Figure 3.

Heterologous SIM Sequences Permit huANP32A to Promote PB2-627E vPol Activity

(A) Panel of constructs used and their respective SIM-like sequences.

(B and C) In vitro SUMO binding assays. Recombinant 6X-His tagged SUMO1 (B) or SUMO2 (C) was added to the indicated pre-immobilized FLAG-tagged constructs. Subsequently, precipitated proteins were detected by western blot.

(D) Sequence alignment of huANP32A chimeric constructs generated to include the 33 amino-acid insertion of chANP32A or heterologous SIM sequences from modified PIASxα or PIAS4. Hydrophobic residues (pink) and acidic stretches (blue), which comprise SIM-like sequences, are highlighted.

(E) Impact of heterologous SIM sequences on the ability of huANP32A to promote PB2-627E vPol activity. Experiment performed as in Figure 1B. Bars represent mean values from three independent experiments (±SD). Significance was determined by Student’s t test (^∗p < 0.05; ^∗∗p < 0.01).

(F) Western blot analysis of 293T cell lysates from (E).

(G) In vitro SUMO-binding assay performed with the huANP32A chimeric constructs as described in (B).