Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells

EAV PLP2 Adopts a Compact OTU-Domain Fold with a Unique Integral Zinc Finger.

Previously, EAV PLP2 was identified and characterized by a combination of bioinformatics analysis and site-directed mutagenesis, and two residues in particular were implicated in catalysis: Cys270 and His332 (16). Throughout this paper, amino acid numbers refer to the sequence of full-length EAV pp1a. The crystal structure of EAV PLP2 (residues 261–392; 13.6 kDa) was determined as a covalent complex with the mechanism-based inhibitor Ub_(1–75)-3-bromopropylamine (Ub–3Br) (38, 39). Because the conservation of multiple cysteine residues and their demonstrated importance for protease function suggested that PLP2 could bind zinc (16), the crystal structure of the complex was determined by a multiwavelength anomalous dispersion (MAD) phasing experiment using X-ray diffraction data collected over the zinc absorption edge (Table S1). The resulting electron density map revealed residues 261–387 of PLP2 bound to a complete Ub molecule and allowed a model of the complex to be built and refined (R_work= 0.16, R_free= 0.18) to 1.45-Å resolution (Fig. 1 A and B).

The protease adopts a compact, two-domain fold with a shallow Ub-binding surface that directs the C terminus of the bound Ub molecule (the distal Ub in a Ub dimer) toward a solvent exposed active site that includes Cys270 and His332 (Fig. 1 A and B). Domain I of PLP2 (residues 267–307 and 365–387) consists of a three-helix bundle (α1, α2, α4) packed against a two-stranded antiparallel sheet (β2↑ β6↓). Domain II centers on a four-stranded β-sheet (β1↑ β5↓ β4↑ β3↑) and an α-helix (α3) that together pack against helices α1 and α2 of domain I. Domain II comprises the majority of the Ub-binding surface, which is stabilized by four cysteine residues (Cys319, 349, 354, and 356) that coordinate a zinc ion with tetrahedral geometry (Fig. 1C). Their arrangement forms a C4 zinc finger that resembles a C-terminal type zinc necklace motif (40). A large insertion between positions C1 (Cys319) and C2 (Cys349), which includes His332, appears to extend the stabilizing effect of the zinc finger throughout much of domain II. A fifth cysteine (Cys344) is located near the zinc ion but does not coordinate with it, consistent with other zinc necklace motifs that have been described (40) and with previous findings showing that a Cys344 to alanine mutation had no effect on catalytic activity of PLP2 (16). Three of the cysteines (Cys319, 349, and 354) are fully conserved in arteriviruses, and mutational analysis of these residues and Cys356 demonstrated zinc binding to be essential for catalytic activity (16). Given its distance from the active site, however (∼25 Å; Fig. 1B), the zinc atom appears to play a structural role as opposed to participating in catalysis. Expression of PLP2 in Escherichia coli grown in the absence of zinc (M9 medium) yielded insoluble protein, supporting the idea that the role of the zinc finger is structural and is likely required for correct folding of the protease.

Consistent with OTU DUBs and papain-like cysteine proteases in general, the PLP2 active site contains a catalytic cysteine nucleophile (Cys270) and histidine (His332) residue, along with an asparagine (Asn263) that hydrogen bonds with the imidazole ring of His332 (Fig. 1D). As expected, the side chain of Cys270 is covalently coupled to the C terminus of Ub via the 3-propylamine (3CN) modification, mimicking the acyl-enzyme intermediate step of the catalytic reaction and confirming the identity of Cys270 as the catalytic nucleophile.

Fold analysis of the PLP2 structure using the DALI server (41) revealed that its closest structural homologs indeed belong to the OTU superfamily of DUBs (29, 42). The most significant matches were to yeast Otu1 (38) (Z-score: 5.1) and the viral OTU protease from CCHFV (43–45) (Z-score: 4.6; Fig. 1E), followed by Otubain1 from human (46) and Caenorhabditis elegans (47) (Z-scores: 4.1 and 4.0, respectively) and the OTU domain of human DUBA (48) (Z-score: 3.9). The sequence identity of the PLP2 regions that aligned with these OTU proteases was low (ranging from 9% for yeast Otu1 to 21% for human Otubain1); however, they accounted for ∼60% of the total PLP2 structure and superposed well with the equivalent regions in the above proteins, with an average rmsd of ∼2.8 Å. The greatest structural similarity between PLP2 and members of the OTU superfamily occurs at the active site and adjoining channel that binds the C-terminal RLRGG-tail of Ub.

