Stable transfectants in HepG2 cell lines expressing core and HCV structural proteins have been described by Harada et al., 1995 35. Stable transfectants of Chang Liver (CHL) cell lines expressing HCV core (CH39) or core-E1-E2-NS2-NS3 (CH352) were established by stable transfection of Chang Liver cells with pCAG39neo, pCAG352neo expression vectors respectively, that expressed HCV structural and non-structural proteins under the control of mammalian CAG promoter. Clonal selection of liver cell transfectants was performed in the presence of 1 mg/ml of G418 antibiotic. Expression levels of HCV proteins were monitored by Western blotting. Positive clones were cultured and maintained under G418 selection; however experiments were performed in G418-free medium to avoid the possible influence of the antibiotic on functions under study as well as to maintain homologous conditions with the parental and control cell lines. Experimental control cell lines, Hepswx and CHwt were transfected with the empty vectors, pcEF321swxneo, described elsewhere by Harada et al. 35 and pcCAGswxneo. This latter plasmid obtained by inserting the CAG promoter cassette in pEF321neo vector linearized by Asp718 digestion; is a derivative of pCAGGS 36, containing the unique Swa I cloning site.

The pCI-NF90 construct (courtesy of Prof. Kumar A, George Washington University) containing the NF90 sequence under the control of CMV promoter has been described 34. Transfection of the established HepG2 and CHL cell lines with pCI-NF90 was performed by using Fugene 6 (Roche) according to manufacturer's instructions and with Fugene 6/plasmid DNA ratio of 3:1.

Detection and quantitation of RANTES protein expression in parental and transfected cell lines was performed by intracellular immunofluorescence and FACS analysis. Briefly, sub-confluent cultures were detached with trypsin-EDTA solution and washed in PBS. After fixation for 20 minutes at room temperature in 4% paraformaldehyde and washing with permeabilization solution (Perm/Wash Buffer, Becton Dickinson), 10⁶ cells were suspended in 0.5 μg of mouse anti-human RANTES phycoerythrin-conjugated monoclonal antibody in Perm/Wash Buffer and were incubated for 15 minutes at room temperature. After washing in Perm/Wash Buffer cells were analysed by FACScalibur flow cytometer using CellQuest software. A minimum of 10,000 events were analysed for each sample.

To detect RANTES mRNA, total RNA was extracted from cultured cell lines by RNeasy kit (Qiagen) according to manufacturer's instructions. 500 ng of total RNA was reverse transcribed using random hexanucleotides and 10 U of transcriptor™ reverse transcriptase (Roche) according to manufacturer's instructions with minor modifications and with the addition of recombinant RNAsin ribonuclease inhibitor. The PCR amplification of the reverse transcribed RNA was carried out with 1.5 unit of Taq DNA polimerase (ABI Prism) in 50 μl reaction mixture containing 200 μM of each dNTPs, 5 mM MgCl₂, 20 μM of specific primers and 1× reaction buffer. Primers used for RANTES mRNA amplification in HepG2 cell lines were 5'-GCTGTCATCCTCATTGCTAC-3' (sense) and 5'-TCCATCCTAGCTCATCTCCA-3' (antisense), which yield a specific 260 (237) bp product; cycling conditions for HepG2 transfected cell lines were 94°C, 5 minutes, 56°C, 90 seconds and 72°C, 120 seconds, followed by 28 cycles at 94°C, 30 seconds, 56°C for 90 seconds and at 72°C for 120 seconds. Primers used to amplify RANTES from Chang Liver cell lines were: 5'-ACGCCTCGTGTCATCCTCATT-3' (sense) and 5'-ACTCTCCATCCTAGCTCATCTC-3' (antisense), which amplify a (226) bp product. PCR conditions were same as those described for HepG2 except MgCl₂ concentration reduced to 1.5 mM and primers concentration to 0.1 μM. Cycling conditions were modified as follows: 94°C 4 minutes, 94°C 1 minute, 55°C 1 minute, 72°C 1 minute, for 40 cycles followed by 72°C 8 minutes. β-actin amplification in parallel samples was performed as controls by using following primers: 5'-ATCTGGCACCACACTTCTACA-3' (sense) and 5'-GTTTCGTGGATGCCAACAGGACT-3' (antisense) (550 bp product). Cycling conditions for β-actin were 94°C 4 minutes, 94°C 1 minute, 50°C 1 minute, 72°C 1 minute, followed by elongation at 72°C 8 minutes. In each experiment, possible DNA contamination was determined by a control reaction in which reverse transcriptase was omitted from the reaction mixture. For semi-quantitative RT-PCR analysis of RANTES, serial dilution of the cDNA samples was used. Amplified PCR fragments were analysed by electrophoresis in a 2% agarose gel.

