Mutagenic and inhibitory effects of ribavirin on hepatitis C virus RNA polymerase

Crotty et al. recently proposed the primary antiviral action of ribavirin to be that of a potent RNA mutagen [Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379]. Here we investigate the effect of ribavirin triphosphate (RTP) on RNA synthesis catalyzed by a full-length hepatitis C virus (HCV) RNA polymerase in vitro. HCV polymerase can use RTP as a nucleotide substrate in a template-dependent manner, incorporating it opposite a pyrimidine (C or U) template residue, but not a purine (A or G). Kinetic analysis revealed that incorporation of ribavirin monophosphate (RMP) across from C is 3 times more efficient catalytically than that across from U, as determined by the k(cat)/K(m) parameter. The efficiency of RMP incorporation, however, is 50-100 fold lower than that of the natural NMP. RMP incorporation does not lead to termination of RNA chain synthesis, as evidenced by the ability of the polymerase to extend its RNA product many nucleotides beyond the site of RMP incorporation. However, multiple-RMP incorporation at low GTP concentrations induced the formation of stalled elongation complexes, particularly at the template region containing consecutive C residues. Most, but not all, such elongation blocks can be relieved by the re-addition of GTP. When ribavirin is present in the RNA template, pyrimidine (but neither purine nor ribavirin) monophosphate is incorporated opposite ribavirin, but at an exceedingly low catalytic efficiency (200-3000-fold lower) compared to the efficiencies of those templated by A or G. Consequently, the level of RNA synthesis on a ribavirin-containing template is significantly reduced. These findings suggest that ribavirin not only is mutagenic but also interferes with HCV polymerase-mediated RNA synthesis.     

A novel G418 conjugate results in targeted selection of genetically protected hepatocytes without bystander toxicity

G418, an aminoglycoside neomycin analogue, is an antimicrobial agent that interferes with protein synthesis and has been used extensively for selection of mammalian cell lines that possess neomycin resistance (NR). It is potent and nonspecific in its effects that occur through tight binding to ribosomal elements. Because of the potent intracellular effect, we wondered whether G418 could be used to select a specific cell type based on receptor-mediated endocytosis. The objective of this study was to target G418 specifically to liver cells via asialoglycoprotein receptors (AsGR) which are known to be highly selective for these cells. A novel G418 conjugate was synthesized chemically by coupling G418 to a galactose-terminating carrier protein, asialoorosomucoid (AsOR), in a molar ratio of 5:1. AsOR-G418 conjugates inhibited viability of AsGR (+) cells by 84.3%, while inhibition in AsGR (-) cells was only by 19%. In AsGR (+) cells, stably transfected with a NR gene, the conjugate decreased viability by less than 9%. Furthermore, incubation of conjugate in cocultures of AsGR (+), and AsGR (-) cells did not result in the loss of viability of neighboring AsGR (-) cells. Our data demonstrate for the first time that G418 can be covalently bound to AsOR to form a conjugate for hepatocyte-specific targeting and toxicity. AsOR-G418 conjugates may be useful tools for genetic manipulation of human liver cells in the presence of nonhepatic cells.     

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase

The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase.     

IMP dehydrogenase and action of antimetabolites in human cultured blast cells

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the Km values for IMP and NAD were 29 and 54 microM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5'-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the Ki values of RMP (0.58 microM) and TAD (0.075 microM) were several orders of magnitude lower than those of XMP (85 microM) and NADH (94 microM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.     

Receptor-mediated gene delivery and expression in vivo

A soluble DNA carrier system was used to target a foreign gene specifically to liver in vivo via asialoglycoprotein receptors. The DNA carrier was prepared consisting of a galactose-terminal (asialo-)glycoprotein, asialoorosomucoid (AsOR), covalently linked to poly-L-lysine. The conjugate was complexed in a 2:1 molar ratio (based on AsOR content of the conjugate) to the plasmid, pSV2 CAT, containing the gene for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Intravenous injection of [32P]plasmid DNA complexed to the carrier demonstrated specific hepatic targeting with 85% of the injected counts taken up by the liver in 10 min compared to only 17% of the counts when the same amount of [32P]DNA alone was injected under identical conditions. Targeted pSV2 CAT DNA was detected at a level of 1.0 ng/g liver by hybridization of a [32P]pSV2 CAT cDNA probe to rat liver DNA extracted 24 h after intravenous injection of AsOR-poly-L-lysine-DNA complex containing 1.0 mg of DNA. Homogenates of livers taken 24 h after injection of the complex revealed that the targeted CAT gene was functional as reflected by the detection of CAT activity (approximately 4 microunits/mg protein). Livers from control animals that received individual constituents of the complex produced no CAT activity. Simultaneous injection of excess AsOR to compete with the AsOR-poly-L-lysine-DNA complex for uptake by the liver inhibited CAT gene expression. Assays for CAT activity in other organs (spleen, kidney, lungs) failed to demonstrate any activity in these organs. This new soluble DNA carrier system can permit targeted delivery of foreign genes specifically to liver with resultant foreign gene expression in vivo.     

Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro

We have developed a system for targeting foreign DNA to hepatocytes in vitro using a soluble DNA carrier that takes advantage of receptor-mediated endocytosis to achieve internalization. The idea is based on the fact that hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo)glycoproteins. To create a targetable carrier system that could bind DNA in a nondeforming manner, we used poly(L-lysine) to bind DNA in a strong but noncovalent interaction. An asialoglycoprotein, asialoorosomucoid (AsOR), was chemically coupled to poly(L-lysine) to form an asialoorosomucoid-poly(L-lysine) conjugate. Various proportions of conjugate to DNA were tested to determine conditions that maximized DNA content in a soluble complex and that limited solubility of complexes. To test the targetable gene delivery system, AsOR-poly(L-lysine) conjugate was complexed to the plasmid pSV2 CAT containing the gene for chloramphenicol acetyltransferase (CAT) driven by an SV-40 promoter. We tested this complex using a model system consisting of human hepatoma cell line Hep G2 [asialoglycoprotein receptor (+)], hepatoma SK-Hep 1, IMR-90 fibroblasts, and uterine smooth muscle [receptor (-)] cells. Each cell line was incubated with 0.2 micron filtered AsOR-poly(L-lysine)-DNA complex or controls consisting of DNA plus AsOR, DNA plus poly(L-lysine), or DNA alone. Cells were assayed for the presence of CAT activity as a measure of gene transformation. SK-Hep 1, IMR-90, and smooth muscle [receptor (-)] cells produced no detectable acetylated chloramphenicol derivatives under any of these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of the phosphoramidate ProTide approach to the antiviral drug ribavirin

Ribavirin is a nucleoside analogue with broad antiviral activity. Here we report the synthesis and biological evaluation of novel ribavirin ProTides designed to deliver the bioactive ribavirin monophosphate into cells. Some of the compounds display activity similar to the parent nucleoside against a range of viruses. Enzymatic, cell lysate and preliminary modeling studies have been performed to investigate the lack of enhancement of potency by the ProTides, and these indicate a failure at the final, amino acid cleavage step in the ProTide activation process, leading to inefficient release of the nucleoside monophosphate.     

DNA condensation for gene therapy as monitored by atomic force microscopy.

The atomic force microscope (AFM) was used to assay the extent of DNA condensation in approximately 100 different complexes of DNA with polylysine (PL) or PL covalently attached to the glycoproteins asialoorosomucoid (AsOR) or orosomucoid (OR). The best condensation of DNA was obtained with 10 kDa PL covalently attached to AsOR, at a lysine:nucleotide (Lys:nt) ratio of 5:1 or higher. These conditions produce large numbers of toroids and short rods with contour lengths of 300-400 nm. Some DNA condensation into shortened thickened structures was seen with 10 kDa PL attached to AsOR at Lys:nt ratios of 1.6:1 and 3:1. Some DNA condensation was also seen with 4 kDa PL at Lys:nt ratios of 3:1 and higher. Little DNA condensation was seen with PL alone or with PL convalently attached to OR at Lys:nt ratios up to 6:1. AsOR-PL enhanced gene expression in the mouse liver approximately 10- to 50-fold as compared with PL alone.     

Crystal structure of Tritrichomonas foetus inosine monophosphate dehydrogenase in complex with the inhibitor ribavirin monophosphate reveals a catalysis-dependent ion-binding site

