Structural and Biochemical Characterization of Endoribonuclease Nsp15 Encoded by Middle East Respiratory Syndrome Coronavirus

Abstract

Nonstructural protein 15 (Nsp15) encoded by coronavirus (CoV) is a nidoviral uridylate-specific endoribonuclease (NendoU) that plays an essential role in the life cycle of the virus. Structural information on this crucial protein from the Middle East respiratory syndrome CoV (MERS-CoV), which is lethally pathogenic and has caused severe respiratory diseases worldwide, is lacking. Here, we determined the crystal structure of MERS-CoV Nsp15 at a 2.7-Å resolution and performed the relevant biochemical assays to study how NendoU activity is regulated. Although the overall structure is conserved, MERS-CoV Nsp15 shows unique and novel features compared to its homologs. Serine substitution of residue F285, which harbors an aromatic side chain that disturbs RNA binding compared with that of other homologs, increases catalytic activity. Mutations of residues residing on the oligomerization interfaces that distort hexamerization, namely, N38A, Y58A, and N157A, decrease thermostability, decrease affinity of binding with RNA, and reduce the NendoU activity of Nsp15. In contrast, mutant D39A exhibits increased activity and a higher substrate binding capacity. Importantly, Nsp8 was found to interact with both monomeric and hexameric Nsp15. The Nsp7/Nsp8 complex displays a higher binding affinity for Nsp15. Furthermore, Nsp8 and the Nsp7/Nsp8 complex also enhance the NendoU activity of hexameric Nsp15 in vitro Taking the findings together, this work first provides evidence on how the activity of Nsp15 may be functionally mediated by catalytic residues, oligomeric assembly, RNA binding efficiency, or the possible association with other nonstructural proteins.IMPORTANCE The lethally pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) pose serious threats to humans. Endoribonuclease Nsp15 encoded by coronavirus plays an important role in viral infection and pathogenesis. This study determines the structure of MERS-CoV Nsp15 and demonstrates how the catalytic activity of this protein is potentially mediated, thereby providing structural and functional evidence for developing antiviral drugs. We also hypothesize that the primase-like protein Nsp8 and the Nsp7/Nsp8 complex may interact with Nsp15 and affect enzymatic activity. This contributes to the understanding of the association of Nsp15 with the viral replication and transcription machinery.

Keywords: MERS-CoV; crystal structure; endoribonuclease; oligomerization.

FIG 1

Overall structure of MERS-Nsp15. (A) Cartoon representation and schematic diagram of the overall structure of a protomer. A schematic diagram of the domain boundaries in the amino acid sequence is shown above the cartoon. The N-terminal domain, middle domain, and C-terminal domain are colored red, green, and blue, respectively. The surface transparency is set to 20%. (B) Side view of the cartoon representation of the hexamer. Three protomers within a trimer are colored green, yellow and, blue, with the other trimer colored gray. (C) Side view and top view of the distribution of the N-terminal domain, middle domain, and C-terminal domain within the hexamer by surface representation. Six protomers are colored differently. The N-terminal domain, middle domain, and C-terminal domain within one protomer are colored red, green, and blue and are labeled N, M, and C, respectively. Six protomers form a hexamer through the N- to N-terminal interaction.

FIG 2

Comparison of MERS-Nsp15 with SARS-Nsp15 and MHV Nsp15. (A) The structures of MERS-Nsp15, SARS-Nsp15 (PDB code 2H85), and MHV (PDB code 2GTH) Nsp15 are superimposed together. Monomers and trimers are shown separately, and RMSD of Cα atoms are listed. (B) Three domains of the monomer for MERS-Nsp15, SARS-Nsp15, and MHV Nsp15 are aligned, and RMSD of Cα atoms are listed.

FIG 3

Sequence alignment of MERS-Nsp15 with Nsp15s of coronaviruses and arteriviruses. Key residues within the catalytic center are marked by red arrowheads, and residues in the subunit interfaces are marked by blue circles. Strictly conserved residues are depicted in white characters on a black background. Secondary-structure elements are shown above the alignment (helices are represented by squiggles, β-strands by arrows, and turns by the letters TT). Sequences are aligned using ClustalW (30), and the figure was drawn with ESPript (31). NCBI accession numbers are as follows: YP_009047226 for Middle East respiratory syndrome-related coronavirus (MERS-CoV), AGT21317 for SARS coronavirus (SARS-CoV), NP_740619 for murine hepatitis virus strain A59 (MHV), AGT21366 for human coronavirus 229E (HCoV-229E), AIM47753 for porcine epidemic diarrhea virus (PEDV), AGZ84515 for feline infectious peritonitis virus (FIPV), ABG89333 for transmissible gastroenteritis virus virulent Purdue (TGEV), and NP_705592 for equine arteritis virus (EAV).

