Mechanism of action of ribavirin in the combination treatment of chronic HCV infection

PERSPECTIVES IN CLINICAL HEPATOLOGY Mechanism of Action of Ribavirin in the Combination Treatment of Chronic HCV Infection Johnson Y. N. Lau,1 Robert C. Tam,1 T. Jake Liang,2 and Zhi Hong1 “In Science the credit goes to the man who convinces the world, not to the man to whom the idea first occurs” William Osler

The story of ribavirin (1-␤-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a purine nucleoside analogue, began when Drs. Joseph T. Witkowski and Roland K. Robins synthesized the compound in 1970.1 The broadspectrum antiviral activity was reported in 1972.2 The aerosol form of ribavirin was approved for the treatment of respiratory syncytial virus infection in children with respiratory distress in mid-1980, before the discovery of hepatitis C virus (HCV) reported in 1989.3 The ribavirin/ interferon alpha (IFN-␣) combination therapy was approved by the United States Regulatory Authorities in 1998. Three decades of research efforts, including the life-long effort of a number of unsung scientists, finally allowed ribavirin to establish its position in the history of medicine. The success of ribavirin/IFN-␣ combination therapy for chronic HCV infection, a viral infection affecting 170 million people worldwide, has provoked a renewed and intense effort to further explore the mechanism(s) of action of ribavirin, with the belief that such information will guide the design of better therapies against HCV. Such a task is not straightforward. IFN-␣ has been shown to possess direct antiviral, antiproliferative, and immunomodulatory activities. The mechanistic pathways inAbbreviations: HCV, hepatitis C virus; IFN-␣, interferon alpha; HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; ALT, alanine transaminase; IMPDH, inosine monophosphate dehydrogenase; RdRp, RNA-dependent RNA polymerase; Th, T helper; CTL, cytotoxic T lymphocyte; IL, interleukin; TNF-␣, tumor necrosis factor ␣; PBMC, peripheral blood mononuclear cell; SEB, staphylococcal enterotoxin B; RMP, ribavirin monophosphate; GTP, guanosine triphosphate; MPA, mycophenolic acid; RTP, ribavirin triphosphate. From 1Research and Development, ICN Pharmaceuticals Inc., Costa Mesa, CA; and 2Liver Disease Section, NIDDK, National Institutes of Health, Bethesda, MD. Received December 5, 2001; accepted January 29, 2002. Address reprint requests to: Johnson Y. N. Lau, M.D., ICN Pharmaceuticals Inc., 3300 Hyland Avenue, Costa Mesa, CA 92626. E-mail: [email protected]; fax: 714-641-7276. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3505-0003$35.00/0 doi:10.1053/jhep.2002.32672 1002

volved in the mediation of IFN-␣ effects, including 2⬘-5⬘ oligoadenylate synthase and induction of Mx and PKR proteins, are well established.4 However, the exact mechanism of action of IFN-␣ in the treatment of chronic HCV infection is not known. In chronic hepatitis B virus (HBV) infection, the presence of hepatitis flare before hepatitis B e antigen (HBeAg) seroconversion during IFN-␣ monotherapy suggested that immune-mediated mechanisms may be involved. Similar hepatitis flare was not observed in patients with chronic HCV infection who respond to IFN-␣ monotherapy. However, a number of studies showed the involvement of the host immune system in the response to IFN-␣ therapy.5 Whether the host immune response is induced directly by IFN-␣ or secondary to treatment response is not clear. Our knowledge on the mechanism of action of ribavirin is no better than that of IFN-␣. Ribavirin is not a potent direct antiviral agent in vitro with EC50 in the high micromolar (␮mol/L) range against most viruses. The most interesting clinical observation is that ribavirin monotherapy had minimal effect on HCV viremia despite the fact that serum alanine transaminase (ALT) levels were reduced significantly in a considerable proportion of patients with chronic HCV infection.6,7 Hence, it was proposed that ribavirin may exert its effect on the host immune response. Another important clinical observation is that the combination of ribavirin and IFN-␣ provides a clinically synergistic effect, i.e., the clinical efficacy of this combination exceeds that of the summation of the individual monotherapies. However, the exact nature of this synergism will not be elucidated until the mechanism of action of each agent is firmly established. At present there are 4 proposed mechanisms of action of ribavirin. They can be divided into 2 groups. The first group consists of 2 possible indirect mechanisms: (1) enhancement of host T-cell–mediated immunity against viral infection through switching the T-cell phenotype from type 2 to type 1 and (2) inhibition of the host enzyme inosine monophosphate dehydrogenase (IMPDH). The second group consists of 2 other hypotheses: (1) direct inhibition of HCV, including NS5B-encoded RNA-

