FIG. 2.

Purification and endoribonucleolytic activity of Nsp15. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the recombinant SARS-CoV purified from a Ni-NTA column. Nsp15 was loaded at two concentrations, and the gel was stained with Coomassie brilliant blue. The positions (in kilodaltons) of molecular mass markers (M) are shown to the left of the gel. (B) A gel image demonstrating the cleavage of a radiolabeled 10-nt RNA and nucleotide resolution of the cleavage products. Nsp15 was added to one master reaction mixture, and aliquots of the reaction mixture were removed at the times shown above the gel image for electrophoresis in a 7.5 M urea-20% polyacrylamide gel. The sequence of the RNA substrate, R10.2, is shown above the gel, and the sizes of the bands (in nucleotides) are indicated to the left of the gel. The percentage of the 10-nt R10.2 that remains after each treatment (% FL) is indicated under the gel. The smaller gel at the right shows that R12, which lacks uridylates, is not cleaved to the same extent as R10.2. (C) Copurification of the endoribonucleolytic activity with the recombinant protein. The protein enriched from the metal affinity column was fractionated over a Mono Q column, with Nsp15 eluting primarily in fractions 17 and 18. The amount of endoribonucleolytic activity in the fraction (% Activity) is indicated. The activities in these column fractions were normalized to that of fraction 17 (set at 100%). (D) Sequences of SK21 and potential products that can be generated from a DNA template coding for SK21 by the T7 RNA polymerase when UTP is absent. (E) Cleavage of a radiolabeled RNA SK21. The percentage of the full-length substrate RNA remaining after each treatment (% FL) is indicated under the gel. Lanes where the input RNA was not treated with Nsp15 (φ) are indicated above the gel. (F) Analysis of the cleavage site preferred by Nsp15. The final product generated by Nsp15 from SK21 should be 5 nt long if cleavage occurs 5′ of uridylates. If cleavage occurs 3′ of uridylates, the stable product should be 6 nt long. To examine the length of the stable Nsp15 product, DNA template T7-SK21 was used to transcribe RNA products in the absence of UTP. T7 polymerase should generate an abundant 5-nt RNA product. Other slightly longer products could be accounted for by terminal nucleotide addition and/or nucleotide misincorporation. End-labeled SK21 RNA was treated with Nsp15 for 1 h (+) or left alone (−) (lanes 1 and 2) as described above. To confirm the length of the Nsp15 cleavage product, T7 transcription was performed in the absence of UTP using template T7-SK21 as described above for panel F (lane 3). Samples from lanes 1 and 3 were mixed together and loaded in lane 4 to show that both products are the same length.