The Baculoviridae family of viruses encode a viral Ubiquitin (vUb) gene. Though the vUb is homologous to the host eukaryotic Ubiquitin (Ub), its preservation in the viral genome indicates unique functions that are not compensated by the host Ub. We report the structural, biophysical, and biochemical properties of the vUb from Autographa californica multiple nucleo-polyhedrosis virus (AcMNPV). The packing of central helix α1 to the beta-sheet β1–β5 is different between vUb and Ub. Consequently, its stability is lower compared with Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin-binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54), and the deubiquitinating enzymes are ineffective against the K54-linked polyubiquitin chains. We propose that the modification of host/viral proteins with the K54-linked chains is an effective way selected by the virus to protect the vUb signal from host DeUbiquitinases.

## Introduction

Ubiquitin (Ub), a 76 residue protein, regulates multiple cellular pathways and is a major way of controlling protein levels in the cell [1–3]. Ub or Ub polymers are covalently attached to the target protein substrate via an isopeptide bond by post-translation modification (PTM). The process of conjugating Ub to substrates is a multi-step process involving three sets of enzymes: Ub-activating enzyme E1, Ub-conjugating enzymes E2s, and Ub ligase enzymes E3s. After conjugation, the C-terminal glycine residue of Ub forms an isopeptide bond with the ε-amino group of the substrate's lysine residue. Furthermore, Ub can extend the conjugation to polymeric chains via the seven lysine sidechains in Ub or the N-terminal amine group. The fate of the substrate is dependent on the specificity of lysine used to form the polymeric Ub (poly-Ub) chain [4]. For example, substrates conjugated with lysine 63 (K63) linked poly-Ub chains are involved in DNA repair pathways or inflammatory response [5], whereas lysine 11 (K11) and lysine 48 (K48) poly-Ub chains target the conjugated substrate for proteasomal degradation [6–8].

The Ub pathway can also play a significant role in host–virus interactions [9]. Often viruses co-opt the host ubiquitin signaling for productive infection [10]. Viral proteomes also mimic vital enzymes of the ubiquitination signaling [11], which help to degrade the host immune responses, avoid detection during latency, enhance viral genome transcription/replication, and facilitate egress [12,13]. Baculoviridae family of viruses infect a variety of invertebrates such as Arthropods, Lepidoptera, Hymenoptera, Diptera, and are commonly referred to as insect viruses [14]. Alphabaculovirus is the most abundant genera of this virus family, which contains a majority of the 33 nuclear polyhedrosis viruses (NPVs). NPVs survive harsh and non-conducive environments by building an extremely stable polyhedral capsid [14]. Autographa californica multiple nucleo-polyhedrosis virus (AcMNPV) is the most studied virus of this genus containing a 134 Kbp double-stranded genome with 154 ORFs packaged into rod-shaped nucleocapsids, hence the name baculovirus [15]. Interestingly, a large number of NPV viruses, including AcMNPV, express an Ub-like molecule known as viral Ubiquitin (vUb).

Eukaryotic Ub has a 76% sequence identity with vUb [16]. Although the presence of a Ub molecule within the baculovirus genome likely arose by horizontal gene transfer [17], its retention suggests that vUb has critical functions, which cannot be compensated by the host eukaryotic Ub. vUb is expressed along with the viral coat proteins in the late phase of the virus life cycle [18]. The deletion of vUb reduces the maturation of virions, suggesting its role in forming or assembling viral particles [19]. Ubiquitination by vUb determines whether a nucleocapsid will mature into a budded virus or occlusion-derived virus [20]. Other possible vUb functions include proteasomal degradation of coat proteins for uncoating the viral capsid during the early stages of infection and proteasomal degradation of antiviral responses from the host. Despite its importance, the structural details of vUb are unavailable, and its mechanism of action is unknown. It is also unclear if vUb interacts with the proteasome, and if vUb conjugated substrates are targeted to the proteasome. Apart from the seven conserved lysines present in eukaryotic Ub, vUb has an extra lysine at the 54th position (K54). Whether vUb can form a novel K54-linked polyubiquitin chain, and the possible functional role of such a chain is also unknown. Interestingly, apart from baculoviruses, Entomopoxviruses such as Melanoplus sanguinipes, Amsacta moorei, Avian viruses like Canarypox and Flamingopox, and Protozoan virus such as Megavirus chiliensis, Golden Marseillevirus also contains Ub gene. None of them encode a lysine residue at the 54th position, except for an uncharacterized phycodna virus named Orpheovirus.