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Fig. 1. Role of T cells in host adaptive antiviral response. The presentation of viral antigens, in an MHC class II–restricted manner, by professional antigen presenting cells (APC) to virus-specific, naive helper CD4⫹ T cells (Thn) induces differentiation to Th1 cells in early infection and differentiation to Th2 cells in late-stage infection. IL-12 and IL-4 are mediators of the differentiation to Th1 and Th2 cells, respectively. Activated Th1 cells release IL-2, IFN-␥, and TNF-␣ that elicit a number of biologic activities including (1) activation of CD8⫹ CTLs in an MHC class I–resticted manner, (2) recruitment of natural killer (NK) cells and macrophages (M␾) to the site of infection, and (3) cytokineinduced nonlytic elimination of virus-infected cells. Activated Th2 cells release Th2 cytokines (IL-4, IL-5, IL-6, IL-10, and IL-13) that activate virus antigen-specific B cells (Bc) to proliferate and differentiate to virus-specific antibody-secreting plasma cells and memory B cells (mBc, not shown).

dependent RNA polymerase (RdRp) and (2) as an RNA mutagen that drives a rapidly mutating RNA virus over the threshold to “error catastrophe.”

Updates on HCV Immunology and Virology Immune Response to HCV. In patients with chronic HCV infection viral specific humoral (i.e., B-cell) responses are detected in most patients, in particular, nonHCV neutralizing antibodies, which form the basis of some of the currently used diagnostic assays. Neutralizing antibodies have also been reported but appeared to be strain/isolate specific. HCV-specific cellular (both CD4⫹ and CD8⫹ T cells) immune responses are also detected in a proportion of patients based on the currently available assays. However, HCV persistence occurs in these patients. In addition, the CD4⫹ and CD8⫹ T-cell responses to HCV appear to be less vigorous and more focused than those seen in patients who have recovered from acute HCV infection. The continuous battle between the virus and the host immune response often results in damage to the battleground, the liver. The high mutation rate of HCV is also believed to be one of the key factors contributing to the virus’ escape from the host immune response. HCV Replication. HCV is a nonsegmented singlestranded positive sense RNA virus. It encodes a single large polyprotein, which is further processed into at least 10 viral proteins. The nonstructural protein 5B (NS5B) at

the carboxyl terminus is an RNA-dependent RNA polymerase (RdRp) responsible for viral replication. HCV replicates through a negative strand intermediate template. Therefore, both terminal untranslated regions of HCV, 5⬘UTR and 3⬘UTR, are essential for viral replication.8,9 As HCV lacks repetitive sequence motifs at both termini of the viral genome, it is likely that HCV initiates RNA replication through a de novo pathway, which is confirmed recently through structural and biochemical characterization.10 A recently identified unique ␤-hairpin in the Thumb subdomain of NS5B may play an important role in positioning the 3⬘-terminus of the HCV genome for terminal initiation.11 Inhibitors that interfere with the replication process will be of great therapeutic value and further assist our understanding of the replication mechanism.

