Structure and Function of Viral Deubiquitinating Enzymes

Abstract

Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate and adaptive immune responses. Ubiquitin-mediated control over these processes can be reversed by cellular deubiquitinating enzymes (DUBs), which remove ubiquitin from cellular targets and depolymerize polyubiquitin chains. The importance of protein ubiquitination to host immunity has been underscored by the discovery of viruses that encode proteases with deubiquitinating activity, many of which have been demonstrated to actively corrupt cellular ubiquitin-dependent processes to suppress innate antiviral responses and promote viral replication. DUBs have now been identified in diverse viral lineages, and their characterization is providing valuable insights into virus biology and the role of the ubiquitin system in host antiviral mechanisms. Here, we provide an overview of the structural biology of these fascinating viral enzymes and their role innate immune evasion and viral replication.

Keywords: innate immune evasion; papain-like protease; ubiquitin; viral deubiquitinating enzyme; viruses.

Graphical abstract

Fig. 1

Structure of Ub and ISG15. (A) Ub (PDB ID: 1UBQ) is shown in cartoon representation, with the residues forming the Ile44 patch shown as sticks. (B) Crystal structure of the compact, Lys48-linked diUb (PDB ID: 1AAR) is shown as a cartoon with transparent surface, with the isopeptide bond between Lys48 and Gly76 indicated. (C) Crystal structure of the extended, Lys63-linked diUb (PDB ID: 2JF5) is shown as a cartoon with transparent surface, with the isopeptide bond between Lys63 and Gly76 indicated. (D) Crystal structure of ISG15 (PDB ID: 1Z2M) is shown as cartoon, with the N- and C-terminal UBL domains indicated. All structural images were generated using PyMOL .

Fig. 2

Illustration of the activation of the innate immune response and its manipulation by human or viral DUBs. White boxes highlight the cytoplasmic receptors RIG-I, MDA5, and cGAS that can sense viral RNA or DNA via adaptor proteins MAVS or STING, in light gray, which in turn activate kinase complexes (partly depicted in dark gray). Ultimately, transcription factors IRF3, IRF7, p50, and p65 (black boxes) are activated and translocate to the nuclease to induce the transcription of type I IFNs and pro-inflammatory cytokines. Differently linked polyUb chains involved in the activation of the innate immune response are shown by the different colored polyUb chains. Dashed boxes placed next to innate immune signaling factors contain human or viral DUBs that remove Ub chains from these specific targets. DUBs placed below the type I IFN or NF-κB pathway (inducing the expressing of pro-inflammatory cytokines) interfere with these pathways without knowing their exact substrate(s). Human DUBs are shown in black, vDUBs in red, and vDUBs having deISGylating activity in red italic.

Fig. 3

Crystal structure of adenovirus Avp. (A) Superposition of Avp bound to activating peptide pVIc (PDB ID: 1AVP; yellow and red, respectively) and Avp in the absence of pVIc (PDB ID: 4EKF; transparent gray). Active site residues are shown as sticks in both structures, and the disulfide bridge between Avp and pVIc is indicated. (B) Surface representation of Avp (PDB ID: 1AVP). Dashed white lines highlight the active-site cleft. (C) Crystal structure of papain (PDB ID: 1PPN). The left (L) α-helical domain and right (R) β-sheet domain are indicated by green and yellow boxes, respectively. (D) Superposition of the active-site residues of Avp (yellow) and papain (cyan). Active site residues of papain are shown as gray sticks, and those of Avp are shown as orange sticks. Avp residues are indicated, followed by the equivalent residues of papain. Papain and Avp structures were aligned using the SPalign-NS server .

Fig. 4

Crystal structure of MCMV M48^USP in complex with Ub. MCMV M48^USP (PDB ID: 2J7Q; blue cartoon) shown in covalent complex with Ub (orange cartoon with transparent surface representation). The unique β-hairpin of M48^USP interacting with the Ile44 patch of Ub is colored in red. Dashed box contains a close-up of the M48^USP active site, with catalytic residues shown as sticks, including the two catalytic bases His158 and His141. Also shown is Gln10 suggested to take part in the formation of the oxyanion hole.

Fig. 5

Crystal structures of zoonotic SARS- and MERS-CoV PL^pro domains. (A) Superposition of SARS-CoV PL^pro (PDB ID: 2FE8; red) with HAUSP (PDB ID: 1NB8; transparent gray). Thumb, fingers, palm, and UBL domains are indicated with blue, yellow, orange, and gray shading, respectively. Inset is a close-up of the SARS-CoV PL^pro (left panel) and HAUSP (right panel) active sites. Backbone atoms are shown as ribbons, and active-site residues are shown as sticks. The BL2 loop is indicated with an arrow. (B) Crystal structure of the SARS-CoV Lys48-linked diUb complex (PDB ID: 5E6J). SARS-CoV PL^pro is shown in red, bound to Lys48-linked diUb, shown as a slate cartoon with transparent surface. Inset is a close-up on the hydrophobic interactions occurring between PL^pro and the distal domain of Lys48-linked diUb, with relevant residues shown as sticks. (C) Crystal structure of the MERS-CoV PL^pro domain in complex with Ub (PDB ID: 4RF0). MERS-CoV PL^pro is depicted in green cartoon, and Ub is shown in orange cartoon with transparent surface. Dotted box depicts a close-up of the interaction between MERS-CoV PL^pro residue Val1691 and Ub residue Ile44, with residues depicted as sticks.

Fig. 6

Crystal structure of the EAV PLP2–Ub complex. EAV PLP2 (PDB ID: 4IUM) is shown in slate, covalently bound to Ub (orange, transparent surface). Depicted inset is the 4C Zn finger (left panel), with Zn-coordinating residues shown as sticks and Zn shown as a gray sphere. EAV PLP2 residue Ile353 forms hydrophobic interactions with the indicated residues on Ub (inset, right panel), and targeted disruption of Ile353-mediated interactions selectively abrogated PLP2 DUB activity.

Fig. 7

CCHFV OTU interacts with Ub and ISG15 in a rotated orientation with respect to yOTU1. (A) Comparison of the Ub-binding orientations of the CCHFV OTU and yeast OTU1 OTU (yOTU1) domains. CCHFV OTU (PDB ID: 3PT2; violet) is shown bound to Ub (PDB ID: 3PT2; orange), and the β-hairpin is indicated with an arrow. yOTU1 in complex with Ub (PDB ID: 3BY4) was superposed onto the CCHFV OTU structure, and yOTU1 was removed for clarity. The yOTU1-bound Ub domain is shown in transparent yellow. (B) Crystal structure of CCHFV OTU (PDB ID: 3PSE; purple) in complex with ISG15 (PDB ID: 3PSE; green). CCHFV OTU binds ISG15 in a comparable orientation to Ub.

Fig. 8

Crystal structure of FMDV Lb^pro. FMDV Lb^pro (PDB ID: 1QOL) is shown in cartoon, and an adjacent monomer in the asymmetric unit is depicted in gray, with the C terminus leading toward the active site of the adjacent Lb^pro monomer.

Fig. 9

Crystal structure of TYMV PRO. The TYMV PRO domain (PDB ID: 4A5U; deep blue, transparent surface) is shown in cartoon. A symmetry mate (gray) is also shown, with the C terminus bound in the active site of the adjacent PRO monomer. The N-terminal lobe is colored red in the adjacent symmetry mate. Dotted box shows a close-up of the pared-down PRO active site, with catalytic residues shown as sticks.