PLP2 Zinc Finger Motif Plays a Central Role in Ub Binding.

Arterivirus PLP2 and nairovirus OTU enzymes differ from eukaryotic OTU DUBs in that they also remove the Ub-like antiviral protein IFN stimulated gene 15 (ISG15) from target proteins, a process also known as deISGylation (18, 49). ISG15 conjugation has been postulated to interfere with proper viral protein function, possibly through steric hindrance, although the exact mechanism underlying its antiviral activity is unknown (50). For CCHFV OTU, cross-reactivity with ISG15 arises primarily from a unique β-hairpin on the Ub-binding surface. The hairpin modifies the surface so that the viral enzyme binds the β-grasp folds of Ub and the C-terminal Ub-like domain of ISG15 in an orientation that is rotated nearly 75° with respect to that observed for Ub bound to a representative eukaryotic OTU DUB from yeast (Otu1) (43, 44). Surprisingly, the β-hairpin is absent in EAV PLP2 and replaced by helix α3 of the zinc finger motif (Fig. 2A). However, in keeping with the role of the β-hairpin in CCHFV OTU, residues of helix α3 bind to the hydrophobic Ile44 patch of Ub, a site commonly targeted by Ub-binding proteins (51), and they also assist in positioning Ub in a rotated manner equivalent to that observed for CCHFV OTU (Fig. 2 B and C).

Structure-Guided Decoupling of PLP2 Deubiquitinase and Polyprotein Cleavage Activities.

Given the distance of helix α3 from the PLP2 active site (Fig. 1B), we hypothesized that mutations could be introduced into this region of the Ub-binding surface that would selectively disrupt PLP2 DUB activity without affecting EAV polyprotein cleavage at the nsp2|nsp3 junction (presumably RLIGG↓WIY). Although this sequence closely resembles the C-terminal tail of Ub (RLRGG↓), we postulated that the nsp2 sequence immediately upstream of the nsp2|nsp3 junction does not adopt a Ub-like fold and that the majority of the PLP2 Ub-binding surface is therefore not required for its cleavage. To test our hypothesis, we used the crystal structure to select three positions within the PLP2 Ub-binding surface (Thr312, Ile313, and Ile353) and engineered a panel of (combined) mutations (Fig. 2 B and C). Ile353 is located at the C-terminal end of helix α3 next to C3 (Cys354) of the zinc finger motif. It projects directly into the Ile44 patch of Ub where it makes extensive van der Waals interactions with Ile44, Val70, and Leu8. Given that Ile353 is located on the Ub-binding surface, we aimed to disrupt Ub binding by introducing various other residues at this position, including large bulky residues such as arginine and tryptophan. Thr312 and Ile313 are located closer to the active site, where they make additional hydrophobic interactions with Leu8, Leu71, and Leu73 of Ub. In an attempt to further disrupt the interaction between PLP2 and Ub, the mutations Thr312Ala and Ile313Val were also combined with changes at position Ile353. Given the close proximity of Ile313 to Leu73 of the RLRGG motif of Ub, mutation to valine was chosen to minimize adverse effects on nsp2|nsp3 cleavage.

Before proceeding to infection experiments with mutant viruses, we used ectopic expression of PLP2 and an in vitro enzymatic assay to characterize the effect of various mutations at the positions described above on polyprotein processing and DUB activity. To this end, mutations were introduced into a mammalian expression vector encoding a self-cleaving nsp2-3 polyprotein carrying an N-terminal HA-tag. On expression of nsp2-3 in HEK293T cells, WT PLP2 mediated efficient cleavage of the nsp2|nsp3 junction (Fig. 3A). As expected, a PLP2 active site mutant (C270A/H332A) did not display any processing of the nsp2|nsp3 site, and only the nsp2-3 precursor was detected. Seven single-site mutants, in which the Ub-binding surface was targeted by replacement of Thr312 or Ile353, displayed WT levels of nsp2|nsp3 cleavage, suggesting that their polyprotein processing activity was not notably affected. In addition, combinations of mutations at positions Thr312, Ile313, and Ile353 were tested, with similar results (Fig. 3A). Longer exposure times did reveal some nsp2-3 precursor, even in the case of WT PLP2, but this amount only marginally increased when two or three mutations were combined (Fig. 4B).