Here, we report the three-dimensional structure of vUb. While the overall fold of Ub is conserved in vUb, there are differences in core packing and salt-bridges between the two proteins. A comparison of the stability between Ub and vUb confirms that these differences reduce the stability of vUb. Binding studies with proteasome receptors suggested that vUb and Ub bind to these receptors similarly. The efficiency of polyubiquitination was also comparable between Ub and vUb. Finally, we confirmed that vUb could assemble K54-linked polyubiquitin chains, which are not effectively cleaved by the host DeUbiquitinases (DUBs). The substrates conjugated with K54-linked chains can be targeted for proteasomal degradation. Our study proposes that the virus encodes vUb to create a unique architecture of the K54-linked polyubiquitin chain. Conjugation of viral protein substrates by such poly-Ub chains may protect it from the host antiviral responses such as DUBs.

## Materials and methods

### Reconstruction of vUb gene tree

Thirty-nine baculoviruses containing the vUb encoding gene were selected for tree reconstruction, and only the amino acid sequences for Ubiquitin-like protein were selected, whereas the non-ubiquitin amino acid sequences were discarded. The gene tree was reconstructed using the maximum likelihood (ML) method. Sequences were aligned using the Muscle algorithm provided by MEGA X [21]. To reconstruct the ML phylogeny, we used the Partition finder [22] to obtain the evolutionary model for codon evolution (GTR + I + γ, GTR + γ, and HKY + I + γ for three codon positions, respectively). Additionally, RaxML Blackbox [23] was used in the CIPRES platform [24] to get the ML tree.

### Cloning

The DNA sequence encoding vUb and its K54o-vUb mutant (in which all lysine were substituted to arginine, except K54) were synthesized commercially from Life Technologies and then subcloned into pET3a vector without any tag using NdeI and BamHI restriction sites. The ligation reaction was carried out by T4 DNA ligase, and the plasmid containing the insert, from the ligated colonies were further verified by Sanger sequencing method using T7 promoter and terminator primers.

The LHP938-Gal1-HA-Ub was a gift from Linda Hicke (Addgene plasmid # 32175). The HA-tagged viral Ubiquitin (HA-vUb) and K54o-vUb (HA-K54vUb) were synthesized from Life Technologies. The HA-Ub, HA-vUb, HA-K54vUb genes were subcloned into a p417 plasmid under a constitutive TEF promoter. They were also cloned into a yeast integration plasmid between two flanking HO locus sequences [25]. The recombinant plasmids were then transformed into ubi4Δ pdr5Δ yeast cells. The transformants were selected using drug selection plates and confirmed by colony PCR.

### Purification of vUb and vUb-K54

Plasmid encoding vUb and K54o-vUb mutant were transformed into BL21-DE3* Escherichia coli expression strain. Cells were grown at 37°C initially, followed by IPTG induction, and incubated further at 37°C for 5–6 h. Harvested cell pellet of vUb was dissolved in sodium acetate buffer (50 mM) without any salt, followed by sonication and centrifugation. The soluble fraction containing vUb was loaded onto the SP FF column (GE), and the protein was eluted with a 300–400 mM salt gradient. Eluted fractions were concentrated and injected in a Superdex SD75 16/600 Prep grade (GE) column using the AKTA-pure machine (GE). The pure fractions of vUb were concentrated and stored at −20°C in 25 mM sodium acetate (pH 5.0) and 100 mM NaCl.

NMR samples of vUb were prepared by growing BL21-DE3* cells in M9 media containing isotopic ammonium chloride (15NH4Cl) or isotopic glucose (13C-glucose). Protein was purified in the same manner as mentioned above. The final buffer for NMR structural experiments contained 25 mM sodium acetate (pH 5.0) and 100 mM NaCl.

Unlike vUb, K54o-vUb mutant protein was insoluble after sonication. Hence the pellet was incubated in 8 M urea buffer at 4°C for 12–14 h. After centrifugation, the unfolded supernatant fraction was kept for serial dialysis from 6 M, 3 M, 1.5 M, 0.75 M, 0.375 M, and finally at 0 M, for 4–6 h each, for gradual refolding of protein. The refolded protein was loaded in the SP FF column and eluted with a 300–400 mM salt gradient. Finally, size exclusion chromatography was carried out to get the pure protein, which was stored at −20°C in 25 mM sodium phosphate buffer (pH 7.5) and 100 mM NaCl.

### Purification UbE1, E2s, E3s, and Ub proteins

UbE1 clone was a generous gift from Dr. Cynthia Wolberger. The Ube1 purification protocol is given elsewhere [26]. All the E2s, UbcH5b, E2-25k, Ubc13, and its cofactor Mms2 used in this study were tagged with 6×His at their N-terminus. UbcH5b plasmid was a gift from Wade Harper (Addgene #15782). All his-tagged proteins were purified using IMAC FF (GE) columns, followed by their elution with a 200–300 mM Imidazole linear gradient. Eluted fractions were loaded on Superdex SD75 16/600 Prep grade column, which was pre-equilibrated in 50 mM Tris buffer (pH7.5) and 250 mM NaCl, to get the pure protein and finally stored at −20°C. Purification of Ubiquitin proceeded in the same manner as described for vUb. GST-RNF38 clone was a generous gift from Prof. Danny Huang. Purification of GST-RNF38 and GST-RNF4 was done using GST beads (Protino) and eluted out with 10 mM reduced Glutathione. Pure protein was eluted after SEC in 50 mM Tris buffer with 250 mM NaCl and store at −20°C. Clones of proteasome receptors Rpn10/S5a (UIM motifs: 196–306 aa) and hRpn13 (Pru domain: 1–150 aa) were a kind gift from Dr. Kylie Walters laboratory. Both the clones were tagged with N-terminus 6×His-tag, and their respective purification was performed as followed for other his-tagged E2 proteins mentioned above.

