The severe acute respiratory syndrome coronavirus Nsp15 protein is an endoribonuclease that prefers manganese as a cofactor

Abstract

Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5' of uridylates of RNAs. Blocking either the 5' or 3' terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn(2+) at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg(2+) and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn(2+) needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.

FIG. 1.

Schematic of the SARS-CoV genome and the sequence of Nsp15. The approximate locations of polyproteins 1a (pp1a) and 1ab (pp1ab) in the SARS-CoV genome are indicated. Proteins made from subgenomic messages encoded by the 3′ portion of the SARS-CoV genome are shown. For clarity, the individual coding sequences are not shown. The Nsp15 sequence is from the Urbani isolate and is represented by the standard one-letter code. Comparable sequences from MHV and IBV were aligned with the CLUSTAL program (28). Residues that have identical side chains (asterisks), functionally similar side chains (colons), and somewhat similar side chains (periods) are indicated under the sequences. Gaps introduced to maximize alignment are indicated by dashes. The central portion of the MHV protein is more divergent than the comparable sequence from the SARS-CoV and IBV proteins. To help demonstrate the possible relevance of the alignments, the PHD program (21) was used to predict the positions of secondary structures predicted within each polypeptide. The residues putatively involved in the formation of β-strands or α-helices are underlined or shaded, respectively. Sequences under the thick black lines represent the region with similarities to the putative XendoU active site, as described by Snijder et al. (25).

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.

FIG. 3.

Interaction of Nsp15 with DNA and heparin. (A) PhosphorImage of 7.5 M urea-20% polyacrylamide gel showing 5′-end-labeled DNA oligonucleotide (D23.2) incubated in the presence (+) or absence (−) of Nsp15 under standard assay conditions. Each treatment was done in triplicate. (B) Effects of heparin and DNA on cleavage of R10.2 RNA by Nsp15. R10.2 digestion was performed in the absence or presence of various concentrations of either heparin or DNA (indicated above the bars). After the RNAs were resolved on urea-polyacrylamide gels, uncleaved R10.2 was quantitated and shown as a percentage [FL (%)]. Substrate RNA incubated in the absence (−) of Nsp15 was set at 100%. Each error bar represents 1 standard deviation from the mean for each treatment.

FIG. 4.

Requirements for divalent metal ions for SARS-CoV endoribonucleolytic activity. (A) Mg²⁺ and Mn²⁺ concentrations needed for endoribonucleolytic activity of Nsp15. RNA R10.2 was used as the substrate in the titrations. All divalent metals used in this figure were in the form of chloride salts. (B) Examination of the effects of other divalent metals on cleavage of R10.2 by Nsp15. The effects of several divalent metals (at 0.5 or 5 mM) were quantified and are shown below the gel. Only the full-length RNA (FL) is shown. φ indicates no divalent ion. (C) Effects of Mn²⁺ on the spectroscopic properties of the SARS-CoV Nsp15. Change in intrinsic fluorescence (FU) of Nsp15 in response to increasing Mn²⁺ concentration. The emission maximum of Nsp15 was determined to be at 337 nm. Scans at increasing concentrations of divalent metal were taken, and values at 337 nm were plotted. (D) Modeling of the binding constant for Mn²⁺. A 1:1 model was fitted to the binding isotherm to derive K_d using equation 1 (see Materials and Methods).

FIG. 5.

Substrate requirements for endoribonucleolytic activity of SARS-CoV Nsp15. (A) RNA substrates with chemically modified 3′ and 5′ termini. RNA LE19P, with a covalently attached 3′ puromycin is a weakly base-paired RNA that could form a weakly stable intramolecular hairpin or partially base-paired dimer (19). F16.2 is a 16-nt RNA that was synthesized with a 5′ fluorescein. The amount of cleavage was normalized to the percentage of full-length RNA remaining after the treatment (% FL). The RNA used in each experiment and whether Nsp15 was added (+) or not (−) are indicated above the gels. Digestion of LE19P is shown as a PhosphorImage of radiolabeled RNA, while F16.2 was from a toluidine blue-stained gel. (B) Comparison of the rate of cleavage of single- and double-stranded RNAs. ds32 is a double-stranded RNA with complete complementarity in the two strands. LE21, a single-stranded RNA with minimal intramolecular base pairing, was used to generate ds34. The amount of each input RNA was normalized to 100%. SL13, a 13-nt RNA was produced by in vitro transcription by use of T7 polymerase. 5′-end phosphate was replaced with radiolabeled phosphate by sequential treatment with alkaline phosphatase and T4 polynucleotide kinase. Radiolabeled RNA was gel purified before use.

FIG. 6.

Comparison of endoribonuclease activities of the SARS-CoV Nsp15 and orthologs from other coronaviruses. (A) Activities of the IBV ortholog. An image of the cleavage products from R10.2 and R12 are shown. The proteins and RNA substrates used in each reaction mixture are indicated above the gel. The contents of all lanes were exposed to Nsp15, but an aliquot was removed and placed in the denaturing gel loading buffer within seconds of addition of the enzyme (<1 min). The sizes of the products (in nucleotides) are indicated to the left of the gel. Quantification of endoribonuclease activity, shown below the gel, refers to the percentage of the full-length RNA (% FL) remaining in each lane. (B) Activity of the MHV ortholog.