Enhancement of Host Antiviral Immunity by Ribavirin Role of T Cells in Host Adaptive Antiviral Response (Fig. 1). The host adaptive antiviral immune response consists of virus-specific CD4⫹ helper (Th) and CD8⫹ cytotoxic T lymphocytes (CTLs). MHC class II–restricted CD4⫹ Th cells provide virus-specific T-cell help (to CD8⫹ CTLs and B cells), and MHC class I–restricted CD8⫹ CTLs provide cytotoxic killing of virus-infected cells. In addition, both T-cell subsets secrete type 1 cyto-

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kines including inteleukin 2 (IL-2), IFN-␥, and tumor necrosis factor ␣ (TNF-␣) in response to viral pathogens. These cytokines recruit additional natural killer cells and macrophages to the site of infection. In the HBV transgenic mouse model, IFN-␥ and TNF-␣ have been shown to suppress HBV replication, and IFN-␥ has been shown to clear HBV replication through a noncytolytic mechanism.12 A type 1 cytokine response is, therefore, important in the early phase of viral infection with the aim of controlling the infection. However, when CD4⫹ and CD8⫹ T cells differentiate into type 2 T cells, usually late in the viral infection, they produce IL-4, IL-5, IL-6, IL13, and IL-10, provide optimal help for humoral immune responses, and limit and control the host type 1 response. Ordinarily this type 2 response is to protect the host from a continual inflammatory, cell-mediated type 1 response and to provide long-term humoral immunity. Therefore, for a given immune response to pathogens such as viruses type 1 or type 2, cytokines play separate but defined regulatory roles and a balance exists between the timing of secretion, level of expression, and numbers of T cells that secrete each subset of cytokines. If this balance is altered and polarized early to type 2, type 1 responses may be suppressed and the internal milieu will favor viral persistence in the absence of the development of neutralizing antibodies. In fact, a dominant type 2 T-cell response has been associated with the development of chronicity in a number of viral infections.13 Host T-Cell Response in Chronic HCV Infection. Several lines of evidence suggest that the host anti-HCV response during chronic HCV infection may be compromised: (1) in acute HCV infection, activation of type 2 cytokine response has been shown to associate with the development of chronicity14; (2) a reduced number of peripheral blood mononuclear cells (PBMCs) showing type 1 cytokine (IL-2 and IFN-␥) expression was observed in patients with persistent HCV viremia compared with patients with self-limited HCV infection15; (3) high serum levels of type 2 cytokines including IL-4 and IL-10 (Th0/Th2) were observed in a proportion of patients with chronic HCV infection16,17; (4) a reduced capacity of HCV NS3 antigen-specific T cells to produce IL-2, but not IL-4 and IL-10 in vitro, are typically found in patients with chronic HCV infection18; (5) a favorable clinical response to IFN-␣ therapy is associated with a reduction in serum type 2 cytokine levels17; and (6) a vigorous, multispecific T-cell response is seen in the majority of patients who clear their HCV infection, either spontaneously or in response to IFN-␣ treatment.15,19 Such an immune response is much less frequent in patients who failed to respond to treatment. These observations highlight the importance of the host immune response on the