### NMR assignments and structure calculations

13C, 15N-labeled vUb protein in 25 mM sodium acetate pH 5.0 buffer with 100 mM NaCl was concentrated up to 1 mM, dissolved in 90–10% v/v H2O–D2O buffer. NMR spectra were recorded at 298 K on 600 MHz Bruker Avance III HD spectrometer equipped with a cryoprobe head. 2D 1H-15N-HSQC and 3D CBCA(CO)NH, HNCACB, HNCA, HN(CO)CA, HNCO experiments were used for backbone assignments. Sidechain assignments were determined by recording 3D H(CCO)NH, (H)CC(CO)NH, and HCCH-TOCSY experiments. 2D (HB)CB(CGCD)HD experiment data along with 2D 13C aromatic HSQC spectra were used for assigning aromatic resonances. Standard 3D 15N-NOESY-HSQC and 13C-NOESY-HSQC experiments (τmix = 100 ms) were used to get NOE restraints. All NMR data were processed in NMRpipe [27] and analyzed by NMRFAM-SPARKY software [28]. Following peak-picking of the backbone and sidechain resonances in SPARKY, the peaks were assigned by PINE and confirmed manually. Automatic peak picking was performed for NOESY spectra. NOESY peak assignments were performed in CYANA [29] and corrected over multiple cycles of structure building. TALOS+ [30] was used to predict the phi and psi torsion angles from the assigned chemical shifts of backbone atoms. Phi/psi torsion angles and NOESY based distance restraints were used to determine the solution structure of vUb by CYANA software. One hundred starting structures were calculated, and out of which twenty lowest energy structure models were chosen to represent the final structural ensemble. The backbone dihedral angles of the final converged structures were evaluated by the Molprobity [31] and PSVS [32] suite of programs. Details of the NMR restraints and structure evaluation are provided in Table 1.

Table 1.
NMR and refinement statistics of vUb
 Distance restraints (NOE) Short range (|i − j|< = 1) 1206 Medium range (|i − j| < 5) 136 Long range (|i − j| => 5) 204 Total 1344 Other restraints Dihedral angles (Ψ,Φ) 130 CYANA target function (Å2) 0.27(±0.04) Average pairwise rmsd1 (Å) All backbone atoms 0.6 All heavy atoms 1.1 Deviations from idealized geometry Bond angles (°) 0.2 Bond lengths (Å) 0.001 Close contacts 0 Ramachandran statistics1 Most favoured regions (%) 97 Allowed regions (%) 3 Disallowed regions (%) 0
 Distance restraints (NOE) Short range (|i − j|< = 1) 1206 Medium range (|i − j| < 5) 136 Long range (|i − j| => 5) 204 Total 1344 Other restraints Dihedral angles (Ψ,Φ) 130 CYANA target function (Å2) 0.27(±0.04) Average pairwise rmsd1 (Å) All backbone atoms 0.6 All heavy atoms 1.1 Deviations from idealized geometry Bond angles (°) 0.2 Bond lengths (Å) 0.001 Close contacts 0 Ramachandran statistics1 Most favoured regions (%) 97 Allowed regions (%) 3 Disallowed regions (%) 0
1

Calculated for ordered residues (2–72) in an ensemble of 20 lowest energy structures.

### NMR dynamics

For NMR dynamics studies, uniformly labeled 15N-vUb protein (500 µM) was dialyzed in 20 mM sodium phosphate buffer, 100 mM NaCl with pH 6.0. All the relaxation datasets were obtained at 298 K on the Bruker Avance 800 MHz instrument equipped with a cryoprobe. longitudinal (T1), transverse (T2) time constraints, and heteronuclear Overhauser enhancement (het-NOE) experiments were carried out using standard pulse sequences in Bruker. For T1 measurements, HSQC data were recorded at following relaxation delays: 0.004, 0.03, 0.06, 0.1, 0.15, 0.2, 0.4, and 0.8 s. For T2 measurements, the relaxation delays were set to 0.002, 0.004, 0.008, 0.016, 0.032, 0.064, 0.096, and 0.128 s. 15N-heteronuclear NOE values were calculated from the ratio of Isat/Iunsat. Order parameter (S2) was calculated using T1, T2, and het-NOE experiments and then fitted to spectral density function [33,34] in Bruker Dynamic Centre software. The error of the fits was generated using the Monte Carlo simulation.

