Presence Of Ribavirin Research Articles

Error-prone replication of West Nile virus caused by ribavirin

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15 μg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.

Effects of ribavirin on cytokine production of recall antigens and phytohemaglutinin-stimulated peripheral blood mononuclear cells. (Inhibitory effects of ribavirin on cytokine production)

Aim: To evaluate the effects of ribavirin on cytokine production of recall antigen and PHA-stimulated PBMC obtained from healthy individuals.Materials and methods: PBMC were challenged with tetanus toxoid (5μg/mL) and PHA (10 μmL) in absence or presence of ribavirin at different concentrations (1, 10 and 100 (μM). Parallel sets of wells containing PBMC exposed to medium alone were used as negative controls. On day 3 after initiation of the cultures, IL-2, IFN-γ, IL-4, IL-10, TNF-α content were determined in supernatants of PBMC from the different individuals.Results: The effects of ribavirin on cytokine released by human PBMC in response to PHA and TT showed a great variation among individuals. No significant changes were observed between 1-10μ M concentrations in the production of TNF-α, IFN-γ and IL-10 by both PHA and TT-stimulated PBMC. Ribavirin inhibited TNF-α, IFN-γ, and IL-10 in both PHA and TT-stimulated PBMC at 100 μM (p <0.05). At this concentration, ribavirin induced an increase of 124% in the production of IL-2 by PHA-stimulated PBMC (p <0.05).Conclusions: The present data suggest that ribavirin may cause diverse effects on immunoregulatory cytokine secretion with changes in the Th1/Th2 balance.

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity.

Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirin's antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirin-resistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.

Ribavirin treatment up-regulates antiviral gene expression via the interferon-stimulated response element in respiratory syncytial virus-infected epithelial cells.

Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. RSV replication is a potent activator of the epithelial-cell genomic response, influencing the expression of a spectrum of cellular pathways, including proinflammatory chemokines of the CC, CXC, and CX(3)C subclasses. Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nontoxic antiviral agent currently licensed for the treatment of severe RSV lower respiratory tract infections. Because ribavirin treatment reduces the cytopathic effect in infected cells, we used high-density microarrays to investigate the hypothesis that ribavirin modifies the virus-induced epithelial genomic response to replicating virus. Ribavirin treatment administered in concentrations of 10 to 100 micro g/ml potently inhibited RSV transcription, thereby reducing the level of RSV N transcripts to approximately 13% of levels in nontreated cells. We observed that in both the absence and the presence of ribavirin, RSV infection induced global alterations in the host epithelial cell, affecting approximately 49% of the approximately 6,650 expressed genes detectable by the microarray. Ribavirin influences the expression of only 7.5% of the RSV-inducible genes (total number of genes, 272), suggesting that the epithelial-cell genetic program initiated by viral infection is independent of high-level RSV replication. Hierarchical clustering of the ribavirin-regulated genes identified four expression patterns. In one group, ribavirin inhibited the expression of the RSV-inducible CC chemokines MIP-1 alpha and -1 beta, which are important in RSV-induced pulmonary pathology, and interferon (IFN), a cytokine important in the mucosal immune response. In a second group, ribavirin further up-regulated a set of RSV- and IFN-stimulated response genes (ISGs) encoding antiviral proteins (MxA and p56), complement products, acute-phase response factors, and the STAT and IRF transcription factors. Because IFN-beta expression itself was reduced in the ribavirin-treated cells, we further investigated the mechanism for up-regulation of the IFN-signaling pathway. Enhanced expression of IFI 6-16, IFI 9-27, MxA/p78, STAT-1 alpha, STAT-1 beta, IRF-7B, and TAP-1-LMP2 transcripts were independently reproduced by Northern blot analysis. Ribavirin-enhanced TAP-1-LMP2 expression was a transcriptional event where site mutations of the IFN-stimulated response element (ISRE) blocked RSV and ribavirin-inducible promoter activity. Furthermore, ribavirin up-regulated the transcriptional activity of a reporter gene selectively driven by the ISRE. In specific DNA pull-down assays, we observed that ribavirin enhanced RSV-induced STAT-1 binding to the ISRE. We conclude that ribavirin potentiates virus-induced ISRE signaling to enhance the expression of antiviral ISGs, suggesting a mechanism for the efficacy of combined treatment with ribavirin and IFN in other chronic viral diseases.

Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons.

