Fig. 1.

Mutation of the nsP3 macro domain does not affect binding of PAR. Homology modeling of the SINV nsP3 macro domain using Af1521 (a macro domain protein from Archaeoglobus fulgidus) as a template (A,B). The predicted structure of the nsP3 macro domain (blue) and the published structure of Af1521 (green) are superimposed, and overall front and rear view images (A) and close view of mutated amino acids (B) are presented. (C) Mutagenesis of the nsP3 macro domain. Amino acids for mutagenesis were chosen based on identification of conserved residues and homology modeling with the Archaeoglobus fulgidus macro domain. One amino acid at position 10 or 24 in nsP3 was mutated from asparagine (N) to alanine (A) for SINV single mutant (SM10 and SM24, respectively), and amino acids at positions 10 and 24 were mutated from N to A for SINV double mutant (DM). (D) PAR binding assay. The nsP3 macro domain WT, SM10, SM24 and DM, and lysozyme (negative control) were dot-blotted onto nitrocellulose membrane, and the membrane was incubated with or without PAR, followed by incubation with antibody to PAR for detection.