Figure 1.
HCV infection inhibits mRNA expression of C2 complement component.
(A) Real-time PCR analysis for C2 mRNA expression from HCV infected IHH and mock infected control cells. Results were normalized to endogenous 18S RNA. Asterisks denote: *p<0.05; **p<0.01; ***p<0.001 when compared to controls. (B) Real-time PCR analysis displaying HCV genotype 1a infection represses C2 expression in chronically infected liver biopsy specimens from patients (n = 12). Liver biopsy samples are each marked by 3-digit numbers at the bottom. Results are compared with non-HCV infected 3 control liver tissues arbitrarily set at 1. The error bars in individual samples are shown as variations form triplicate assays. All samples used in this study were significantly inhibited (p<0.01) as compared to negative controls.
Figure 2.
C3 convertase level reduces in HCV infected patient sera.
(A) Comparison of serum C3 convertase level in randomly chosen HCV infected patient sera (n = 12) and normal human sera (NHS) from healthy donors (n = 12) using a commercially available ELISA kit. (B) HCV infected patient sera (n = 12) and NHS (n = 12) were subjected to analysis for comparison of C3b deposition on E. coli surface in the presence or absence of purified C3 protein. Asterisks denote: *p<0.05; **p<0.01; ***p<0.001 when compared to control. (C) Complement mediated lysis was inhibited in HCV infected patient sera. Normal sera (n = 6) and HCV infected patient sera (n = 6) were incubated with sheep RBC after treatment with EDTA. Sera from each test group were studied, with lysis of erythrocytes completed using EDTA treated guinea pig serum. (D) Sheep RBCs were incubated with sera in the presence or absence of anti-factor B antibody to separate out alternate pathway mediated lysis from classical antibody mediated lysis, and lysis was carried out as described above.
Figure 3.
Factor H and Factor I expression are impaired in HCV infected patient sera.
Real-time PCR analysis from HCV genotype 1a infected liver biopsy specimens from patients (n = 12) exhibiting Factor H (A) and Factor I (B) expression status are shown. Results were compared with non-HCV infected control liver RNAs (n = 3) arbitrarily set at 1. The error bars in individual samples are shown as variations form triplicate assays. Increase of Factor H was significant (p<0.05 in #186, 171, and 181; p<0.01 in #158, 173, and 165) as compared to control liver. Factor I inhibition was significant (p<0.05) in all tested samples.
Figure 4.
iC3b level decreases in HCV infected patient sera.
(A) Undiluted NHS were incubated at room temperature for 1, 3, and 6 h. Purified C3 protein (50 µg/ml) was added to preincubated serum at 1∶10 dilution at 37°C for 30 min. The α2'iC3b protein band was detected in samples by Western blot analysis. (B) Comparison of serum iC3b level in NHS (n = 12) and HCV infected patient sera (n = 12) using a commercially available ELISA kit. (C) NHS and HCV infected patient sera incubated with purified C3 protein (50 µg/ml) at 1∶5 and 1∶20 dilution. The protein band representing α2'iC3b was detected by Western blot and a representative figure is shown. (D) Densitometry analysis for α2'iC3b protein band intensity was done by ImageJ software after repeated experiments with NHS (n = 6) and HCV infected sera (n = 6).
Figure 5.
Schematic presentation of complement regulation by HCV.
HCV infection inhibits C4 and C3 components expression [3], [4] as well as C2 production (current study). Reduction of C4 and C2 synthesis by HCV affects C3 convertase formation in classical pathway, which results in the inhibition of C3 cleavage. Limited C3b generation affect C3b deposition onto pathogenic surface, and impairs another C3 convertase formation of alternative pathway and C5 convertase formation in both pathways. Together with the reduction of membrane attack complex formation [5], the impairment of C3 convertase activity may be a key event for HCV persistence. CP = classical pathway, AP = alternate pathway.