The polymerase of RNA viruses lacks the proofreading capacity found in the polymerase of DNA viruses, leading to high error rates and low replicative fidelity. Although the high mutation rate of RNA viruses enables them to readily adapt to environmental changes, they also risk 'lethal mutagenesis' when accumulating mutations corrupt essential functions. Here, Smith et al. reveal that, in the case of severe acute respiratory syndrome coronavirus (SARS-CoV), an exoribonuclease domain (ExoN) in non-structural protein 14 provides proofreading activity that protects the virus from mutagenesis.

Previous work in coronaviruses, the largest RNA viruses, had shown that ExoN deletion leads to reduced replicative fidelity and attenuation of virulence. Thus, Smith et al. speculated that ExoN is a proofreading enzyme. To test this hypothesis, they infected cells with wild-type (ExoN+) or ExoN-deleted (ExoN) SARS-CoV in the presence of the mutagenic pyrimidine analogue 5-fluorouracil (5-FU). Indeed, loss of ExoN sensitized the virus to 5-FU, as shown by a 160-fold reduction in viral replication for ExoN SARS-CoV compared with ExoN+ SARS-CoV. Furthermore, genome sequencing of viral populations after 5-FU treatment revealed that ExoN SARS-CoV harboured 3,648 mutations, whereas ExoN+ SARS-CoV accumulated only 259 mutations. Of these mutations, 3,304 and 197 were U-to-C and A-to-G transitions, respectively, which are characteristic for nucleotide mismatches caused by the incorporation of 5-FU metabolites. Taken together, these results show that ExoN proofreading protects SARS-CoV from the deleterious effects of mutagens, such as 5-FU.

ExoN proofreading protects SARS-CoV from the deleterious effects of mutagens

To date, no antiviral treatment exists for coronaviruses; neither ribavirin nor interferon-α, the only two broadly acting antivirals available in the clinic today, have a consistent antiviral effect against SARS-CoV. However, because ribavirin is a mutagenic nucleoside analogue, it might have similar effects to 5-FU in the absence of ExoN. Consistent with this, ExoN murine CoV was more sensitive to ribavirin than ExoN+ murine CoV, although further experiments indicated that other mechanisms besides mutagenesis (for example, reduced RNA synthesis) contributed to this ExoN-dependent effect. As the cells used for SARS-CoV infections failed to take up ribavirin, the sensitivity of ExoN SARS-CoV to ribavirin remains unclear.

Nevertheless, coronavirus ExoN is the first proofreading protein reported in an RNA virus, and targeting ExoN is a promising strategy to develop new coronavirus inhibitors and sensitizers to existing antivirals.