Baloxavir acid (BXA), derived from the prodrug baloxavir marboxil (BXM), 
potently and selectively inhibits the cap-dependent endonuclease within the 
polymerase PA subunit of influenza A and B viruses. In clinical trials, single 
doses of BXM profoundly decrease viral titers as well as alleviating influenza 
symptoms. Here, we characterize the impact on BXA susceptibility and replicative 
capacity of variant viruses detected in the post-treatment monitoring of the 
clinical studies. We find that the PA I38T substitution is a major pathway for 
reduced susceptibility to BXA, with 30- to 50-fold and 7-fold EC50 changes in A 
and B viruses, respectively. The viruses harboring the I38T substitution show 
severely impaired replicative fitness in cells, and correspondingly reduced 
endonuclease activity in vitro. Co-crystal structures of wild-type and I38T 
influenza A and B endonucleases bound to BXA show that the mutation reduces van 
der Waals contacts with the inhibitor. A reduced affinity to the I38T mutant is 
supported by the lower stability of the BXA-bound endonuclease. These 
mechanistic insights provide markers for future surveillance of treated 
populations.