Next, we assayed the effect of these mutations on PLP2 DUB activity by transfecting mammalian cells with plasmids encoding FLAG-tagged Ub and nsp2-3 carrying WT or mutant PLP2. FLAG-tagged ubiquitination of a wide range of cellular targets could be visualized by Western blot analysis using an anti-FLAG antibody (Fig. 3B). As expected, expression of WT PLP2 strongly decreased the accumulation of Ub-conjugates, whereas expression of the active site mutant (C270A/H332A) had a negligible effect. Fig. 3B presents the results obtained with a selection of PLP2 Ub-binding surface mutants with the most pronounced effect on DUB activity, some of which approached the level of the active site mutant. In contrast, these Ub-binding surface mutations had only minor effects on the deISGylating activity of PLP2 (Fig. S1).

To corroborate our findings from the expression system showing that PLP2 DUB activity could be selectively removed without disturbing nsp2|nsp3 cleavage, an in vitro activity assay of recombinant PLP2 produced in E. coli was performed using the fluorescently labeled substrates Ub-aminomethylcoumarin (Ub-AMC) or RLRGG-AMC, representing the C-terminal peptide motif of Ub. By comparing the activity of PLP2 mutants against Ub-AMC (which requires the Ub-binding surface) versus their activity against RLRGG-AMC (which binds to the active site region only), PLP2 mutants with a selective reduction in DUB activity could be identified. Indeed, compared with WT enzyme, mutants I353W and I353R exhibited ∼10- and 40-fold reductions in specificity (k_cat/K_m) toward Ub, respectively; in contrast, their activity toward RLRGG-AMC was essentially unaltered (Table 1). Expression of PLP2 containing additional mutations at Thr312 and Ile313 yielded insoluble protein in E. coli, preventing analysis of these mutants. Nevertheless, the results of the in vitro activity assay further confirmed the successful decoupling of the polyprotein processing and DUB activities of EAV PLP2 by specifically targeting key residues of the Ub-binding surface.

Ability of PLP2 to Suppress Innate Immune Signaling Depends on Its DUB Activity.

The effect of the various PLP2 Ub-binding surface mutations on innate immune signaling was initially assessed in the context of ectopic nsp2-3 expression using a luciferase-based IFN-β promoter activity assay. For this, HEK293T cells were transfected with a combination of plasmids encoding firefly luciferase under control of the IFN-β promoter, renilla luciferase as an endogenous control, constitutively active RIG-I_(2CARD) to induce innate immune signaling (52), and EAV nsp2-3 containing either WT or mutant PLP2. In this assay, reporter gene expression was reduced to ∼20% on coexpression of WT PLP2, whereas 80% of the level of the untreated control was retained on expression of the PLP2 active site mutant (C270A/H332A; Fig. 4A). Compared with WT PLP2, all mutants included in Fig. 4A were significantly impaired in their inhibitory activity (P < 0.01), with mutants I353R, I353W, T312A/I313V/I353R, and T312A/I313V/I353W displaying inhibition levels similar to that of the active site mutant (P > 0.05). Western blot analysis confirmed equal expression of WT and mutant nsp2-3 (Fig. 4B). These results demonstrated that, at least in the context of the nsp2-3 expression system, the selective removal of PLP2 DUB activity significantly disrupted its ability to suppress IFN-β promoter activity.

Arteriviruses Lacking PLP2 DUB Activity Elicit an Enhanced Host Innate Immune Response.

Having identified mutations in PLP2 that reduce its ability to suppress Ub-mediated innate immune signaling (Fig. 4A) without adversely affecting nsp2|nsp3 cleavage (Fig. 3A), we were in a position to directly evaluate the importance of PLP2 DUB activity for immune evasion during arterivirus infection. The six (combinations of) mutations used in Figs. 3B and 4 were introduced into an EAV full-length cDNA clone, and mutant viruses were launched by electroporation of in vitro transcribed RNA into BHK-21 cells. Immunofluorescence microscopy subsequently confirmed expression of both nsp2 and the nucleocapsid (N) protein, followed by virus spread to initially untransfected cells. These findings indicated that viruses carrying these (combinations of) mutations were replication competent.