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pathogenesis of chronic HCV infection as well as host response to treatment. Ribavirin as a Modulator of Type 1/Type 2 Cytokine Response. A number of studies have shown that ribavirin can mediate immunomodulation by altering the type 1/type 2 cytokine bias in favor of type 1 during an immune response. (1) Ribavirin at 1 to 20 ␮mol/L inhibits immune responses known to be mediated by type 2 cytokines such as antibody-mediated cytotoxicity in vivo.20 (2) Ribavirin treatment of respiratory syncytial virus infection not only eradicates the virus but also reduces the prevalence of airway hyper-reactivity, which is believed to be associated with a polarized type 2 cytokine response.21 (3) Ribavirin has been shown to enhance antiviral type 1 and suppress type 2 cytokine expression in human T cells.22 Ribavirin, at 5 to 10 ␮mol/L (doses previously reported to be clinically achievable23), augmented type 1 cytokines IL-2, IFN-␥, and TNF-␣ and suppressed type 2 cytokines IL-4 and IL-5 levels in stimulated (phorbol myristate acetate plus ionomycin, or staphylococcal enterotoxin B [SEB]) T cells or in CD4⫹ or CD8⫹ T-cell subsets derived from healthy subjects. Further analysis in SEB-stimulated T cells showed that ribavirin induced a type 1 cytokine bias at the transcriptional level. Animal Model. In animal models, ribavirin significantly increase the inflammatory responses in contact hypersensitivity responses to dinitrofluorobenzene and bacterial superantigen, SEB. In both models the enhanced immune response induced by ribavirin was associated with a type 1 cytokine bias in lymphoid organs in vitro.22,24 A similar cytokine profile switch was also seen in HCV antigen-immunized mice or in an HBV transgenic mouse model. Immunization of BALB/c mice with HCV core antigen and 0.5 mg ribavirin daily resulted in increased IL-2 and IL-12 expression, core-specific cytotoxic T-cell activity and core-specific IgG2a secretion by corestimulated spleen cells.25 In transgenic mice expressing HBeAg, immunization with a peptide determinant derived from HBeAg resulted in a predominantly IgG1, type 2 response. When ribavirin (1 mg) was coadministered, a shift to an IgG2a, type 1 response was observed.26 Finally in a murine fulminant hepatitis model, ribavirin reduced murine macrophage production of inflammatory cytokines and inhibited type 2 cytokine responses in the absence of a substantial direct antiviral action.27 These data showed that ribavirin can augment a type 1 cytokine expression in favor of immune-mediated antiviral activities in vivo. Effect of Ribavirin Ex Vivo. Three studies evaluated the effect of ribavirin on HCV-specific or mitogen-stimulated type 1 and type 2 cytokine expression in PBMCs

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derived from patients with chronic HCV infection. He et al.28 showed that ribavirin (1-10 ␮mol/L) enhanced phytohemagglutinin A–induced IFN-␥ expression and HCV-specific TNF-␣ production in an ex vivo study. Rigopoulou et al. showed that ribavirin (2 and 10 ␮mol/L) augmented HCV-specific T-cell reactivity associated with an increase in IFN-␥ and a decrease in IL-10 mRNA expression in a proportion of patients with chronic hepatitis C.29 Martin et al. observed that ribavirin plus IFN-␣ treatment in vitro induced a higher level of IL-2 and IFN-␥, compared with IFN-␣ treatment alone.30 These results suggest that ribavirin, at the therapeutic level, can bias the host immune response toward a type 1 phenotype in PBMCs derived from patients with chronic HCV infection. Effect of Ribavirin in Patients. In patients receiving ribavirin monotherapy, serum viral level was not affected but ALT levels were reduced in a considerable proportion of patients.6,7 This observation, while having been attributed to the immunosuppressive effect of ribavirin, cannot be completely explained by our current knowledge on the mechanisms of action of ribavirin (see below). The combination of ribavirin and IFN-␣ was found to be associated with a biased type 1 cytokine response in nonresponders. In a study of anti-HBe patients, patients who developed sustained virologic response to the combination therapy had a reduction of viremia associated with induction of intrahepatic HBV-specific CD4⫹ Tcell proliferation and Th1 cytokine production (elevated IFN-␥ and IL-12 responses), in contrast to nonresponders who showed a decrease in IFN-␥ and IL-12 and an increase in IL-10 expression.31 Similar profiles were also observed in patients with chronic HCV infection treated with ribavirin and IFN-␣ combination therapy. Cramp et al. showed that HCV-specific T-cell reactivities increased at treatment weeks 4 to 8 (with either IFN-␣ alone or ribavirin and IFN-␣), and this was associated with a higher rate of sustained virologic response.32 The addition of ribavirin was associated with significantly lower IL-10 production. With regard to the timing of events in relation to treatment response, 2 studies showed that a favorable treatment response with ribavirin and IFN-␣ was associated with activation of an HCV-specific T-cell response during treatment rather than at the end of treatment.33,34 These data are consistent with the hypothesis that ribavirin serves as an immunomodulator to enhance type 1 cytokine production. The observation that ribavirin and IFN-␣ combination therapy is less effective in liver transplant recipients with recurrent HCV is in further support that an immunosuppressive environment may reverse the beneficial effect of the combination therapy.