Real-Time quantitative PCR assessment of RANTES gene expression in Hep39 cell lines was performed using the ABI PRISM 7000 Sequence Detector system (Applied Biosystem). 800 nanograms (ng) of total RNA were in vitro reverse transcribed as described above for RT-PCR analysis. A portion of the resulting cDNA corresponding to 80 ng of in vitro transcribed RNA was subjected to Real-Time quantitative PCR that was performed in triplicate using TaqMan chemistry with primers and probe sets from the Assay-on-Demand list (Applied Biosystem). The standard curve of the target RANTES gene was compared to the standard curve of β-microglobulin gene used as endogenous control reference; calculation of the slope of the log (ng RNA) vs ΔCt was always <0.1. Fold reduction was then calculated by the ΔΔCt method after normalization of the mRNA level to the values of β-microglobulin.

2.5 μg of human RANTES reporter construct, pRANTES-Luc, (courtesy of Dr. Michael Gale), was transiently transfected by Fugene6 (Roche) in HepG2 and Chang Liver cell lines constitutively expressing HCV core protein, as well as in parental and the empty vector transfected control cells. To examine the effect of NF90 on RANTES activity 2.5 μg of the pCI-NF90 plasmid DNA was co-transfected with pRANTES-Luc into the liver cell lines. As transfection control, pSV β-galactosidase vector (1.0 μg) was co-transfected into the cells to monitor transfection efficiency. 48 h post-transfection, cell lysate was prepared and luciferase quantifications were performed using commercial reagents (Promega) and measuring in a Lumat LB 9501 Berthold Luminometer. Results are expressed as relative light units (RLU), calculated as the ratio of luciferase to β-galactosidase activities within each sample.

Total proteins (50 μg) were diluted in 2× sample buffer, separated by 10% SDS-PAGE and transferred onto PVDF membranes. After incubation in blocking buffer containing 5% milk, membranes were incubated overnight at 4°C with specific polyclonal antibodies anti-IRF-1 or IRF-7 (Santa Cruz). The membranes were subsequently incubated with HRP-conjugated anti-rabbit IgGs and immunodetection was realized by ECL (Pierce) followed by autoradiography.

The Student's t test and Fisher's exact test were used for comparison of the difference in RANTES expression between Hep39 and CH39 and their relative controls Hepswx and CHWT as well as to compare the luciferase activity of RANTES promoter in the two cell lines expressing HCV core protein. Differences were considered significant when p values were <0.05. Statistical analyses were performed using SPSS software (SPSS Inc).

Considering the important role of chemokine signaling in regulating physiological events associated with chronic HCV infection, the effect of HCV core protein on RANTES expression was evaluated by quantitative immunofluorescence and flow cytometry in the two liver derived cell lines, HepG2 and Chang Liver, constitutively expressing HCV core protein. Results shown in Figures 1A and 1B indicated a twofold decrease in fractions of cells expressing RANTES protein for cells transfected with HCV core protein, as indicated by comparing percentage of positive HepG2 cell line controls (93.77% ± 0.8%) or the cells transfected with the empty vector (65.63% ± 1.1% in Hepswx) with the HCV core protein expressing cells (45% ± 2.6% in Hep39). Reduction in RANTES expression was detected also in Hepswx cells as compared to the parental cell line, suggesting a possible contribution of constitutive transfection on RANTES level. However the differences in percentage of cells positive for RANTES expression in Hep39 as compared to Hepswx cell lines was statistically significant (p < 005). However in Hep352 cell line (expressing core-E1-E2-NS3 proteins) percentage of RANTES positive cells (78,5 ± 1.5%) was close to that in control cell lines. In contrast, flow cytometry quantitation of RANTES protein in CH39 cell lines showed (Figures 2A and 2B) a statistically significant 2.5 fold increase in percentage of cells positive for RANTES expression, as compared with the CHWT control cells (16.6% ± 2.9% vs. 6.7% ± 1.2%, p < 0.05). In CH352 cell lines the fraction of RANTES positive cells (7.5% ± 0.7%) was similar to the controls. This latter result and that from Hep352 cells suggest a possible role of HCV structural E1 and E2 proteins or non-structural proteins NS2-NS3 in counteracting the effect of the core protein in RANTES induction. The results described above suggest that the effect of HCV core protein on the expression of RANTES may be regulated differently in liver cells derived from transformed hepatocytes and the non-transformed fibroblast cell lines.