We next focused on the two mutants showing the greatest decrease in inhibitory activity in the IFN-β promoter activity assay: I353R and T312A/I313V/I353R (Fig. 4A). We first characterized the replication kinetics of these mutants in a time course experiment in primary equine lung fibroblasts (ELFs), which are derived from the natural host species of EAV and are likely to maintain an intact innate immune response. ELFs were infected with WT or mutant EAV at a multiplicity of infection (m.o.i.) of 0.5 or 5. The first-cycle replication kinetics as determined by real-time quantitative reverse transcriptase PCR (qRT-PCR) measurement of viral genome RNA levels did not notably differ between WT and mutant EAV (Fig. 5 A and B). In addition, viral titers in cell culture supernatants harvested from infected ELFs at 24 h postinfection (hpi) revealed no significant difference between WT and mutant EAV (Fig. 5C). Finally, immunofluorescence microscopy of ELFs infected with m.o.i. 5 revealed expression of nsp2 from 6 hpi onward, and by 9 hpi, expression of N protein could be seen in all cells for both WT and mutant EAV (Fig. S2). These results demonstrated that the replication kinetics of the PLP2 Ub-binding surface mutants and WT control were essentially the same, in line with our previous conclusion that cleavage of the nsp2|nsp3 site is hardly affected by these mutations. At the same time, Western blot analysis showed that, compared with WT EAV, the DUB activity of both mutants was severely impaired during infection (Fig. 5D).

Subsequently, we used real-time qRT-PCR to measure the levels of mRNAs encoding IFN-β, the IFN-stimulated protein MX1, and the proinflammatory cytokine IL8 and investigate the effect of mutations I353R and T312A/I313V/I353R on innate immune signaling during infection of ELFs. Initially, we infected ELFs with WT or mutant EAV at m.o.i. 5, but in this setup, IFN-β mRNA levels remained below the detection limit at all time points analyzed (from 3 to 12 hpi), suggesting that the innate immune response triggered by EAV on initial infection is very limited. Therefore, we next infected ELFs with WT or mutant EAV at m.o.i. 0.25, resulting in infection of ∼20% of cells. We hypothesized that this would allow for IFN-β–mediated priming of uninfected cells as a result of paracrine signaling from cells infected during the first round. The expression of IFN-stimulated genes would then result in a more potent response during the second cycle of infection. Indeed, following such a low m.o.i. infection with WT EAV, low but detectable levels of IFN-β mRNA were induced by 20 hpi (Fig. 6A). Interestingly, at both 20 and 24 hpi, cells infected with either mutant showed significantly increased levels of IFN-β mRNA compared with WT virus-infected cells (P < 0.05), with mutant T312A/I313V/I353R showing the most pronounced difference. In addition, the levels of MX1 mRNA differed significantly between WT and mutant EAV-infected cells at 24 hpi (Fig. 6B). In contrast, at 20 and 24 hpi, no significant difference in the levels of IL8 mRNA was observed on infection with WT or mutant EAV (Fig. 6C). Equally efficient replication of WT and mutant EAV was corroborated by comparing viral genome RNA levels, for which no significant differences were measured (Fig. 6D). In summary, these data show that the PLP2 DUB function is indeed involved in the inhibition of innate immune signaling during EAV infection in primary equine cells and that it is possible to specifically inactivate this function to suppress immune evasion of otherwise fully replication-competent viruses.

PLP2 Plasmids.

For bacterial expression of EAV PLP2, a cDNA fragment encoding residues 261–392 of EAV pp1a and an in-frame C-terminal His₆ purification tag were inserted downstream of a Ub fusion partner in the pASK3 vector (76), yielding plasmid pASK3-ePLP2. A mammalian expression construct encoding an EAV nsp2-3 polyprotein was made by cloning residues 261–1,064 of EAV pp1a in-frame with an N-terminal HA tag in the pcDNA3.1 vector (Invitrogen). All mutants were engineered by site-directed mutagenesis using Pfu DNA polymerase (Fermentas). Primer sequences are available on request. All constructs were verified by sequence analysis.