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Possible Hypotheses. One could postulate that the enhancement of antiviral cytokines by ribavirin treatment in patients with chronic HCV infection requires the antiviral activity of IFN-␣ to be more effective or that IFN-␣ can antagonize the immunosuppressive effects of ribavirin in the liver. Indeed end-of-treatment responses to IFN-␣ monotherapy are associated with enhanced CTL activity and enhanced CD4⫹ T-cell–proliferative responses.35,36 Collectively, the current data favor the hypothesis that a strong antiviral immune response is the primary component of the antiviral effect of ribavirin/IFN-␣ therapy. The enhancement of type 1 cytokine responses mediated by ribavirin may influence the number of sustained responders by accentuating the immunostimulatory effects of IFN-␣. It is important to point out that the exact mechanism of action of ribavirin in relation to the synergistic antiHCV effect with IFN-␣ observed in clinical studies may be different from that projected from the ex vivo and monotherapy data. Next Step. How can we prove this mechanism? Certainly, any therapy that selectively biased type 1 versus type 2 cytokine profile, if found to be effective in combination with IFN-␣ in patients with chronic HCV infection, should provide the strongest support of these hypotheses. A number of immunomodulating agents, including IL-2, IL-12, Thymosin-alpha, and Maximine are currently being studied in patients with chronic HCV infection, but their mechanisms of action are not identical to that of ribavirin. Levovirin, the L-enantiomer of ribavirin, was found to possess similar immunomodulatory activities as ribavirin.24 However, because of the stereostructural differences, levovirin cannot be metabolized to the mono-, di-, and triphosphate derivatives and, hence, has no direct antiviral activities and the associated toxicity of the metabolites but retains the immunostimulatory function.37 The clinical data generated by these agents may provide further insights into the anti-HCV immunomodulatory activities of ribavirin.

Inhibition of Host IMPDH by Ribavirin The cellular conversion of ribavirin to its 5⬘-monophosphate (RMP) is mediated by adenosine kinase.38 RMP, mimicking inosine 5⬘-monophosphate, is a competitive inhibitor of IMPDH and has been cocrystallized with this host enzyme.39 As IMPDH is the rate-limiting step in the de novo synthesis of guanosine triphosphate (GTP), inhibition of this enzyme may have the following pharmacologic effects. Effect on Viral RNA Replication. GTP is a critical building block for viral RNA synthesis; thus depletion

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of intracellular GTP pool may in theory lead to suppressed viral RNA replication. Based on this theory, IMPDH inhibitors such as mycophenolic acid (MPA)40 and VX-49741 have been tested for antiviral activities. Using the HCV subgenomic replicon system, ribavirin, MPA, and VX-497 all showed inhibitory activity of HCV replication. The addition of 200 ␮mol/L guanosine completely abolished the antiviral activities of MPA and VX-497. However, only partial reversibility of ribavirin’s antiviral activity was observed, suggesting that ribavirin possesses additional antiviral components against HCV replication.42 This is also supported by a previous observation that the 1,4,5-triazole derivative of ribavirin, also an IMPDH inhibitor, is devoid of antiviral activity.43 These data suggest that the antiviral activities of ribavirin cannot be completely explained for by IMPDH inhibition. Immunosuppressive Effect. Ribavirin has been shown to have an antiproliferative effect on lymphocytes at high concentrations.44 The mechanism of this antiproliferative effect is presumably caused by the inhibition of de novo nucleic acid synthesis following the depletion of the GTP pool as a result of IMPDH inhibition. This leads to the hypothesis that this may be the mechanism responsible for the observed reduced inflammation in the liver. However, at lower concentrations (5 to 10 ␮mol/L) ribavirin modulates cytokine production in peripheral blood lymphocytes with a concomitant increase in T-cell proliferation.22 Therefore, this dose range of ribavirin should be distinct from that shown to be immunosuppressive. As the clinically relevant dose of ribavirin in the plasma is in the concentration range that induces a type 1 cytokine bias,23 it is reasonable to assume that the immunomodulatory activity, rather than the immunosuppressive property of ribavirin, is predominant in patients treated with ribavirin. Clinical Correlates. In transplant recipients, MPA is sometimes used as a second line antirejection immunosuppressive therapy. In patients with HCV infection after heart or liver transplantations, the use of MPA did not show any beneficial effect in the clinical outcome. Another IMPDH inhibitor VX-497 was found to reduce serum ALT levels but had no effect on serum HCV RNA levels in a short-term monotherapy study, consistent with an immunosuppressive effect.45 The results of a currently ongoing phase II VX-497 and IFN-␣ combination treatment study will provide the critical information on the role of ribavirin as an IMPDH inhibitor in the treatment of chronic HCV infection.