### Denaturation melts

#### Chemical melt

A total of 40 µM of protein was incubated overnight in 20 mM sodium phosphate buffer (pH 6) containing 100 mM salt and different concentrations of guanidium hydrochloride from 0 to 6 M at room temperature. The unfolding of protein was probed by monitoring the far-UV CD signal at 222 nm using Jasco J-815 spectropolarimeter. The data were analyzed and fitted to a two-state (N→U) unfolding process using Sigma plot software where the raw data were converted into fraction unfolded (Fu) values using the following equation [35]:
$Fu={y−(p∗[GdnCl]+q)}/{(r∗[GdnCl]+s)−(p∗[GdnCl]+q)}$
Here, y is the CD value at a particular concentration of [GdnCl]; p and r represent slopes whereas q and s represent intercepts of the native and unfolded protein baselines, respectively. Further fitting the curve of Fu vs. [GdnCl] using monomeric 2-para melt equation, the free energy of unfolding, ΔGunfolding, and slope of the transition curve, m, were obtained. Finally, the free energy (ΔG0) at 0 M [GdnCl] is calculated by fitting ΔGunfolding and m in the following 2-state unfolding equation [36]: ΔGunfolding, [GdnCl] = ΔG0 − m[GdnCl].

#### Thermal melt

A total of 40 µM of protein in sodium phosphate buffer and 100 mM salt was used for thermal melt. The mean residual ellipticity at 222 nm was measured using Jasco J-815 spectropolarimeter, which is connected to a Peltier. The data were recorded from 30 to 95°C after every 1°C per minute rise in temperature with 32 s of data integration time and finally fitted with a two-state folding curve to obtain Tm.

#### Temperature coefficient

Temperature coefficients experiments were performed using uniformly labeled 15N-vUb and 15N-Ub samples dialyzed in 25 mM NaOAc, 50 mM NaCl, and pH 5.8. A total of 9 HSQC spectra were collected for both the proteins from 283 to 323 K after every 5 K increment. All the NMR HSQC experiments were processed by NMRPipe and analyzed in Sparky. Only shifts in 1H-dimension were considered and later analyzed using linear regression by MATLAB. The slope of each plot between ΔδNH and temperature is termed as temperature coefficients (ΔδNHT).

### Binding with proteasome receptors

#### NMR titrations

For NMR titration experiments, 110 µM 15N-vUb was titrated with 700 µM of S5a-UIMs at following molar ratios 0.25, 0.5, 1, 2, 3, 4, and 5. The reverse titration was performed using and 105 µM 15N-S5a and 1.3 mM vUb in 0.25, 0.5, 1, 2, 3, 4, and 5 molar ratios. Both the titration was performed in 20 mM sodium phosphate buffer (pH 6) with 100 mM NaCl. Chemical shift assignments of S5a was taken from the BMRB server (ID: 6233). All the titration data were processed in NMRpipe and analyzed in Sparky software. Using MATLAB, the titration data were fit in either 1 : 1 or 1 : 2 protein : ligand model using the equation CSPobs = CSPmax {([P]t + [L]t + Kd) − [([P]t + [L]t + Kd)2 - 4[P]t[L]t]1/2}/2[P]t, where [P]t and [L]t are total concentrations of protein and ligand at any titration point. NMR titration experiments for 15N-vUb (100 μM) and hRpn13 (400 μM) were performed at following molar ratios 0.5, 1, 2, 3, and 4 in 20 mM sodium phosphate buffer (pH 7.5) with 250 mM NaCl. However, due to intermediate exchange and high affinity, the Kd values could not be calculated between vUb and Rpn13.

#### ITC binding assay

vUb and S5a were co-dialyzed in 20 mM sodium phosphate buffer (pH 7.5) containing 100 mM NaCl. A total of 4 mM vUb was injected into the sample cell containing 80 µM of S5a at 25°C. The measurement was performed on a Microcal ITC200 (GE Healthcare), and binding isotherm was plotted and analyzed using Origin (v7.0). Integrated interaction heat values from individual experiments were normalized, and the data were fit, omitting the first point, using a single binding site model.

#### Affinity measurements by fluorescence spectroscopy

The tryptophan fluorescence of Rpn13 was monitored during titration with vUb to determine the Kd. The intrinsic fluorescence emission scan from 300 to 400 nm was measured using Horiba Fluromax-4 after excitation at 295 nm. The slit width for excitation and emission was kept at 3 nm. A decrease in fluorescence was observed after adding vUb with increasing molar quantities from 0.5 nM to 10 μM. The change in fluorescence intensity at 340 nm at different vUb molar ratios was used to generate the binding curve. The total bound fraction was calculated (for n = 3) and normalized and then fitted to a one-site specific binding curve in GraphPad Prism 8 to calculate Kd.