The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-alpha), IFN-gamma, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-alpha, IFN-gamma, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-alpha and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.

Ribavirin causes error catastrophe during Hantaan virus replication.

Except for ribavirin, no other antiviral drugs for treating hantaviral diseases have been identified. It is well established that ribavirin will inhibit the production of infectious Hantaan virus (HTNV); however, its mechanism of action is unknown. To characterize the inhibitory effect of ribavirin on HTNV, the levels of viral RNAs, proteins, and infectious particles were measured for 3 days posttreatment of HTNV-infected Vero E6 cells. HTNV-infected cells treated with ribavirin showed a slight reduction in the levels of cRNA, viral RNA, and mRNA populations on the first day postinfection. The amount of cRNA and viral RNA increased to that observed for untreated HTNV-infected cells on day 2, whereas mRNA levels were more greatly reduced on days 2 and 3. Despite the finding of S-segment mRNA, albeit low, three of the viral proteins-nucleocapsid (N) protein and glycoproteins G1 and G2-could not be detected by immunohistochemistry in ribavirin-treated cells. To test the hypothesis that these effects were caused by incorporation of ribavirin into nascent RNA and a resultant "error catastrophe" was occurring, we cloned and sequenced the S-segment cRNA/mRNA from ribavirin-treated or untreated cells from day 3. We found a high mutation frequency (9.5/1,000 nucleotides) in viral RNA synthesized in the presence of ribavirin. Hence, the transcripts produced in the presence of the drug were not functional. These results suggest that ribavirin's mechanism of action lies in challenging the fidelity of the hantavirus polymerase, which causes error catastrophe.

RNA virus error catastrophe: direct molecular test by using ribavirin.

RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. We recently showed that the important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and we proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here we describe a direct demonstration of error catastrophe by using ribavirin as the mutagen and poliovirus as a model RNA virus. We demonstrate that ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is observed after a single round of virus infection in ribavirin concentrations sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, we have constructed a graph of error catastrophe showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.

Hemolytic anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage.

The antiviral drug ribavirin (RBV) is widely used in combination with interferon (IFN) in the treatment of chronic hepatitis C virus (HCV) infection. A major side effect of RBV is a reversible hemolytic anemia. We have evaluated the in vitro effects of RBV on erythrocyte adenosine triphosphate (ATP) content and on hexosemonophosphate shunt (HMS). The ATP levels were significantly decreased in the presence of RBV and the HMS was increased, suggesting the presence of red cell susceptibility to oxidation. In vivo, we have studied the hematologic effects of treatment with RBV alone or in combination with IFN in 11 patients with chronic hepatitis C: 6 were treated with RBV (1,000-1,200 mg/d) and 5 were treated with a combination of RBV and IFN (5 million U thrice weekly). Patients were studied at semi-monthly intervals from 0 to day 60 of therapy. Both treatments were associated with a significant reduction in hemoglobin levels (steady state level at day 45) and a marked increase in absolute reticulocyte counts. Erythrocyte Na-K pump activity was significantly diminished, whereas K-Cl cotransport and its dithiotreitol-sensitive fraction, malondialdehyde and methemoglobin levels were significantly increased. RBV-treated patients showed an increase in aggregated band 3, which was associated with a significantly increased binding of autologous antibodies and complement C3 fragments indicating an erithrophagocytic removal by reticuloendothelial system.

Activation-associated necrosis in human immunodeficiency virus infection.

Mitogenic stimulation of lymphocytes from persons infected with human immunodeficiency virus (HIV) resulted in massive cell death. In addition to early apoptosis, a second wave of cell death occurred 48-72 h after stimulation. At that time, the cells were enlarged, leaked content, and had plasma membrane damage-all indicative of necrosis. Furthermore, DNA fragmentation as determined by TUNEL assay was virtually absent. This activation-associated necrosis could not be prevented by interfering with CD95/CD95-ligand interactions or by blocking caspase activity and was unaffected by neutralizing antibodies to tumor necrosis factor-alpha or interferon-gamma. Necrosis was also induced by activation of normal lymphocytes in the presence of ribavirin, which inhibits the de novo pathway of nucleotide synthesis. Lymphocytes from HIV-infected persons are defective in their ability to synthesize nucleotides via this pathway, indicating one possible mechanism for the activation-associated necrosis seen in HIV infection.

Effect of ribavirin on zidovudine efficacy and toxicity in vitro: a concentration-dependent interaction.