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Direct Inhibition of HCV Replication by Ribavirin Ribavirin, like other antiviral nucleoside analogues, undergoes intracellular phosphorylation to ribavirin mono- (RMP), di- (RDP), and triphosphates (RTP) with RTP as the major intracellular metabolite.43 Direct Inhibiton of HCV Polymerase. A number of nucleoside analogue triphosphates have been shown to work as substrate mimics for misincorporation by viral polymerases, leading to primer chain termination and inhibition of viral replication. In vitro enzymatic studies have shown that RTP exhibits a weak inhibitory effect on several viral polymerases such as those of vesicular stomatitis virus, influenza virus, Lacrosse virus, reovirus, and human immunodeficiency virus. Recently, RTP was found to possess weak inhibitory activity against NS5Bencoded RdRp of bovine viral diarrheal virus (a virus closely related to HCV) with an IC50 of 50 ␮mol/L.45 In an optimized HCV RdRp assay using poly(C) as the template, RTP was shown to weakly inhibit RdRps derived from all 6 genotypes of HCV with IC50 ranging from 50 to 150 ␮mol/L (Hong et al., unpublished data). Further characterization showed that ribavirin was misincorporated by HCV RdRp into the RNA products, suggesting that RTP binds to HCV polymerase and can be recognized as a substrate mimic (Maag et al., unpublished data). Weak Inhibitory Effects Against HCV in the Replicon System. Using the HCV replicon model, ribavirin was found to be a weak inhibitor, with an IC50 between 12 to 40 ␮mol/L (Zhong et al., unpublished data, and Laxton et al.42). This weak inhibitory activity on HCV replication may be directly correlated with its inhibition of HCV polymerase as ribavirin and its metabolites have been tested to be inactive against other HCV targets such as NS3 protease/RNA helicase/NTPase and the 5⬘ internal ribosomal entry site.

Ribavirin as an RNA Virus Mutagen HCV is an RNA virus that exists as “quasispecies” with marked genetic variation caused by the lack of fidelity of its viral polymerase. These quasispecies provide the flexibility to HCV for rapid adaptation to adverse environment and for evading host immune responses.46 However, the increased mutation rate also puts the virus in potential jeopardy at the threshold of “error catastrophe,” a theory hypothesizing that minor increases in the mutation rate could mutagenize the viral genomes and drastically reduce the viral fitness.47 Crotty et al. proposed recently that ribavirin may act as an RNA mutagen, an effect that mutates the virus and

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Fig. 2. Possible mechanisms of action of ribavirin. (1) Immune clearance—ribavirin can induce a Th2 to Th1 bias in favor a host antiviral response through either CTLs or through the Th1 cytokines. (2) RMP can inhibit the host enzyme IMPDH, thus reducing GMP and the GTP pool. (3) RTP has been shown to have weak inhibitory activity against HCV NS5B RdRp. (4) Ribavirin may also act as an RNA mutagen to introduce mutations in the HCV genome, thus inducing the production of defective HCV particles. Also noted are the conversion of ribavirin to its phosphorylated metabolites (RMP, di-DP RDP, and RTP) and its reversion back to ribavirin through dephosphorylation. The transporter on cell membrane is believed to be the es transporter.

