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, 5 (6), e11265

Cell Type Mediated Resistance of Vesicular Stomatitis Virus and Sendai Virus to Ribavirin

Affiliations

Cell Type Mediated Resistance of Vesicular Stomatitis Virus and Sendai Virus to Ribavirin

Nirav R Shah et al. PLoS One.

Abstract

Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus). Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of RBV on viral replication in seven cell lines.
(A) The organization of the negative-sense RNA genomes of the recombinant viruses used in this study. (B) The panels show photographs of cells pretreated for 24 h with increasing concentrations of RBV as indicated (or mock-treated), infected with VSV-GFP (left) or SeV-GFP (right) at MOI 3 CIU/cell (or mock-infected, upper row), and then the same concentrations of drugs as in the pretreatment was added to each well after virus absorption. Fluorescence (upper panels) and light (lower panels) microscopy images were captured at 10× magnification. The photographs are typical representations of at least three independent experiments and an average field for each well is shown. (C) Media from the experiments described in B was collected at 24 h p.i for VSV (left) or at 48 h p.i for SeV (right) and virus titer was determined by standard plaque assay on BHK21 (for VSV) or Vero cells (for SeV). The data represent the mean ± standard deviation of two independent experiments (done in duplicates). Statistical analysis was done using one-way ANOVA with Tukey's post hoc test (GraphPad Prism 4, San Diego, CA). RBV treatments without significant decrease in viral titer at any tested RBV concentrations as compared to mock-treated cells (“0 µM RBV”) are indicated as P>0.05.
Figure 2
Figure 2. Effect of RBV on cell viability of seven cell lines.
To determine the relative toxicity of increasing concentrations of RBV in different cell lines, 80%-confluent uninfected cells were treated with RBV for 24 h and tested for viability using MTT cell viability assay (A) or CellTiter-Glo Luminescent Cell Viability Assay (B) or by cell counting using trypan blue dye exclusion as an indicator of live cells (C) as described in Materials and Methods. To determine the sensitivity of the MTT assay, serial dilutions of A549 and HeLa cells were plated [lower left and right graphs in (A)], grown for 24 h, cells from separate wells were trypsinized and counted using a hemocytometer (36,000 cells for HeLa and 38,000 cells for A549 formed 100% confluent monolayers), and MTT assay was conducted as described in Materials and Methods. (A–C) The data (done in triplicate) represent the mean ± standard deviation and are expressed as a percentage of the untreated control. Statistical analysis was done using one-way ANOVA with Tukey's post hoc test (GraphPad Prism 4, San Diego, CA). ***P<0.001, **P<0.01, *P<0.05, as compared to mock-treated cells (indicated as 0 µM RBV).
Figure 3
Figure 3. Plaque reduction assay to determine RBV inhibitory concentrations.
Cell monolayers were infected with VSV-GFP or SeV-GFP (or mock-infected; 0 µM RBV) using virus dilutions producing about 100 virus (“100%”) on each cell line in the absence of RBV, overlaid with SFM containing 1.2% Avicel RC-581 and increasing concentrations of RBV (note that different RBV concentrations were used for each virus-cell type combination). Cells were then incubated for 24 h (VSV) or 48 h (SeV), and plaques were counted with the aid of fluorescence and bright field microscopy. “0%” indicates that no fluorescent infectious foci were detected. Each experiment was performed at least twice (done in duplicates) and data points represent the mean ± standard deviation.
Figure 4
Figure 4. RBV uptake and its inhibition in different cell lines.
(A) Cell monolayers on 24-well plates (done in triplicates) were pretreated for 15 minutes with 15 or 100 µM NBMPR/DMSO or mock-treated with the same amount of DMSO as contained in the treated wells. Cells were then treated with SFM containing 50 µM RBV 1% of which was [3H]RBV for 15 minutes at 37°C. Nucleotide pools were isolated and measured for [3H] as described in Materials and Methods. Uptake values represent CPM divided by number of cells in a 24-well plate and normalized to the uptake by DMSO-treated BHK21 cells (defined as 100%). The mean ± standard deviation is shown for four independent experiments (done in triplicates). (B) Cells monolayers (done in triplicates) on 12-well plates were treated with SFM containing 50 µM RBV 1% of which was [3H]RBV (without uptake inhibitors) at 37°C for 1 h, 16 h or 24 h. Nucleotide pools were isolated and measured for [3H] as described in Materials and Methods. Uptake values represent CPM divided by number of cells in a 12-well plate and normalized to the uptake by BHK21 cells for 1 h (defined as 100%). The mean ± standard deviation is shown for two independent experiments (done in triplicates). (A–B) Statistical analysis was done using one-way ANOVA with Tukey's post hoc test (GraphPad Prism 4, San Diego, CA). ***P<0.001, **P<0.01, *P<0.05, as compared to RBV only treated cells (A) or cells treated with RBV for 1 h (B).
Figure 5
Figure 5. Effect of exogenously added guanosine on antiviral activity of RBV.
Cells were mock infected or infected with either VSV-GFP or SeV-GFP at MOI of 3 CIU/cell, and then mock-treated or treated with SFM containing 500 µM RBV, 50 µM guanosine, or both. The intensity of GFP fluorescent signal at 18 h p.i for VSV (A) and 24 h p.i for SeV (B) was quantified using a 96-well plate reader, as described in Materials and Methods. Each of these experiments was performed twice (done in triplicates) and data points represent the mean ± standard deviation. (A–B) Statistical analysis was done using one-way ANOVA with Tukey's post hoc test (GraphPad Prism 4, San Diego, CA). ***P<0.001, **P<0.01, *P<0.05 are shown to compare RBV plus guanosine treatment against RBV treatment only.
Figure 6
Figure 6. Effect of ActD on antiviral activity of RBV.
Cell monolayers were infected with SeV-GFP (A) or VSV-GFP (B) at MOI 3 CIU/cell in the absence of drugs, or with 5µg/ml ActD, 500 µM RBV, or both. Fluorescence (upper panels) and light (lower panels) microscopy images were captured at 10× magnification. The photographs are typical representations of at least three independent experiments and an average field for each well is shown. (C) The number of new infectious VSV-GFP particles generated in the wells photographed in (B) was determined by analysis of SFM collected from each well by plaque assay on BHK21 cells (done in duplicates, average is shown).
Figure 7
Figure 7. Viral infectivity and replication kinetics in the seven cell lines.
(A) Cells were infected with serial dilutions of VSV-GFP (left) or SeV-GFP (right), and infectious foci were counted to calculate the infectivity of the viral stock for each cell line. (B) One-step kinetics of viral replication in seven cell lines. Cells were infected in parallel with VSV-GFP or SeV-GFP at MOI of 3 CIU/cell (1 h absorption), washed 3 times with PBS, and kept in SFM. The media containing newly generated virions was collected at the indicated time points and viral titrations were performed on BHK21 (for VSV) or Vero cells (for SeV).

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