Establishment of Hepatitis C Virus RNA-Replicating Cell Lines Possessing Ribavirin-Resistant Phenotype

<div><p>Background</p><p>Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells.</p><p>Methodology/Principal Findings</p><p>By repetitive RBV (100 μM) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 μM). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor.</p><p>Conclusions/Significance</p><p>These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.</p></div

Base substitutions occurring in genome-length HCV RNAs during long-term cell culture.

<p>Base substitutions were counted by comparison with the sequence of genome-length HCV RNA (ON/C-5B/QR,KE,SE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091156#pone.0091156-Kato5" target="_blank">[20]</a>).</p><p>Average numbers of base substitutions per cDNA clone are shown.</p><p>Ts: Transition; Tv: Transversion; dN: Nonsynonymous; dS: Synonymous.</p

HCV Core is targeted to lipid droplets even in the ESCRT knockdown cells.

<p>The RSc cells transduced with a control lentiviral vector (Con), the TSG101 knockdown (TSG101i), the Alix knockdown (Alixi), the Vps4B knockdown (Vps4Bi), or the CHMP4b knockdown (CHMP4bi) cells were infected with HCV-JFH1. Cells were fixed 60 hrs post-infection and were then examined by confocal laser scanning microscopy. Cells were stained with anti-HCV Core (CP-9 and CP-11 mixture) and were then visualized with Cy3 (red). Lipid droplets and nuclei were stained with BODIPY 493/503 (green) and DAPI (blue), respectively. Images were visualized using confocal laser scanning microscopy. Colocalization is shown in yellow (Merged).</p

Genes differentially expressed in the RBV-resistant cell lines compared to those in the parental OL8(3.5Y) cell line.

<p>(A) Genes whose expression levels were upregulated (left panel) or downregulated (right panel) at ratios of more than 4 or less than 0.25 in the case of each RBV-resistant cells versus OL8(3.5Y) cells were selected. Five genes upregulated and six genes downregulated in all three comparisons were obtained. (B) Expression profiles of representative genes upregulated or downregulated commonly among the RBV-resistant cells. Quantitative RT-PCR analysis was performed using the total RNAs prepared from the cells treated with (gray bar) or without (black bar) 50 μM RBV for 3 days. The data are expressed as the means±standard deviation of three independent experiments. Relative level of each gene expression is shown with assignment to 1 in the nontreated OL8(3.5Y) cells.</p

Amino acid substitutions detected in HCV RNAs obtained from the RBV-resistant cells.

<p>The number in parentheses shows the number of indicated amino acids found among seven clones in OL8(3.5Y) or three clones in each R200 series cells. The amino acid without parentheses means that it was found in all clones. Conserved aa substitutions among three R200 cell species are shown by bold letters. Amino acid substitutions found only in one or two R200 cell species are shown by italic letters.</p><p>Amino acid substitutions detected in HCV RNAs obtained from the RBV-resistant cells.</p

Mutation rates of genome-length HCV RNAs in long-term cell culture.

<p>The mutation rates of three regions (5′-terminus-EMCV-IRES, Core-NS2, and NS3-NS5B) of genome-length HCV RNAs (OL, OL8, OL11, and OL14) were calculated using the sequence data obtained from 2- or 4-year cell culture. The vertical line indicates the means of the mutation rates calculated using the nucleotide sequences of 10 clones (OL) or 3 clones (OL8, OL11, or OL14) of genome-length HCV RNAs, by comparison with the original sequence (ON/C-5B/QR,KE,SR RNA) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091156#pone.0091156-Kato6" target="_blank">[21]</a>.</p

Characterization of OL8(3.5Y)-derived cell lines possessing RBV-resistant phenotype.

<p>(A) R200#1, R200#8, R200#11, and OL8(3.5Y) cells were treated with RBV (25, 50, and 100 μM) for 3 days, followed by real-time LightCycler PCR for intracellular HCV RNA. The data are expressed as the means±standard deviation of four independent experiments. Asterisks indicate significant differences compared to R200#11 cells at each RBV concentration. *<i>P</i><0.05; **<i>P</i><0.01; <i>NS</i>, not significant. The relative value (%) calculated at each point, when the level in nontreated cells was assigned to 100%, is presented here. (B) Production of NS5B in the cells with or without treatment of 50 μM RBV for 3 days was analyzed by immunoblotting using anti-NS5B antibody. ß-actin was used as a control for the amount of protein loaded per lane.</p

Sensitivity to telaprevir of the 4-year cultured genome-length HCV RNA-replicating cells.

<p>(A) Telaprevir sensitivities on genome-length HCV RNA replication in OL(4Y), OL8(4Y), OL11(4Y), and OL14(4Y) cells. OL(0Y) cells were used as a control. The cells were treated with telaprevir for 72 h, and then the levels of intracellular genome-length HCV RNA were quantified by LightCycler PCR. (B) Telaprevir-treated OL(0Y) and OL(4Y) cells (designated as OL(0Y)T and OL(4Y)T, respectively) became telaprevir-resistant easily. Telaprevir treatment and quantitative RT-PCR were preformed as shown in (A).</p

Genetic deletions occurred in the first half of genome-length HCV RNAs during the long-term cell culture.

<p>The conservative deleted portions in the genome-length HCV RNAs derived from OL8(2Y), OL8(4Y), OL11(2Y), OL11(4Y), OL14(2Y), or OL14(4Y) cells were shown by boxes. The original sequence was from ON/C-5B/QR,KE,SR RNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091156#pone.0091156-Kato6" target="_blank">[21]</a>.</p

Base substitution patterns occurred in genome-length HCV RNAs during the long-term cell culture.

<p>Base substitutions were counted by the comparison with the sequence of genome-length HCV RNA (ON/C-5B/QR,KE,SR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091156#pone.0091156-Kato5" target="_blank">[20]</a>).</p><p>*Data from the previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091156#pone.0091156-Kato5" target="_blank">[20]</a>.</p