Figure 2.

Avian ANP32A₂₉ Is Sufficient to Restore SpeciesRestricted Avian Polymerase Activity and Replication Activity of human-style (PB2 K627) and avian-style (PB2 E627) polymerases was measured in the presence of increasing concentrations of the indicated ANP32A proteins. Protein expression was assessed via western blot. ANP32A₃₃ selectively rescues restricted avian polymerase activity in human 293T cells. ANP32A₂₉ is sufficient to restore polymerase activity in human cells (left) but does not significantly affect human viral polymerase in human cells oreither polymerase in chicken LMH cells (right). Insertion of the avian 29 amino repeat into huANP32A (huANP32A₊₂₉) enhances activity, whereas deletion of the repeat in chANP32A (chANP32A_D) disables its function. chANP32A₃₃ is the most potent enhancer of avian polymerase activity in human cells compared with chANP32A₂₉ or chANP32A_33mut. For all assays, polymerase activity was normalized to an internal control and compared with PB2 K627 polymerase in the absence of ANP32A. Data are shown as mean (columns) of n = 3 technical replicates (dots) ± SD derived from representative results of at least three independent biological replicates. C, empty vector control. In (A)–(D), pairwise comparisons between PB2 K627 and E627 at each condition were significant (p < 0.05, Student’s t test) except where indicated as not significant (ns). Replication kinetics of influenza virus encoding the avian S009 RNP (WT) ora human human-adapted mutant (SRK), WSN, or B/Brisbane. WT A549 cells or those stably expressing chANP32A were infected (MOI = 0.1), and viral titers were determined at the indicated time points. Data are shown as average of n = 3 ± SD. *p < 0.05, one-way ANOVA with post hoc Tukey honestly significant difference (HSD) test compared with WT A549 cells. See also Figures S1 and S2.