Zidovudine (ZDV) is converted to its active triphosphate (ZDVTP) by intracellular kinases. The intermediate ZDV monophosphate (ZDVMP) is believed to play a major role in ZDV toxicity. Manipulation of ZDV phosphorylation is a possible therapeutic strategy for altering the risk-benefit ratio. Here we investigate whether combining RBV with ZDV is able to modulate efficacy and toxicity of ZDV. We have measured the intracellular activation of ZDV (0.3 microM) in the absence and presence of ribavirin (RBV; 2 and 20 microM) in Molt 4 and U937 cells. MTT cytotoxicity of ZDV (10-1000 microM) was also measured with and without RBV (2 microM) in Molt 4 and U937 cells. Measurement of endogenous deoxythymidine triphosphate (dTTP) allowed investigation of the dTTP/ZDVTP ratio. The antiviral efficacy of ZDV in combination with RBV (2 microM) was assessed by HIV p24 antigen measurements. In the presence of RBV (2 and 20 microM) a decrease in total ZDV phosphates was observed, owing mainly to an effect primarily on ZDVMP rather than the active ZDVTP. RBV also increased endogenous dTTP pools in both cell types, resulting in an increase in the dTTP/ZDVTP ratio. ZDV alone significantly reduced p24 antigen production, with an IC50 of 0.34 microM. Addition of RBV increased the IC50 approximately fivefold (1.52 microM). However, at higher concentrations of ZDV (10 and 100 microM) the antagonistic effect of RBV (2 microM) on ZDV was lost. The RBV-mediated decrease in ZDVMP may explain the reduction in ZDV toxicity when combined with RBV (2 microM). Cytotoxicity of ZDV was reduced in the presence of RBV (2 microM) at all concentrations in both cell lines, probably owing to saturation of ZDVTP formation. The interaction of ZDV and RBV is concentration dependent.

Ribavirin inhibits protein synthesis and cell proliferation induced by mitogenic factors in primary human and rat hepatocytes.

Ribavirin, a guanosine analog, used in combination with interferon alpha (IFN-alpha) in the treatment of chronic hepatitis induced by hepatitis C virus (HCV) infection, has been shown to improve liver histology and to decrease transaminases even when administered alone. We analyzed the direct effects of ribavirin on the liver by using primary cultures of human and rat hepatocytes. Between 10 to 60 micromol/L, ribavirin was found to inhibit both the synthesis and secretion of whole proteins in a time- and dose-dependent fashion. Such an effect was confirmed by the measurement of albumin and haptoglobin secretion rates. [3H]-Thymidine incorporation was suppressed both in hepatocyte growth factor-stimulated human hepatocytes and in epidermal growth factor (EGF)-stimulated rat hepatocytes in the presence of ribavirin. The inhibitory effect on DNA synthesis was associated with a delayed progression to S phase of the cell cycle, as determined by flow cytometry and detection of cyclin A and cdc2 which are two proteins expressed during the S phase. The inhibition of DNA synthesis, caused by 50 micromol/L ribavirin, was completely restored by the addition of 80 micromol/L guanosine. These observations demonstrate that ribavirin at concentrations close to those found in plasma of treated patients can directly affect hepatic functions in vitro. Its effects could, however, be reduced in vivo by guanosine salvage supply.

Effects of drugs on 2',3'-dideoxy-2',3'-didehydrothymidine phosphorylation in vitro.