reduces its infectivity.47 Using the poliovirus model, ribavirin induces lethal mutational events to poliovirus genome as a result of RTP utilization by the viral RdRp, inducing misincorporation of RMP into viral RNA.47,48 Using a primer extension assay with a symmetrical primer/template substrate, RTP was recognized by the poliovirus polymerase 3Dpol and misincorporated without termination of elongation. The pseudobase of ribavirin (1,2,4-triazole carboxamide) was able to base pair equivalently with cytidine and uridine, provided that rotation of the carboxamide moiety was not restricted. This misincorporation of ribavirin into the viral RNA genome could be mutagenic by promoting transitions of A to G and G to A. Using a poliovirus replicon in which the capsid coding sequence is replaced by a luciferase gene, ribavirin was shown to decrease replicon RNA replication only modestly but at the same time to cause significant reduction on viral specific infectivity in a single round of infection.47 In a poliovirus guanidine resistance (guar) assay a point mutation in the 2C open reading frame at nucleotide position 4605 (C4605U) confers viral resistance to guanidine. A ribavirin dose-dependent increase in the frequency of guar virus was observed, providing further support to the mutagenic potential of ribavirin in vivo. Subsequent DNA sequencing analysis of the viral capsid gene also showed a ribavirin-dependent increase of mutational rate in ribavirin-treated polioviruses.48 Although this effect has not been shown in HCV, a similar effect was observed in GB virus-B in a primary tarmarin hepatocyte model, a virus closely related to HCV.49 Thus

ribavirin may induce an antiviral state by pushing the RNA virus over the edge to “error catastrophe.” Whether ribavirin is an effective mutagen for HCV remains to be determined.

Ribavirin and Hemolytic Anemia Hemolytic anemia is a known side effect of ribavirin therapy. The mechanism is not firmly established. However, it is known that ribavirin is converted to its phosphorylated metabolites in all cell types but the conversion back to ribavirin through dephosphorylation occurs mainly in nucleated cells (Fig. 2). Erythrocytes, which appear to lack the dephosphorylation enzymes, are not able to convert the phosphorylated ribavirin metabolites back to ribavirin, resulting in extensive accumulation of phosphorylated ribavirin compared with nucleated cells. It is postulated that ribavirin triphosphate, which resembles ATP from a structural perspective, may competitively inhibit ATP-dependent utilization in cells. With this effect on oxidative respiration, the half-life of erythrocytes is reduced due to extravascular hemolysis.50

Conclusions Ribavirin appears to be a pleiotropic agent with many intrinsic mechanisms that can influence its overall antiviral properties (Fig. 2). Many investigators are favoring the immunomodulatory mechanism, although the possibility that ribavirin is a viral RNA mutagen is also gaining attention. Understanding the key mechanism of action of

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ribavirin in the ribavirin and IFN-␣ combination therapy will certainly help design better therapy for patients with chronic HCV infection. It is possible that the combination of a number of ribavirin’s activities is the key to its anti-HCV activities. What we have learned so far is perhaps the tip of the iceberg for ribavirin. The saga of ribavirin continues. “Knowledge of truth alone does not suffice, on the contrary, it has to be continuously renewed by ceaseless effort if it is not to be lost” Albert Einstein

Acknowledgment: The authors thank Drs. Roberts A. Smith, Jane W. S. Fang, Chin-chung Lin, Nanhua Yao, Weidong Zhong, James Z. Wu, Robert K. Hamatake, Jingfan Huang, Craig Cameron, Paul Glue, Janice K. Albrecht, and Nikolai Naoumov for their guidance to our understanding of this subject; Leslie Sphar for her editorial assistance, and Milan Panic for never failing to believe in this technology platform.

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