Multiple polymerase acidic (PA) I38X substitutions in influenza A(H1N1)pdm09 virus permit polymerase activity and cause reduced baloxavir inhibition

Background Baloxavir marboxil is an antiviral drug that targets the endonuclease activity of the influenza virus polymerase acidic (PA) protein. PA I38T/M/F substitutions reduce its antiviral efficacy. Objectives To understand the effects of the 19 possible amino acid (AA) substitutions at PA 38 on influenza A(H1N1)pdm09 polymerase activity and inhibition by baloxavir acid, the active metabolite of baloxavir marboxil. Methods Influenza A(H1N1)pdm09 viral polymerase complexes containing all 19 I38X AA substitutions were reconstituted in HEK293T cells in a mini-replicon assay. Polymerase complex activity and baloxavir inhibitory activity were measured in the presence or absence of 50 nM baloxavir acid. Results Only three substitutions (R, K, P) reduced polymerase activity to <79% of I38-WT. When compared with the prototypical baloxavir marboxil resistance marker T38, 5 substitutions conferred 10%–35% reductions in baloxavir acid inhibitory activity (M, L, F, Y, C) and 11 substitutions conferred >50% reductions (R, K, S, N, G, W, A, Q, E, D, H), while two substitutions (V, P) maintained baloxavir acid inhibitory activity. Conclusions Most PA 38 substitutions permit a functional replication complex retaining some drug resistance in the mini-replicon assay. This study provides a targeted approach for virus rescue and analysis of novel baloxavir marboxil reduced-susceptibility markers, supports the consideration of a broader range of these markers during antiviral surveillance and adds to the growing knowledge of baloxavir marboxil resistance profiles.