We analysed RANTES mRNA expression in the liver cell lines by RT-PCR using specific primers. Results showed (Figures 3 and 4) expected size band of RANTES cDNA, 257 and 268 bp respectively in HepG2 and in Chang Liver cell lines. Using similar quantity of cDNA (500 ng) for each sample from PCR amplification we made serial dilutions of RANTES cDNA to demonstrate that RANTES transcripts levels were decreased in Hep39 cell lines compared to the controls (Hepswx and HepG2) (Figure 3(A) upper panel). In order to corroborate these results, Real-Time quantitative PCR analysis was performed to determine relative RANTES mRNA levels in Hepswx and Hep39 cell lines. Results shown in Fig 3(B) indicated that in Hep39 cell lines RANTES mRNA level was 50% less than in Hepswx cell lines. Conversely, RANTES transcripts levels were increased by fourfold in CH39 cell lines, expressing HCV core protein (Figure 4), as compared to the control CHWT, and up to eightfold compared to parental CHL cells (Figure 4, upper panel). These results are consistent with the data presented above which examined the effect of core on RANTES protein expression, and suggest that HCV core protein affects RANTES chemokine expression in liver cell lines differentially.

We asked whether the modulation in RANTES expression by HCV core protein could be regulated at the transcriptional level. We carried out transient co-transfections with RANTES promoter-luciferase plasmid DNA in HepG2 and Chang Liver cell lines constitutively expressing core and core-E1-E2-NS2-NS3 proteins of HCV. Inhibition of RANTES promoter activity (Figure 5A) was observed in two different clones of Hep39 cells, with fourfold reduction (in RLU values) detected in Hep39 cells as compared to Hepswx cell lines. Interestingly, in Hep352 cell line a 3,5 fold induction of RANTES promoter was detected in comparison to Hep39 cell line that suggested a counteracting effect of the structural E1-E2 proteins or non-structural proteins NS2-NS3 toward the core protein inhibition on RANTES promoter also in HepG2. Conversely, in CH39 cells about twofold induction of RANTES promoter was detected (Figure 5B). The differences in RLU values among the cell lines in comparison to the controls were statistically significant according to the p value (<0.05) in a t Test's. Consistent to RANTES protein expression in CH352 the promoter of RANTES was not induced as in CH39, suggesting that the E1-E2 structural proteins or non-structural proteins NS2-NS3 may counteract the effect of the core protein on RANTES promoter activity.

These results are consistent with the effect of HCV core protein on RANTES mRNA levels and suggest that the core protein regulates RANTES expression by influencing its promoter activity.

It has been reported that interferon response factors (IRF-1, -3, -7) influence RANTES promoter activity and RANTES expression in different cell lines 2837. We examined IRF-1 and -7 expression in the two liver cell lines (HepG2 and CHL) in order to correlate differential expression of the IRFs with the differences in RANTES induction between HepG2 and Chang Liver cells mediated by HCV core protein. The results of immunoblotting analysis (Figure 6) indicate that IRF-7 was equally expressed in either Hepswx and parental CHL cell lines (Figure 6, lower panel, lanes 1 and 3, respectively) and IRF-7 expression and phosphorylation were unaffected by the HCV core protein in the two cell lines under study, since no differences in the pattern of IRF-7 bands were detected in Hep39 or in CH39 cell transfectants (Figure 6 lower panel, lanes 2 and 4, respectively). With regard to IRF-1 expression, results of immunoblotting analysis indicated increased IRF-1 level in Hep39 cell lines (Figure 6, upper panel, lane 3); in contrast, IRF-1 was barely detectable in Chang Liver cell transfectants (Figure 6, lanes 5–7).

These results suggest that stable expression of HCV core protein induces IRF-1 expression in human hepatoblastoma cell line. Differential basal expression of IRF-1 between HepG2 and Chang Liver cell could be, at least in part, responsible for differential modulation of RANTES.

Since NF90 is known to induce the transcriptional program of IFN response genes which is partly responsible for its interference with virus replication, we sought to examine whether the expression of NF90 affected the modulation of RANTES promoter in HCV core protein expressing cell lines as part of the anti-viral effect of NF90. The results in Chang Liver cells showed that NF90 countered the RANTES promoter activation mediated by the core protein, either in CH39 (1.5 times reduction as compared to NF90 controls, Figure 7), or in CH352 cells (the NF90-mediated inhibition was about 7 folds, Figure 7, right panel). In HepG2 cells or in Hepswx cell lines, the effect of NF90 on RANTES promoter was irrelevant (Figure 8). In Hep39 cells however, co-transfection with NF90 increased RANTES-promoter luciferase activity by twofold (Figure 8, right panel). The differences observed were statistically significant according to the t Test's (p < 0.05). The results suggest that NF90 expression could reverse the effect of HCV core protein on RANTES promoter either in HepG2 or in Chang Liver cell lines.