Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50 The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.

Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease

Since the first antiviral drug, idoxuridine, was approved in 1963, 90 antiviral drugs categorized into 13 functional groups have been formally approved for the treatment of the following 9 human infectious diseases: (i) HIV infections (protease inhibitors, integrase inhibitors, entry inhibitors, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and acyclic nucleoside phosphonate analogues), (ii) hepatitis B virus (HBV) infections (lamivudine, interferons, nucleoside analogues, and acyclic nucleoside phosphonate analogues), (iii) hepatitis C virus (HCV) infections (ribavirin, interferons, NS3/4A protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors), (iv) herpesvirus infections (5-substituted 2'-deoxyuridine analogues, entry inhibitors, nucleoside analogues, pyrophosphate analogues, and acyclic guanosine analogues), (v) influenza virus infections (ribavirin, matrix 2 protein inhibitors, RNA polymerase inhibitors, and neuraminidase inhibitors), (vi) human cytomegalovirus infections (acyclic guanosine analogues, acyclic nucleoside phosphonate analogues, pyrophosphate analogues, and oligonucleotides), (vii) varicella-zoster virus infections (acyclic guanosine analogues, nucleoside analogues, 5-substituted 2'-deoxyuridine analogues, and antibodies), (viii) respiratory syncytial virus infections (ribavirin and antibodies), and (ix) external anogenital warts caused by human papillomavirus infections (imiquimod, sinecatechins, and podofilox). Here, we present for the first time a comprehensive overview of antiviral drugs approved over the past 50 years, shedding light on the development of effective antiviral treatments against current and emerging infectious diseases worldwide.

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Approved Antiviral Drugs over the Past 50 Years

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.

https://www.pnas.org/content/pnas/98/12/6895.full.pdf

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

Favipiravir (T-705), a novel viral RNA polymerase inhibitor.

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.

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

The antiviral activity of the synthetic nucleoside, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), against measles virus in Vero cell cultures was substantially reversed by xanthosine, guanosine, and to a slightly lesser extent by inosine. Virazole 5'-phosphate was subsequently found to be a potent competitive inhibitor of inosine 5'-phosphate dehydrogenase (IMP:NAD(+) oxidoreductase, EC 1.2.1.14) isolated from Escherichia coli (K(m) = 1.8 x 10(-5) M) with a K(i) of 2.7 x 10(-7) M. Guanosine 5'-phosphate (GMP) was a competitive inhibitor of this enzyme with a K(i) of 7.7 x 10(-5) M. Virazole 5'-phosphate was similarly active against IMP dehydrogenase isolated from Ehrlich ascites tumor cells, with a K(i) of 2.5 x 10(-7) M. The K(m) for this enzyme was 1.8 x 10(-5) M, and the K(i) for GMP was 2.2 x 10(-4) M. These results suggest that the antiviral activity of Virazole might be due to the inhibition of GMP biosynthesis in the infected cell at the step involving the conversion of IMP to xanthosine 5'-phosphate. This inhibition would consequently result in inhibition of the synthesis of vital viral nucleic acid.

Mechanism of Action of 1-β-D-Ribofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole), A New Broad-Spectrum Antiviral Agent

The synthetic nucleoside ribavirins has been shown to have broad spectrum antiviral activity both in vitro and in vivo. and recent clinical studies indicate that it is also efficacious in man.I-l2 Streeter et al.I3 have shown that the 5’-phosphate of ribavirin is a potent inhibitor of IMP-dehydrogenase, and have suggested that its antiviral effect may be mediated in part by the inhibition of this crucial biosynthetic enzyme. Streeter et aI.,l3 have also shown that ribavirin-5’-phosphate is found in the liver of rats after oral dosing, and that the enzyme most probably responsible for the phosphorylation of ribavirin is deoxyadenosine kinase. Further investigations, which are reported here, were directed towards determining if ribavirin-5’-phosphate could be further phosphorylated to the corresponding diand tri-phosphates, and towards finding out if any additional metabolites of ribavirin are produced in the whole animal. The primary purpose for studying the metabolism of ribavirin is to gain further understanding of the mechanism of action of the drug. Metabolites of ribavirin other than the 5’-phosphate may be acting at sites in the cell other than IMP-dehydrogenase. Knowledge of what metabolites are formed may give some insight into what those other sites may be.

The relationship between the metabolism of ribavirin and its proposed mechanism of action.

Philosophy. Present address, Department of Biology, University of California, San Diego. 1 Sypherd, P. S., N. Strauss, and H. P. Treffers, Biochem. Biophys. Res. Comm., 7, 477 (1962). 2 Spiegelman, S., H. 0. Halvorson, and R. Ben-Ishi, in Amino Acid Metabolism (Baltimore: Johns Hopkins Press, 1955), pp. 124-170. 3Torriani, A.-M., Biochem. Biophys. Acta, 19, 224 (1956). 4Kameyama, T., and G. D. Novelli, Arch. Biochem. Biophys., 97, 529 (1962). 1 Lowry, O., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 6 Dische, Z., in The Nucleic Acids (New York: Academic Press, 1955), vol. 1, pp. 285. 7Rickenberg, H. V., C. Yanofsky, and D. M. Bonner, J. Bact., 66, 683 (1953). 8 Cohn, M., and J. Monod, Biochem. Biophys. Acta, 7, 153 (1951). 9 Jacob, F., and J. Monod, in Cellular Regulatory Mechanisms, Cold Spring Harbor Symposia on Quantitative Biology, vol. 26 (1961). 10 Pardee, A. B., F. Jacob, and J. Monod, J. Mol. Biol., 1, 165 (1959).

Induction by an RNA phage of an enzyme catalyzing incorporation of ribonucleotides into ribonucleic acid.

Topical application of 1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole) significantly inhibited the development of herpetic keratitis in the eyes of rabbits, as determined by both infectivity and Draize scoring parameters. Significant inhibition of the infection was demonstrated with 10% concentrations of Virazole; a 1% solution had a moderate effect, whereas doses of 0.1 and 0.01% had little activity in this system. A 5% concentration of Virazole similarly inhibited vaccinia keratitis in rabbits. Encephalitis-induced mortality in hamsters initially infected intraocularly with herpesvirus was significantly prevented or inhibited by topical application of 5, 10, and 20% concentrations of Virazole. Surviving, treated hamsters had no signs of herpes keratitis. The 20% concentration was the approximate LD 50 in hamsters. Virazole administered subcutaneously or intraperitoneally to mice did not appreciably alter the course of herpes virus- or vaccinia virus-induced encephalitis in these animals, although in a herpesvirus experiment direct injection of the drug into the brains 3 hr prior to virus inoculation resulted in a significant survivor increase.

Effect of 1-β-d-Ribofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole, ICN 1229) on Herpes and Vaccinia Keratitis and Encephalitis in Laboratory Animals

Lionel N. Simon

Papers

Mechanism of Action of 1-β-D-Ribofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole), A New Broad-Spectrum Antiviral Agent

The relationship between the metabolism of ribavirin and its proposed mechanism of action.

Induction by an RNA phage of an enzyme catalyzing incorporation of ribonucleotides into ribonucleic acid.

Effect of 1-β-d-Ribofuranosyl-1,2,4-Triazole-3-Carboxamide (Virazole, ICN 1229) on Herpes and Vaccinia Keratitis and Encephalitis in Laboratory Animals

3-Substituted 5,7-dimethylpyrazolo(1,5-a)pyrimidines, 3',5'-cyclic-AMP phosphodiesterase inhibitors. 1.