#### In vitro ubiquitination

In vitro ubiquitination reactions were carried out using 1 µM of UbE1, 5 µM of E2s, 10 µM of E3s and 50 µM of Ub and vUb. The reaction was activated by reaction buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 2.5 mM MgCl2 and 5 mM ATP and incubated for 1 h at 37°C. The ubiquitination reaction was quenched with 25 mM EDTA and further ran on 15% SDS gel. For probing polyubiquitin chains, immunoblotting was performed using Ubiquitin monoclonal antibody (P4D1 from ENZO) at 1 : 5000 dilution, and HRP conjugated anti-mouse secondary antibody at 1 : 10 000 dilutions. The chemiluminescence was observed by ECL (Bio-Rad) staining in Image Quant LAS 4000 (GE). The time point based assay was carried out with 5 µM of Ub and vUb. The SDS–PAGE gel was stained overnight with SYPRO® Ruby protein gel stain and imaged in UVITEC Cambridge.

#### In vitro deubiquitination assay

For the deubiquitination assays, 5 µl for Ub and vUb and 10 µl for K54o-vUb of the quenched ubiquitination reaction mixture, which was carried out by UbcH5b and RNF38, was incubated with different DUBs such as USP2CD, GST-AMSH, OTUBAIN, Cezzane, Trabid, OTUD3, and YOD1. All DUBs except GST-AMSH were activated in DUB activation buffer (25 mM Tris, 150 mM NaCl, and 10 mM DTT) for 15 min at RT before initiating their respective deubiquitination reactions. The deubiquitination reaction was carried out at 37°C for 1 h, quenched with SDS loading dye and immunoblotted onto the PVDF membrane, and finally probed using the Ubiquitin antibody (as mentioned above).

#### Mammalian cell culture experiments

1XFLAG-Ub, 1XFLAG-vUb, and 1XFLAG-K54o-vUb were cloned in the pcDNA3.2 vector. Approximately 50 000 cells were seeded in each of 12-well plates and incubated for 12 h at 37°C with 5% CO2. Subsequently, the cells were transfected with 1XFLAG-Ub, 1XFLAG-vUb, and 1XFLAG-K54o-vUb using Lipofectamine 3000. Post transfection, the cells were incubated for 24 h. The cells were then treated with MG132 for 12 h before lysing the cells in 1×TBS containing 0.5% NP40 and 1× protease inhibitor cocktail. Total protein was estimated using BCA reagent (Thermo), and the final amount of 3 µg of FLAG-Ub and 15 µg of FLAG-vUb and FLAG-K54o-vUb were loaded on 10% SDS–PAGE gel. Immunoblots were probed with a Flag antibody. Quantification of poly-Ub and poly-vUb conjugates (n = 3) were done using ImageJ and plotted in GraphPad Prism 8.

#### Yeast strains, growth conditions, protein extraction, and detection

The prototrophic yeast strain CEN.PK was used in the experiments involving Saccharomyces cerevisiae. Since the experiments involved MG132 treatment, the multidrug transporter PDR5 was deleted in all the strains to prevent the efflux of MG132. The standard YPD (1% yeast extract, 2% peptone, and 2% dextrose) growth media was used in all experiments. Cells were grown at 30°C in every experiment except for the heat stress experiment. Cells were grown at 30°C to an OD600 ∼ 0.8 and then shifted to 40°C in a water bath for the heat stress experiments. In the experiments involving MG132 treatment, a final concentration of 100 μM MG132 was used. The gene deletions were done using a standard PCR based gene deletion strategy [37].

Ten OD600 cells were pelleted, and protein extraction was done using the TCA (trichloroacetic acid) method. Briefly, the pellets were resuspended in 400 µl 10% TCA and bead beaten using glass beads (3 × 20 s, with 1 min intermittent cooling). The lysate was collected and centrifuged at 14 000 rpm, 10 min at 4°C. The protein pellets were resuspended in 400 µl SDS–Glycerol buffer (7.3% SDS, 29.1% glycerol, and 83.3 mM Tris base) and heated at 95°C for 10 min. The supernatant collected after centrifugation (14 000 rpm, 10 min at RT) was used to perform a BCA protein estimation (BCA assay kit, G-Biosciences). Samples were normalized and run in 4–12% bis-tris gels (Invitrogen). Western blots were developed using an anti-HA antibody (Sigma, H6908). Horseradish peroxidase-conjugated secondary antibodies (mouse and rabbit) were obtained from Sigma. Chemiluminescence reagent (Western Bright ECL HRP substrate, Advansta) was used to develop the blots. Coomassie-stained gels showing total protein was used as the loading control. Quantifications of the blot (n = 3) were done using ImageJ.

