significant difference with p<0.05. Data are presented as the mean of three separate experiments with error bars representing standard deviation. CM, conditioned media.

Figure 4. Schematic representation of MTA fluctuations during HCV infection.

‘↑’ indicates that the mean metabolite level was significantly higher (p<0.05) in HCV-infected cells compared to control. ‘↓’ indicates that the mean metabolite level was significantly lower (p<0.05) in HCV-infected cells compared to control. ‘–’ indicates that there was no change in the mean metabolite level between HCV-infected and mock-infected cells. Metabolites coloured in grey were not measured during the analysis.