X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions

Abstract

Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication.

Keywords: MHV PLP2; crystal structure; deISGylation; deubiquitylation (deubiquitination); enzyme kinetics; protein-protein interaction; viral protease.

FIGURE 1.

Domain organization of the MHV and SARS-CoV coronavirus polyprotein and nsp3. A, domain organization of the MHV polyprotein. The locations of the different nsps resulting from the processing of polyproteins are numbered 1–16. The two papain-like protease domains of MHV within nsp3, PLP1 and PLP2, are represented in orange and red, respectively, whereas the 3C-like protease (3CLpro) within nsp5 is represented in blue. The corresponding cleavage sites for each protease (PLP1, PLP2, and 3CLpro) are indicated by arrows and are colored according to the protease that performs the cleavage. B, domain architecture of SARS-CoV nsp3 in comparison with MHV nsp3. Domains with known structures are shown as ribbons, whereas domains with unknown structures are represented as boxes. For SARS-CoV, only one PLpro is encoded in nsp3, and it catalyzes cleavage of the polyprotein at all three cleavage sites. The structures of the three MHV nsp3 domains determined in this study are underscored with dashed lines. PDB codes are as follows: SARS-CoV nsp3, 2IDY, 2ACF, 2W2G, 2KQW, 2FE8, and 2K87; MHV nsp3, 2M0A and 4YPT (from this study). C, a summary of the currently known structures of various nsp3 domains within CoVs studied to date (13–18, 22, 30, 53, 54, 63, 64). The currently known nsp3 domains are indicated at the top and are shaded in different shades of red. The PDB codes for the structures of individual or single domains are listed under the domain acronym. The PDB codes for the structures of domains that have been determined from a single polypeptide chain are connected with dashed lines and are shaded according to the color code of the corresponding domains.

FIGURE 2.

PLP2+-mediated processing of ubiquitin chains. A, survey of the hydrolysis of di-Ub by PLP2+. 0.5 μg of di-Ub with different linkages (Lys⁶, Lys¹¹, Lys²⁷, Lys²⁹, Lys³³, Lys⁴⁸, Lys⁶³, and linear) was incubated at 25 °C with 20 nm PLP2+ for 2 h. Di-Ub incubated without PLP2+ serves as the negative control. B and C, time-dependent hydrolysis of Lys⁴⁸-Ub4 (B) and Lys⁶³-Ub6 (C) by PLP2+. The reactions were incubated at 25 °C, and aliquots were removed at five different time points, quenched with sample buffer, and then analyzed by SDS-PAGE. Polyubiquitin chains incubated without PLP2+ serve as negative control (ctrl). Markers were (from top to bottom) 50, 37, 25, 20, 15, and 10 kDa.

FIGURE 3.

X-ray crystal structure of PLP2+. A, ribbon representation of the overall structure of PLP2+. The structure contains five domains: fingers (green), palm (light blue), thumb (orange), Ubl2 (magenta), and DPUP (wheat). The zinc atom in the fingertip of the fingers domain is shown as a gray sphere. The catalytic triad residues and the four zinc-coordinating cysteines from the fingers domain are represented as sticks. B, structural superposition of the DPUP domain (wheat) with SARS SUD-C domain (cyan; PDB code 2KQW). The root mean square deviation between the two structures is 2.1 Å over 63 aligned Cα atoms at 13% sequence identity. C, electron density maps covering the catalytic triad before (left) and after (right) refinement of the sulfonic group at the catalytic Cys. In the right panel, the occupancies of the oxygen atoms in the sulfonic acid group were set to 0.5. In each panel, the residual electron density in F_o − F_c maps are colored green and are contoured at 3σ, whereas the 2F_o − F_c maps are colored blue and are contoured at 1σ.

FIGURE 4.

Structural comparison among coronaviral PLPs. A, structural superposition of unbound PLP2 (light blue), unbound SARS-CoV PLpro (light pink; PDB code 2FE8), and unbound MERS-CoV PLpro (light green; PDB code 4P16). Areas with major differences are indicated with black arrows. B, close-up view of the active site alignments. The Cys-His-Asp catalytic triad is conserved among the three proteins, whereas the oxyanion hole residues are different. The color code is the same as in A. The catalytic cysteine in MERS-CoV PLpro was covalently modified by β-mercaptoethanol. C, conformations of the BL2 loops in different CoV PLPs. The color code is the same as in A with the additional structure of inhibitor-bound SARS-CoV PLpro shown in cyan (PDB code 3E9S). The loop from MERS-CoV PLpro is omitted because it is not visible in the structure. D, sequences of the BL2 loops in different CoV PLPs.

FIGURE 5.

Model of PLP2 in complex with Ubal. A and B, potential interactions between PLP2 and the C-terminal residues of Ubal (A) in comparison with the identified interactions between SARS-CoV PLpro and the C-terminal residues of Ubal (B) as observed in the structure of SARS-CoV PLpro-Ubal complex (PDB code 4MM3). PLP2 is colored as in Fig. 3: thumb (orange), palm (light blue), and fingers (green). Ubal is shown in yellow, and SARS-CoV PLpro is shown in cyan. Hydrogen bonding and salt bridge interactions are represented as dashed lines. The red dashed line in A indicates that a similar interaction is not observed in B, and vice versa. C, potential interactions between PLP2 and Ubal (yellow). Only the interactions that persist for more than 50% of the simulation time are shown. The magenta dashed line indicates that the interaction persists for more than 50% of the simulation time but is not present in the current analyzed frame.

FIGURE 6.

Sequence alignment of various coronaviral PLP2s. Group 1, HCoV-229E and HCoV-NL63; group 2, MHV, SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-HKU1; group 3, IBV. The BL2 loop residues are boxed in green. Purple stars, catalytic triad residues; orange ovals, residues of MHV PLP2 that may be involved in important interactions with Ub. Accession numbers are as follows: MHV PLP2, P0C6V0; SARS-CoV PLpro, AEA10816.1; MERS-CoV PLpro, YP_007188578.1; HCoV-OC43, NP_937947.21; HCoV-HKU1, YP_460024.1; HCoV-229E, AGT21365.1; HCoV-NL63, AFD98832.1; IBV, P0C6V5.1. The alignment was performed using Clustal Omega. The figure was generated using ESPript (67). Same residues are shaded in red, and conserved residues are shown in red.

FIGURE 7.

Packing of MHV PLP2 around residues Tyr¹⁸²⁴ and Phe¹⁸¹². A, the packing of the PLP2 fingers domain around Tyr¹⁸²⁴ shown in a space-filling view (green, carbon; red, oxygen; blue, nitrogen; gray, hydrogen). Ub is shown as a cartoon in yellow. B, the packing of Phe¹⁸¹² from PLP2 against the Ile⁴⁴–Val⁷⁰ patch from Ub shown in a space-filling view. The color code is the same as in A, except carbon atoms of Ile⁴⁴ and Val⁷⁰ are shown in yellow. C, temperature inactivation of PLP2+ mutant Y1824A. The activity of WT PLP2+ and two PLP2+ mutants (Y1824F and Y1824A) were measured after incubation at 30 °C for different time periods and then normalized to the activity at 0 min. Error bars, S.D.

FIGURE 8.

Interferon antagonism activity of MHV PLP2+ wild type and mutants. HEK293T cells were transfected with plasmids expressing either the WT, the catalytic mutant (CA), or the indicated PLP2+ mutants and plasmids expressing IFNβ-luc, Renilla-luc, and N-RIG-I. At 16 h post-transfection, cells were lysed, and luciferase activity was measured. Experiments were performed in triplicate. Error bars, S.D.