## Results

### Sequence conservation of viral Ubiquitin

A gene tree was reconstructed using the ML method [23] from all Ub coding amino acid sequences taken from 39 baculoviruses, out of which 31 are NPV including AcMNPV and 8 are Granulovirus (GV) (Supplementary Table S1). The gene tree suggests all the GVs are monophyletic, while NPV and MNPVs are polyphyletic groups. Interestingly, all the K54 containing viruses share a common ancestry, except EpNPV (Figure 1). The sequence identity of vUb from AcMNPV with human Ub is 76% (Figure 2A). The Ub surface is generally polar, except a hydrophobic surface centered around the C-terminal end of β-strand 5 that includes Leu8, Ile44, and Val70. The hydrophobic surface plays a role in many interactions with Ub binding domains and enzymes in the Ubiquitination reaction. The sequence alignment suggested that the hydrophobic surface is conserved in viral Ub sequences. On the contrary, the buried aliphatic hydrophobic residues like Leu15, Ile23, and Val26 are not conserved. The seven lysines that extend polyubiquitin chains are also conserved. The significant differences in the sequence are observed in helix α1, the loop between β2 and α1, and the loop between β4 and β5.

#### Sequence analysis of vUb.

Figure 1.
Sequence analysis of vUb.

Maximum Likelihood tree of vUb among 39 insect viruses (31 Nuclear Polyhedrosis Virus and 8 Granuloviruses) based on the amino acid sequences. All the insect viruses with extra lysine at the 54th position are highlighted in red. Ub sequence from Bombyx Mandarina (host) is used as an outgroup.

Figure 1.
Sequence analysis of vUb.

Maximum Likelihood tree of vUb among 39 insect viruses (31 Nuclear Polyhedrosis Virus and 8 Granuloviruses) based on the amino acid sequences. All the insect viruses with extra lysine at the 54th position are highlighted in red. Ub sequence from Bombyx Mandarina (host) is used as an outgroup.

#### Solution structure of vUb.

Figure 2.
Solution structure of vUb.

(A) Alignment of human Ub and vUb with secondary structure information given on the top. The identical residues are colored in blue in the background. The non-conserved hydrophobic residues are colored in gray in the background. (B) The 15N-edited HSQC spectrum of vUb is shown. The assigned backbone amide resonances are represented by one-letter amino acid code followed by residue number. The side-chains resonances of glutamines and asparagines are connected with by black dashed lines. (C) The 20 lowest energy structures of vUb are in cartoon representation. The N-termini, C-termini, and secondary structures are labeled. (D) The lowest energy structure of vUb (purple) is superimposed on the structure of Ub (orange, PDB: 1UBQ). (E) The vUb/Ub superposition is shown in pipes-and-planks representation. There is a difference of 20° in the orientation of helix α1 between Ub and vUb. (F) The structures of Ub and vUb are shown as space-filling models, where the atoms are shown as cyan spheres. (G) The important hydrophobic patch L8-I44-V70 is shown for Ub and vUb. The sidechain of the residues is shown in red. (H) The lysines used to extend polyubiquitin chains in Ub and vUb are shown. The sidechains are colored in blue. The lysine residues are colored in black, and the additional lysine K54 in vUb is colored in red.

Figure 2.
Solution structure of vUb.

(A) Alignment of human Ub and vUb with secondary structure information given on the top. The identical residues are colored in blue in the background. The non-conserved hydrophobic residues are colored in gray in the background. (B) The 15N-edited HSQC spectrum of vUb is shown. The assigned backbone amide resonances are represented by one-letter amino acid code followed by residue number. The side-chains resonances of glutamines and asparagines are connected with by black dashed lines. (C) The 20 lowest energy structures of vUb are in cartoon representation. The N-termini, C-termini, and secondary structures are labeled. (D) The lowest energy structure of vUb (purple) is superimposed on the structure of Ub (orange, PDB: 1UBQ). (E) The vUb/Ub superposition is shown in pipes-and-planks representation. There is a difference of 20° in the orientation of helix α1 between Ub and vUb. (F) The structures of Ub and vUb are shown as space-filling models, where the atoms are shown as cyan spheres. (G) The important hydrophobic patch L8-I44-V70 is shown for Ub and vUb. The sidechain of the residues is shown in red. (H) The lysines used to extend polyubiquitin chains in Ub and vUb are shown. The sidechains are colored in blue. The lysine residues are colored in black, and the additional lysine K54 in vUb is colored in red.