Purification and Crystallization of EAV PLP2 Bound to Ub.

E. coli BL21-Gold(DE3) cells were transformed with pASK3-ePLP2 and cultured to an optical density (OD₆₀₀) of 0.7 in lysogeny broth (LB) medium at 37 °C. The culture was then supplemented with 200 ng/mL of anhydrous tetracycline and incubated for 3 h at 28 °C with shaking to induce expression of the Ub-PLP2-His₆ fusion protein. The cells were pelleted and resuspended in ice-cold lysis buffer [20 mM 2-(N-Morpholino)ethanesulfonic acid (MES), pH 7, 500 mM NaCl, 10% (vol/vol) glycerol, 5 mM imidazole, pH 7.4, 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP)] and lysed using a French pressure cell [American Instrument Company (AMINCO)]. The lysate was clarified by centrifugation and loaded onto a Ni-NTA column (Qiagen) preequilibrated with lysis buffer. After washing with lysis buffer supplemented with 15 mM imidazole, recombinant PLP2 was eluted from the column using an equilibration buffer supplemented with 150 mM imidazole and exchanged into 50 mM Tris, pH 8.0, 300 mM NaCl and 5 mM DTT before storing at 4 °C. The endogenous DUB activity of PLP2 resulted in the efficient removal of the N-terminal Ub-tag from the fusion protein during expression in E. coli; therefore, affinity chromatography yielded highly pure PLP2 carrying a C-terminal His₆ purification tag only.

The mechanism-based suicide inhibitor Ub–3Br was prepared according to Messick et al. (38) and Borodovsky et al. (39), as described by James et al. (43). Ub-3Br was covalently bound to purified PLP2 by gently mixing the proteins in a 3:2 molar ratio for 1 h at 37 °C. The resulting PLP2-Ub complex was purified by gel filtration (Superdex 75) followed by anion exchange (Source 15Q) chromatography and then exchanged into 20 mM Tris, pH 8.0, 50 mM NaCl before concentrating to 10 mg/mL and storing at 4 °C.

The PLP2-Ub complex was crystallized by hanging-drop vapor diffusion at 10 mg/mL in mother liquor consisting of 100 mM MES, pH 6.2, 18% (wt/vol) polyethylene glycol (PEG) 20,000. Crystals were flash-cooled and stored in liquid nitrogen after sweeping them through mother liquor supplemented with 20% (vol/vol) glycerol.

X-Ray Data Collection and Crystal Structure Determination.

X-ray diffraction data for a multiwavelength anomalous dispersion (MAD) experiment were collected at the Canadian Light Source (beam line 08ID-1). Data were collected at three different wavelengths over the absorption edge of zinc from a single crystal of the PLP2-Ub complex held at 100 K in an N₂ (g) stream. The data were processed using MOSFLM and SCALA (77), and structure factor phases were determined using phenix.autosol (78). Initial phases generated by SOLVE were improved by density modification using RESOLVE within the PHENIX package. After reserving a random subset of reflections for cross-validation using the free R-factor (79), a model was built using phenix.autobuild (78) and manually completed and refined using COOT (80) and phenix.refine (78). Crystallographic data and model refinement statistics are summarized in Table S1.

In Vitro Enzymatic Assays.

To ensure complete removal of the N-terminal Ub-tag from recombinant PLP2, E. coli C2523 containing (WT or mutant) pASK3-ePLP2 were cotransformed with plasmid pCG1, which encodes the DUB ubiquitin-specific processing protease 1 (Ubp1) (76). The DUB activity of PLP2 (WT and mutants) was assayed using 7-amino-4-methylcoumarin (AMC)–labeled versions of Ub (Ub-AMC) (Boston Biochem) and the C-terminal peptide motif of Ub: RLRGG-AMC (Enzo Life Sciences). The enzymes cleave the AMC label causing a significant increase in the fluorescence quantum yield of the dye. All reactions were performed in 50 mM Tris-Cl buffer at pH 8 and 100 mM NaCl. Time-dependent fluorescence traces were collected by a Fluorolog-3 Horiba Jobin Yvon fluorimeter. The monochromators were set to 360 nm (excitation) and 460 nm (emission). The slits were set between 1 and 3 nm bandpass depending on substrate concentration. Enzyme activities in all mutants were characterized by the specificity constant. At substrate concentrations significantly smaller than K_m, the formation of product follows pseudo–first-order kinetics; therefore, the temporal evolution of product fluorescence F follows the equation