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in GMP biosynthesis. The resulting intracellular pool of guanine nucleotides is of great importance to all cells for use in DNA and RNA synthesis, metabolism, and signal transduction. The enzyme binds IMP and the cofactor NAD(+) in random order, IMP is converted to XMP, NAD(+) is reduced to NADH, and finally, NADH and then XMP are released sequentially. XMP is subsequently converted into GMP by GMP synthetase. Drugs that decrease GMP synthesis by inhibiting IMPDH have been shown to have antiproliferative as well as antiviral activity. Several drugs are in use that target the substrate- or cofactor-binding site; however, due to differences between the mammalian and microbial isoforms, most drugs are far less effective against the microbial form of the enzyme than the mammalian form. The high resolution crystal structures of the protozoan parasite Tritrichomonas foetus IMPDH complexed with the inhibitor ribavirin monophosphate as well as monophosphate together with a second inhibitor, mycophenolic acid, are presented here. These structures reveal an active site cation identified previously only in the Chinese hamster IMPDH structure with covalently bound IMP. This cation was not found previously in apo IMPDH, IMPDH in complex with XMP, or covalently bound inhibitor, indicating that the cation-binding site may be catalysis-dependent. A comparison of T. foetus IMPDH with the Chinese hamster and Streptococcus pyogenes structures reveals differences in the active site loop architecture, which contributes to differences in cation binding during the catalytic sequence and the kinetic rates between bacterial, protozoan, and mammalian enzymes. Exploitation of these differences may lead to novel inhibitors, which favor the microbial form of the enzyme.     

Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv)2 bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2. ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC.     

Host-based ribavirin resistance influences hepatitis C virus replication and treatment response

Many individuals infected with hepatitis C virus (HCV) develop a chronic infection, and of those who are treated with pegylated interferon and ribavirin (RBV), many do not respond. While the nucleoside analog RBV improves treatment outcome, and will likely be an important component of therapy with next-generation viral inhibitors, RBV's mechanism is controversial. Most of RBV's proposed mechanisms require RBV import into cells. Therefore, we explored whether host-based RBV resistance develops through reduced cellular uptake, akin to chemotherapy resistance in some cancers. We examined the effect of host-based RBV resistance on HCV replication in cultured hepatoma Huh7.5 liver cells and whether RBV resistance develops in HCV patients. When Huh7.5 cells were exposed to RBV, resistance developed through reduced RBV uptake via the ENT1 nucleoside transporter and antiviral efficacy was reduced. The uptake defect in RBV-resistant cells was specific to RBV, since transport of another ENT1 substrate, cytidine, was unaffected. Importantly, RBV uptake significantly declined in HCV patient peripheral blood mononuclear cells (PBMCs) following 4 weeks of therapy. Furthermore, maintenance of RBV uptake correlated with rapid treatment response. Our results uncovered a novel form of antiviral drug resistance and suggest that host-based RBV resistance develops in HCV patients undergoing therapy and that maintenance of RBV uptake may contribute to rapid viral clearance.     

Ribavirin augments doxorubicin's efficacy in human hepatocellular carcinoma through inhibiting doxorubicin‐induced eIF4E activation

Activation of eukaryotic translation initiation factor 4E (eIF4E) is a cellular survival mechanism in response to chemotherapy in cancers. In this work, we demonstrate that targeting eIF4E by ribavirin sensitizes hepatocellular carcinoma (HCC) cell response to doxorubicin. Ribavirin inhibits growth and survival of HCC cells, and to a greater extent than in normal liver cells. Its combination with doxorubicin achieves greater efficacy than single drug in vitro and in vivo. Ribavirin suppresses phosphorylation of molecules involved in Akt/mTOR/eIF4E pathway. Overexpression of the phosphomimetic form (S209D) but not the nonphosphorylatable form (S209A) eIF4E significantly reverses the inhibitory effects of ribavirin. Interestingly, doxorubicin significantly increases p‐eIF4E(S209) level in a dose‐ and time‐dependent manner, suggesting that doxorubicin induces eIF4E activation in HCC cells. In addition, eIF4E activation induced by doxorubicin in HCC cells is inhibited by ribavirin. Our work demonstrates the greater efficacy of ribavirin and doxorubicin combination and its underlying mechanisms.     

Interferons and ribavirin effectively inhibit Norwalk virus replication in replicon-bearing cells

The development of effective therapies for noroviral gastroenteritis has been hampered by the absence of a cell culture system. Recently, we reported the generation of Norwalk virus (NV) replicon-bearing cells in BHK21 and Huh-7 cells and demonstrated that alpha interferon (IFN-alpha) effectively inhibited the replication of NV in these cells. In continuing studies for screening potential antinoroviral agents, we tested IFN-gamma and ribavirin for their effects on NV replication in the cells. Like IFN-alpha, IFN-gamma inhibited the replication of NV in the replicon-bearing cells, showing the reduction of the NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED(50)) of the NV genome and protein was calculated to be approximately 40 units/ml. When ribavirin was applied to the cells, it effectively reduced the NV genome and protein with the ED(50) calculated as approximately 40 microM. The combination of IFN-alpha and ribavirin showed additive effects on the inhibition of NV replication. With the addition of guanosine to the ribavirin treatment, moderately reversed antiviral effects were observed, suggesting that the ribavirin effect may be associated with the depletion of GTP in the cells. Sequencing analysis of the conserved polymerase regions of NV in the ribavirin-treated (100 microM) and nontreated groups showed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations. The NV replicon-bearing cells provide an excellent tool for screening potential antinoroviral agents, and our results indicated that IFNs and ribavirin may be good therapeutic options for noroviral gastroenteritis.     