### Solution structure of viral Ubiquitin

The structure of vUb was determined to see if these differences in the sequence have any structural implications for vUb. Uniformly double-labeled (13C, 15N) vUb sample was prepared to determine its solution structure. The sample prepared in 25 mM sodium acetate (pH 5.0) and 100 mM NaCl was concentrated up to 1 mM, dissolved in 90–10% v/v H2O–D2O buffer. All NMR spectra were recorded at 298 K on 600 MHz Bruker Avance III HD spectrometer equipped with a cryoprobe head. The 15N-edited HSQC spectrum of vUb showed that the backbone amide resonances were well-dispersed, suggesting a folded protein (Figure 2B). A series of 3D NMR experiments, including 3D CBCA(CO)NH, HNCACB, HNCA, HN(CO)CA, and HNCO, were recorded and analyzed to assign chemical shifts of backbone 1H, 15N, 13Cα, 13C′ and the sidechain 13Cβ resonances. The dihedral angles were obtained by analyzing the backbone chemical shifts in TALOS+ [30]. Standard 3D H(CCO)NH, (H)CC(CO)NH, and HCCH-TOCSY and HCCH-COSY experiments were carried out to assign the chemical shifts of the side chain atoms. 2D (HB)CB(CGCD)HD experiment and 2D 13C aromatic HSQC spectra were used for assigning the chemical shifts of aromatic sidechains. 15N-edited 3D NOESY-HSQC and 13C-edited 3D NOESY-HSQC experiments with mixing time of 100 ms were recorded to obtain the distance constraints. All NMR data were processed in NMRpipe and analyzed by NMRFAM-SPARKY software [28]. NOESY peak assignments were performed in CYANA [29] and corrected over multiple cycles of structure building. In the final cycle, 100 structures were calculated using dihedral and distance constraints, and the twenty lowest energy structures superimposed with an rmsd of 1.1 Å (Figure 2C). The backbone dihedral angles of the final converged structures were evaluated by the Molprobity and PSVS suite of programs [31,32]. The NMR and refinement statistics are provided in Table 1.

The structure of vUb maintains the conserved beta-grasp fold like Ub, including five beta sheets (β1–β5) and one helix (α1) (Figure 2C). The structures of vUb and Ub (PDB: 1UBQ) superimpose well with an rmsd of 2.0 Å (Figure 2D). A significant difference in structures of Ub and vUb is observed in the orientation of helix α1 (Figure 2E). The substitutions in β-strands β2, β5, and helix α1 altered the sidechain interactions at the buried core of vUb (Supplementary Figure S1A,B). Consequently, the helix α1 in vUb is rotated by twenty degrees away from the α1 in Ub (Figure 2E). The helix α1 in vUb is farther from the β1–β5 sheets than in Ub. As a result, a cavity is observed in vUb, and the two salt-bridges present in helix α1 in Ub are absent in vUb (Figure 2F and Supplementary Figure S1C). The electrostatic surface potential distribution between Ub and vUb is similar, except for an additional negative charge in the loop between β1 and α1 of vUb (Supplementary Figure S1D). In this region, the negatively charged residue D21 is buried in Ub. The corresponding residue E21 is exposed in vUb, giving rise to the differences in surface charge. The surface of the beta-sheet that presents the critical hydrophobic patch (L8-I44-V70) is similar between Ub and vUb (Figure 2G). Apart from the seven conserved lysines, an extra lysine K54 is present in the loop between β4 and α2 in vUb (Figure 2H).

Alanine scan studies on Ub have suggested that hydrophobic contacts present at the buried core are essential to maintain high stability and rigidity [38]. Mutations of buried residues I30A-Ub and L43A-Ub create a cavity and significantly reduce the stability of Ub. I30 on α1 tightly contacts L15 on the β2 strand (Supplementary Figure S1A). However, in the vUb, L15 is substituted to A15, and this disrupts the contact between I30 and L15. I36 is present at the C-terminal loop of α1 and packs between L69 and L71 of the β5 strand (Supplementary Figure S1D). L69 is substituted to M69 in vUb, which may disrupt the interaction between the β5 strand and α1. Overall, structural studies suggest that the stability of vUb and Ub may differ due to differences at the buried core and differences in the number of salt-bridges.

### The backbone dynamics of vUb is similar to Ub

The backbone dynamics in the ps–ns timescale of vUb and Ub was compared by measuring the longitudinal (T1) relaxation, transverse (T2) relaxation, and the het-NOE of the two proteins. The dynamics experiments were carried out at 298 K and pH 6.0 (Figure 3A–D). These measured values were used to analyze the order parameter (S2) using the model-free approach [33,34]. The values of S2 are limited between 0 and 1, where S2 values close to 0 means a higher degree of disorder in the N–H bond vector, and values close to 1 indicate a restricted N–H bond vector. The average value of S2 in vUb is 0.824, suggesting that the protein is significantly rigid. The S2 values dropped by 0.2 in the loop between β1 and β2. The same happened in the loop between β4 and β5, suggesting that these regions are more dynamic than the rest (Figure 3E). For comparison, the T1, T2, and het-NOE experiments were repeated on Ub, and its S2 was calculated. A comparison between the S2 values of Ub with vUb is given in Figure 3F. The β1 and β2 are less dynamic in vUb compared with Ub. In vUb, the residue V5 in β1 is substituted to I and L69 in β5 is substituted to M. The substituted longer sidechains form more local contacts between β1, β2 and β5 in vUb than Ub, which probably decreases the local dynamics. For example, an additional network of contacts between M69(β5)-I13(β2)-T7(β1) is present in vUb but absent in Ub (Supplementary Figure S1E). The regions in vUb that have S2 values higher than the standard deviation in Figure 3F include the region in between β2 and α1, and the region from β4 to β5, indicating that these regions are more dynamic in vUb than Ub. The lower rigidity in the loops is in synchrony with the differences in packing observed in the structure of vUb. Interestingly, the loop between β3 and β5 is dynamic and disordered in a higher-energy partially unfolded intermediate of Ub, which has lower stability than native Ub [39]. The higher dynamics in the β3–β5 region of vUb suggest that Ub may be more stable than vUb.