FIG 4

Identification and characterization of residues within the catalytic center of MERS-Nsp15. (A) Structural superposition of MERS-Nsp15 and SARS-Nsp15 (PDB code 2H85) (13). The structure of MERS-Nsp15 is colored magenta, and the structure of SARS-Nsp15 is colored yellow. The catalytic center of MERS-Nsp15 superimposed with SARS-Nsp15 is enlarged in panel A (the cartoon transparency is set to 20%). Residues discussed in this paper are labeled with stick representations (magenta, MERS-Nsp15; yellow, SARS-CoV Nsp15). Equivalent residues located in the catalytic site of SARS-Nsp15 are listed at the bottom. (B) Protein-RNA binding profiles and the inhibitory effects of the cleavage of the fluorescent substrate by RNAs R1, R2, and R3. R1 contains 20 rU nucleotides and exhibits a K_d value of 818.28 ± 50.39 nM. R2 binds to Nsp15 with a K_d value of 1,190.52 ± 137.91 nM. R3 exhibits a K_d value of 932.49 ± 49.40 nM. The activity of the wild-type Nsp15 in the absence of R1, R2, or R3 is set to 100%. The fluorescence intensity was measured at each RNA (R1 to R3) concentration, and the values shown are the averages from three measurements. (C) DSF profile of wild-type Nsp15. All mutants listed in panel A share melting temperatures and DSF profiles similar to those of wild-type Nsp15. (D) NendoU activity profile for the mutants with alanine substitution of residues located within the catalytic site. FRET-based assays for different mutants were conducted, and the reaction rate was calculated. (E) NendoU activity profile for active-site mutants with alanine substitutions and the corresponding residue in SARS-Nsp15.

FIG 5

Stick representation of residues involved in the interprotomer interaction of MERS-Nsp15. A side view of the surface representation of the interactions within three protomers is shown. Protomers A, B, and C are colored blue, yellow, and magenta, respectively. Residues involved in the subunits interaction are labeled with stick representations (blue, subunit A; yellow, subunit B; and magenta, subunit C [the cartoon transparency is set to 20%]). Four contact regions are boxed and enlarged. The atomic distances were measured with PyMOL.

FIG 6

Biochemical characterization of mutants critical for the oligomeric assembly of MERS-Nsp15. (A) Analytical ultracentrifuge (AUC) analysis of mutants containing mutations located at interaction surfaces. AUC profiles of the wild-type protein and the D39A, E263A, N38A, Y58A, and N157A mutants are shown. The first peak at approximately 40 kDa represents the position of the monomer, and the second peak at approximately 240 kDa represents the position of the hexamer. (B) DSF profiles of wild-type Nsp15 and mutants D39A, E263A, N38A, Y58A, and N157A. (C) Protein-RNA binding profiles of the Nsp15 mutants determined by fluorescence polarization (FP). Alanine substitution was performed at K286 in the wild type and mutants (D39A, E263A, N38A, Y58A, and N157A) for this assay to prevent substrate digestion during the experiment. (D) NendoU activity profiles for the mutants related to oligomeric assembly.

FIG 7

Effects of Mn²⁺ and Mg²⁺ on MERS-Nsp15. (A) Catalytic rate profile for the effects of Mn²⁺ on the activity of MERS-Nsp15. (B) Nsp15 (K286A)-RNA binding profiles for Nsp15 with increasing Mn²⁺ concentration determined via fluorescence polarization. (C) Catalytic rate profile for the effects of Mg²⁺ on the activity of MERS-Nsp15. (D) Nsp15 (K286A)-RNA binding profiles for Nsp15 with increasing Mg²⁺ concentrations determined via fluorescence polarization.

FIG 8

Influence of MERS-CoV Nsp8 and the Nsp7/Nsp8 complex on MERS-Nsp15. (A) Pulldown assays detecting the interaction of the Nsp12/Nsp15, Nsp8/Nsp15, or Nsp8/Nsp15 mutants. (B) MST binding curves for the titration of fluorescently labeled Nsp15 into Nsp8 (green) and Nsp12 (orange). Error bars showing SD were calculated from triplicate experiments. (C) Determination of the binding affinities of Nsp15 with Nsp7 (black) and the Nsp7/Nsp8 complex (blue) via MST assays. (D) Effects of Nsp8 and the Nsp7/Nsp8 complex on the endoribonuclease activity of MERS-Nsp15. (E) Effects of Nsp8 and the Nsp7/Nsp8 complex on the catalytic rate of Nsp15 mutants.