Cytogenetic effects of ribavirin on mouse bone marrow

The micronucleus test and mitotic chromosome analysis were used to study the in vivo mutagenic activity of ribavirin on bone marrow cells of Swiss albino mice. To determine the incidence of micronuclei, mice were injected i.p. twice, at an interval of 24 h. with the drug at doses of 20, 100 and 200 mg/kg. Animals were killed 6 h after the second dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. Ribavirin significantly (P less than 0.05) induced micronuclei in polychromatic erythrocytes at all doses. A study was conducted to investigate the cytogenetic effect of the drug on mitotic chromosomes. Ribavirin at 200 mg/kg/day was administered to mice for 3 and 5 days. Repeated treatment with the high dose of ribavirin produced a highly significant (P less than 0.02) increase in abnormal metaphase spreads. The results indicate that ribavirin is mutagenic to bone marrow cells of mice as evaluated by the micronucleus test and by chromosome analysis.     

The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen

The ribonucleoside analog ribavirin (1-beta-D-ribofuranosyl-1,2, 4-triazole-3-carboxamide) shows antiviral activity against a variety of RNA viruses and is used in combination with interferon-alpha to treat hepatitis C virus infection. Here we show in vitro use of ribavirin triphosphate by a model viral RNA polymerase, poliovirus 3Dpol. Ribavirin incorporation is mutagenic, as it templates incorporation of cytidine and uridine with equal efficiency. Ribavirin reduces infectious poliovirus production to as little as 0. 00001% in cell culture. The antiviral activity of ribavirin correlates directly with its mutagenic activity. These data indicate that ribavirin forces the virus into 'error catastrophe'. Thus, mutagenic ribonucleosides may represent an important class of anti-RNA virus agents.     

An antiviral mechanism investigated with ribavirin as an RNA virus mutagen for foot-and-mouth disease virus

To prove whether error catastrophe/lethal mutagenesis is the primary antiviral mechanism of action of ribavirin against foot-and-mouth disease virus (FMDV). Ribavirin passage experiments were performed and supernatants of Rp1 to Rp5 were harvested. Morphological alterations as well as the levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected using the supernatants of Rp1 to Rp5 and control were measured by microscope, real-time RT-PCR, western-blotting and plaque assays, respectively. The mutation frequency was measured by sequencing the complete P1- and 3D-encoding region of FMDV after a single round of virus infection from ribavirin-treated or untreated FMDV-infected cells. Ribavirin treatment for FMDV caused dramatically inhibition of multiplication in cell cultures. The levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected were more greatly reduced along with the passage from Rp1 to Rp5, moreover, nucleocapsid protein could not be detected and no recovery of infectious virus in the supernatant or detection of intracellular viral RNA was observed at the Rp5-infected cells. A high mutation rate, giving rise to an 8-and 11-fold increase in mutagenesis and resulting in some amino acid substitutions, was found in viral RNA synthesized at a single round of virus infection in the presence of ribavirin of 1000 microM and caused a 99.7% loss in viral infectivity in contrast with parallel untreated control virus. These results suggest that the antiviral molecular mechanism of ribavirin is based on the lethal mutagenesis/error catastrophe, that is, the ribavirin is not merely an antiviral reagent but also an effective mutagen.     

Ribavirin can be mutagenic for arenaviruses

Arenaviruses include several important human pathogens, and there are very limited options of preventive or therapeutic interventions to combat these viruses. An off-label use of the purine nucleoside analogue ribavirin (1-β-d-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is the only antiviral treatment currently available for arenavirus infections. However, the ribavirin antiviral mechanism action against arenaviruses remains unknown. Here we document that ribavirin is mutagenic for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) in cell culture. The mutagenic activity of ribavirin on LCMV was observed under single- and multiple-passage regimes and could not be accounted for by a decrease of the intracellular GTP pool promoted by ribavirin-mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). Our findings suggest that the antiviral activity of ribavirin on arenaviruses might be exerted, at least partially, by lethal mutagenesis. Implications for antiarenavirus therapy are discussed.