Small-Molecule Antiviral β-d- N 4 -Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance.

Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN) β-d-N4-Hydroxycytidine (NHC) (EIDD-1931;Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0 17 μM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0 56 μM) with minimal cytotoxicity NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease However, no antivirals have been approved to treat these infections Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-N4-hydroxycytidine (NHC), against two divergent CoVs Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections [ABSTRACT FROM AUTHOR] Copyright of Journal of Virology is the property of American Society for Microbiology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission However, users may print, download, or email articles for individual use This abstract may be abridged No warranty is given about the accuracy of the copy Users should refer to the original published version of the material for the full abstract (Copyright applies to all Abstracts )