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.