Drugs commonly administered to patients infected with the human immunodeficiency virus (HIV) have been studied for their propensity to alter the intracellular phosphorylation of the anti-HIV nucleoside analog stavudine (2',3'-dideoxy-2',3'-didehydrothymidine; d4T) in peripheral blood mononuclear cells (PBMCs) and U937 cells in vitro. PBMCs isolated from the blood of healthy volunteers were stimulated by the mitogen phytohemagglutinin (10 microg/ml) for 72 h. Stimulated PBMCs (3 x 10(6) cells/plate) were then incubated with [3H]d4T (0.65 microCi; 3 microM) and either acyclovir, dapsone, ddC, ddI, fluconazole, foscarnet, ganciclovir, itraconazole, lobucavir, ranitidine, ribavirin, rifampin, sorivudine, sulfamethoxazole, trimethoprim, lamivudine (3TC), zidovudine, or thymidine (30 and 300 microM) for 24 h. Doxorubicin and drugs showing some evidence of inhibition were also studied at 0.3 and 3 microM. Cells were extracted overnight with 60% methanol prior to analysis by radiometric high-performance liquid chromatography. Additional data for nine of the drugs were obtained by incubation with [3H]d4T in U937 cells for 24 h. The effect of d4T (0.2 to 20 microM) on zidovudine (0.65 microCi; 0.018 microCi) phosphorylation was also studied. Zidovudine significantly reduced d4T total phosphates in PBMCs and U937 cells (in PBMCs to 33% [P < 0.001] and 17% [P < 0.001] of that in control cells at 3 and 30 microM, respectively). A small reduction in zidovudine phosphorylation was seen with d4T but only at d4T:zidovudine ratios of 100 and 1,000. Of the other compounds screened, only thymidine, ribavirin, and doxorubicin produced inhibition of d4T phosphorylation in both PBMCs and U937 cells. However, doxorubicin was cytotoxic at 3 microM. The decrease in d4T phosphorylation in the presence of ribavirin is consistent with previous findings with zidovudine. Although ddC significantly inhibited the phosphorylation of d4T in PBMCs, this was not seen in U937 cells, and it is probable that the findings in PBMCs are related to mitochondrial toxicity [based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cytotoxicity assay]. The only drugs screened which may interfere with d4T phosphorylation at clinically relevant concentrations were zidovudine, ribavirin, and doxorubicin.

Intracellular Metabolism of 3′-azido-3′-deoxythymidine and 2′,3′-dideoxyinosine in Combination in the Absence and Presence of Ribavirin

We examined the intracellular phosphorylation of 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxyinosine (ddl) and the effects on rNTP and dNTP pools when AZT and ddl were incubated separately and in combination in lymphocytes. We also compared the effect of adding ribavirin (RBV) to the two-drug combination of AZT + ddl. AZT and ddl, used singly or in combination, had no effect on the dNTP pools of CEM lymphoblastoid cells. Neither did the combination of AZT + ddl have any effect on the rNTP pools. RBV, a known inhibitor of IMP dehydrogenase, caused a decrease in GTP and an increase in dTTP whether incubated alone or with the drug combination of AZT + ddl. The addition of AZT + ddl therefore did not alter the effects of RBV upon cellular nucleotide pools. AZT was phosphorylated to a much greater extent than ddl. The activation of ddl to ddA-TP was increased 2-fold in the presence of AZT, whereas AZT phosphorylation was unchanged when combined with ddl. This increase in ddl activation may explain in part the synergistic antiviral activity of the combination of AZT + ddl. The increased activation was not due to increased phosphorylation of ddl resulting from IMP dehydrogenase inhibition. The addition of 10 μm RBV to the two-drug combination of AZT + ddl did not change the intracellular phosphorylation of AZT or ddl. The activation of ddl to ddA-TP, when combined with AZT, appeared to be maximal and could not be further increased by addition of RBV to this combination.

Effect of ribavirin on macromolecular synthesis in vesicular stomatitis virus-infected cells.

Ribavirin at 200 micrograms/ml inhibited vesicular stomatitis virus (VSV) growth in Chinese hamster ovary (CHO) cells by 2.5 logs. To determine the mechanism of this inhibition, viral macromolecular synthesis was examined. VSV primary transcription remained unaffected, but overall VSV RNA synthesis decreased by 40 to 60%. When ribavirin was added 1.5 h after infection, inhibition of progeny production was partially lost, indicating that the antiviral effect was on an early stage after primary transcription. Inhibition of RNA polymerization by premature chain termination was not evident. Viral translation, on the other hand, was reduced by 95% with an inhibition of every protein species. Furthermore, viral RNA synthesized in the presence of ribavirin did not translate well in an in vitro translation system. In contrast, uninfected CHO cells treated with ribavirin showed a greater sensitivity in RNA synthesis than in protein synthesis. This suggests that the cellular translational machinery was not directly affected. Short-term treatment of cells resulted in negligible toxicity, but after 24 h there was marked alteration of cellular integrity. These results, taken together with data on other viruses, suggest that in the presence of ribavirin, dysfunctional VSV mRNA was synthesized, resulting in its failure to be translated. The selective antiviral effects of ribavirin and its relative lack of toxicity for host cells may be predicted on the basis of mRNA turnover and the requirements for de novo functional mRNA.