Centrifugal enhancement of hepatitis C virus infection of human hepatocytes

Abstract

Hepatitis C virus (HCV) is a human pathogen associated with chronic liver disease. Recently, the cell culture systems supporting complete replication and production of HCV genotype 2a (JFH1) have been established. This study investigated the effect of low-speed centrifugation on HCV JFH1 infection of human hepatocytes (Huh7.5.1). Higher levels of HCV RNA expression were observed in Huh7.5.1 cells infected with centrifugal inoculation of HCV JFH1 than those in the control cells. This increased HCV RNA expression was associated with the elevated expression of HCV NS3 protein in the hepatocytes. The centrifugal enhancement of HCV infection was time and speed dependent. However, the enhancement was not observed when centrifugation was performed before or after HCV infection. In addition, there was no association between centrifugal enhancement and the expression of HCV entry receptors (CD81 and claudin-1) and intracellular IFN-alpha in the hepatocytes. These data indicate that centrifugal inoculation is a useful tool for increasing the efficiency of HCV infection and replication in the target cells in vitro.

Fig. 1

Effect of centrifugation on HCV JFH1 infection. Huh7.5.1 cells were incubated with HCV JFH1 for 90 min with or without centrifugation (500×g; 1000×g). Total cellular RNA was collected at the indicated time points and subjected to real time RT PCR for HCV and GAPDH mRNA. Data are expressed as HCV RNA levels relative (fold) to control (without centrifugation), which is defined as 1.0. The results shown are the mean ± SD of three independent experiments (*, P<0.05; **, P<0.01, centrifugation vs. control).

Fig. 2

Effect of centrifugation time on HCV JFH1 infection. Huh7.5.1 cells were incubated with HCV JFH1 with or without centrifugation (1000×g) for the indicated times. Total cellular RNA was collected at day 2 and day 6 post infection and subjected to real time RT PCR for HCV and GAPDH mRNA. Data are expressed as HCV RNA levels relative (fold) to control (without centrifugation), which is defined as 1.0. The results shown are the mean ± SD of three independent experiments (*, P<0.05; **, P<0.01, centrifugation vs. control).

Fig. 3

Western blot analysis of HCV NS3 protein expression in HCV JFH1-infected Huh7.5.1 cells. Huh7.5.1 cells were incubated with HCV JFH1 for 120 min with or without centrifugation (500×g; 1000×g). At day 6 post infection, equal amount of proteins extracted from infected cells with or without centrifugation were subjected to Western blot assay using antibodies against HCV NS3 and actin, respectively. The numbers in the right panel are the signal intensities of protein bands of the Western blot expressed as densitometry scanning units (DSUs). One representative of three experiments with similar results is shown.

Fig. 4

Effect of centrifugation on CD81, claudin-1 and IFN-α expression in Huh7.5.1 cells. (A) Total cellular RNA was collected from Huh7.5.1 cells with or without centrifugation (1000×g) for 120 min immediately after centrifugation and subjected to real time RT PCR for CD81, claudin-1, IFN-α and GAPDH mRNA. Data are expressed as mRNA levels relative (%) to control (without centrifugation), which is defined as 100. The results shown are the mean ± SD of three independent experiments. (B) Western blot analysis of CD81 and claudin-1 proteins in Huh7.5.1. Equal amount of proteins extracted from the cells with or without centrifugation immediately after centrifugation were subjected to Western blot assay using antibodies against CD81, claudin-1 and actin. The numbers in the right panel are the signal intensities of protein bands of the Western blot expressed as densitometry scanning units (DSUs). One representative of these experiments with similar results is shown.

Fig. 4

Effect of centrifugation on CD81, claudin-1 and IFN-α expression in Huh7.5.1 cells. (A) Total cellular RNA was collected from Huh7.5.1 cells with or without centrifugation (1000×g) for 120 min immediately after centrifugation and subjected to real time RT PCR for CD81, claudin-1, IFN-α and GAPDH mRNA. Data are expressed as mRNA levels relative (%) to control (without centrifugation), which is defined as 100. The results shown are the mean ± SD of three independent experiments. (B) Western blot analysis of CD81 and claudin-1 proteins in Huh7.5.1. Equal amount of proteins extracted from the cells with or without centrifugation immediately after centrifugation were subjected to Western blot assay using antibodies against CD81, claudin-1 and actin. The numbers in the right panel are the signal intensities of protein bands of the Western blot expressed as densitometry scanning units (DSUs). One representative of these experiments with similar results is shown.