where is the fluorescence when all AMC is liberated, is the background fluorescence, [E] is the total enzyme concentration, and t is the time elapsed. From fitting the fluorescence time traces to Eq. 1, all parameters including the specificity constantare obtained. Our assays indicate that PLP2 exhibits pseudo–first-order kinetics for Ub-AMC at concentrations less than 0.2 µM and for RLRGG-AMC at concentrations less than 150 µM.

Reverse Genetics.

Mutations in the EAV PLP2 coding sequence were engineered in an appropriate shuttle vector and subsequently transferred to pEAN551/AB, a derivative of EAV full-length cDNA clone pEAN551 carrying additional (translationally silent) AflII and BspEI restriction sites (17, 81). The virus derived from pEAN551/AB was used as a WT control in all experiments. All constructs were verified by sequence analysis.

In vitro RNA transcription from XhoI-linearized WT or mutant EAV full-length cDNA clones was performed using the mMESSAGE mMACHINE T7 Kit (Ambion). Five micrograms of full-length EAV RNA was electroporated into 5.0 × 10⁶ BHK-21 cells using the Amaxa Cell Line Nucleofector Kit T and the program T-020 of the Amaxa Nucleofector (Lonza) according to the manufacturer’s instructions. Cells were incubated at 39.5 °C, and virus-containing supernatants were harvested at 24 h after transfection. Titers were determined by plaque assay on primary ELFs essentially as described before (82).

To verify the presence of the correct mutations, RNA was isolated from virus-containing supernatants using the QIAamp Viral RNA Mini Kit (Qiagen) and converted to cDNA using RevertAid H Minus reverse transcriptase (Fermentas) and random hexameric primers. The region of PLP2 encoding the mutations was subsequently PCR amplified using Pfu DNA polymerase (Fermentas) and sequenced.

Real-Time qRT-PCR.

Confluent ELFs were infected with WT or mutant EAV at m.o.i. 5, 0.5, or 0.25 and incubated at 37 °C. At the indicated time points, cell lysates were harvested in TriPure Isolation Reagent (Roche). After the addition of chloroform, the aqueous phase was mixed in a 1:1 ratio with buffer RA1 of the Nucleospin RNA II kit (Macherey-Nagel). RNA was isolated as per the manufacturer’s instructions and reverse transcribed using RevertAid H Minus RT (Fermentas) and the oligo(dT)₂₀ primer. Finally, samples were assayed by real-time qRT-PCR on a CFX384 Touch Real-Time PCR detection system (BioRad) using iTaq SYBR Green Supermix with ROX (BioRad). Primers (Table S2) targeting mRNAs encoding equine GAPDH, β-actin, IFN-β, MX1, and the EAV genome were designed using Primer3 (83) or sequences were kindly provided by Udeni Balasuriya (University of Kentucky, Lexington, KY) in the case of IL8. The real-time PCR was followed by a melting-curve analysis to verify the specificity of the reaction. Results were quantified using the standard curve method and normalized against the geometric mean of the relative quantities of GAPDH and β-actin mRNA. Data from three independent experiments were analyzed with SPSS Statistics software using a one-sample t test or unpaired Student t test, where appropriate. P < 0.05 was considered statistically significant.

Deubiquitination During Infection.

Confluent ELFs were infected with WT or mutant EAV at m.o.i. 5 and incubated for 10 h at 37 °C. Cells were then lysed in 500 µL 2× Laemmli sample buffer [250 mM Tris, 2% (wt/vol) SDS, 20% (vol/vol) glycerol, 0.01% bromophenol blue, 2 mM DTT, pH 6.8], and total ubiquitination was assessed by Western blot analysis as described in SI Materials and Methods.

Ectopic Expression Experiments.

Ectopic expression experiments were performed essentially as described before (31) but are described in detail in SI Materials and Methods, together with a description of the additional plasmids, cells, and antibodies used.