Figure 2.

chANP32A Contains a SIM-like Sequence Important for Promoting PB2-627E vPol Activity

(A) Western blot analysis of lysates from 293T cells transfected with the indicated constructs.

(B) SUMOylation of chANP32A minimally affects PB2-627E vPol activity. chANP32A-K68R and chANP32A-K0 were tested for their abilities to promote PB2-627E/K vPol activities, as described in Figure 1B.

(C) Impact of SUMO proteases on chANP32A-K0 function during PB2-627E vPol activity. Experiment performed as in Figure 1E.

(D) Specificity index of chANP32A-WT- versus chANP32A-K0-mediated PB2-627E vPol activities in the presence of the indicated SUMO protease (original data from Figure 1E and C; values represent 1 − K0/WT ± SD).

(E) (Top) Sequence analysis of the region surrounding the 33 amino-acid insertion in chANP32A. Hydrophobic residues (pink) and acidic stretches (blue), which comprise a SIM-like sequence, are highlighted. (Bottom) Alignment of bona fide SIM sequences from various human proteins.

(F) Panel of chANP32A mutants generated.

(G and H) Impact of chANP32A SIM-like sequence on PB2-627E (G) or PB2-627K (H) vPol activity. Experiment performed as in (B).

(I) Western blot analysis of 293T cell lysates from (G).

(J) Immunofluorescence analysis of the indicated FLAG-chANP32A proteins in transfected MRC5 cells. Scale bars represent 10 μm.

In (B), (C), (G), and (H), bars represent mean values from three independent experiments (±SD). Significance was determined by Student’s t test (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001; NS, non-significant).