#### The ps-ns backbone dynamics of vUb (orange) and Ub (grey).

Figure 3.
The ps-ns backbone dynamics of vUb (orange) and Ub (grey).

The measured values of (A) longitudinal relaxation, T1, (B) transverse relaxation, T2, and (C) the T1/T2 ratio for each residue is plotted against the residue number of vUb and Ub. (D) heteronuclear nuclear Overhauser enhancement, het-NOEs, were plotted for each residue of vUb and Ub. (E) The order parameter S2 was calculated using the Lipari Szabo model and plotted against the residue number of vUb and Ub. (F) The difference in S2 between Ub and vUb (δS2) is plotted. The standard deviation is highlighted with the black dashed line. The secondary structure elements of vUb against its sequence are provided on top of each plot.

Figure 3.
The ps-ns backbone dynamics of vUb (orange) and Ub (grey).

The measured values of (A) longitudinal relaxation, T1, (B) transverse relaxation, T2, and (C) the T1/T2 ratio for each residue is plotted against the residue number of vUb and Ub. (D) heteronuclear nuclear Overhauser enhancement, het-NOEs, were plotted for each residue of vUb and Ub. (E) The order parameter S2 was calculated using the Lipari Szabo model and plotted against the residue number of vUb and Ub. (F) The difference in S2 between Ub and vUb (δS2) is plotted. The standard deviation is highlighted with the black dashed line. The secondary structure elements of vUb against its sequence are provided on top of each plot.

### Ub is more stable than vUb

Chemical-induced and temperature-induced denaturation melts were carried out to compare the stability of vUb and Ub. For the chemical denaturation melt, purified vUb (40 µM) and Ub (40 µM) were incubated overnight in different concentrations of guanidium chloride (ranging from 0 to 6 M). Subsequently, circular dichroism (CD) spectra were collected for the proteins. The concentration of Guanidium chloride required to unfold 50% of vUb was 1.9 M. Alternately, Ub required 3.6 M Guanidium chloride to unfold 50% of the protein, indicating that Ub was more stable than vUb. The ΔG0 reduced from 11.5 kcal mol−1 for Ub to 6.6 kcal mol−1 for (Figure 4A and Table 2). The stability of the proteins was also measured by thermal denaturation. The melting point (Tm) for vUb was 74°C. Ub did not melt up to 90°C, confirming that it is more stable than vUb (Figure 4B).

#### A comparison of the stability of Ub and vUb.

Figure 4.
A comparison of the stability of Ub and vUb.

(A) GdnCl melt curves of vUb and Ub. Normalized mean ellipticity shift is plotted against GdnCl concentration (B) The thermal melt curve of vUb and Ub. Change in ellipticity is normalized and plotted against temperature. (C,D) Overlay of 15N-HSQC of vUb and Ub, respectively, at temperatures ranging from 283 to 323 K. Temperature coefficients are plotted against the residue numbers for (E) vUb and (F) Ub. The secondary structure elements of vUb and Ub are drawn on top of the respective plots. (G) The difference in temperature coefficients of Ub and vUb is plotted against residue numbers. The standard deviation is shown as dashed black lines. Red bars represent the residues in vUb that are significantly destabilized upon increasing temperature. The secondary structure elements of vUb are drawn on top of the plot.

Figure 4.
A comparison of the stability of Ub and vUb.

(A) GdnCl melt curves of vUb and Ub. Normalized mean ellipticity shift is plotted against GdnCl concentration (B) The thermal melt curve of vUb and Ub. Change in ellipticity is normalized and plotted against temperature. (C,D) Overlay of 15N-HSQC of vUb and Ub, respectively, at temperatures ranging from 283 to 323 K. Temperature coefficients are plotted against the residue numbers for (E) vUb and (F) Ub. The secondary structure elements of vUb and Ub are drawn on top of the respective plots. (G) The difference in temperature coefficients of Ub and vUb is plotted against residue numbers. The standard deviation is shown as dashed black lines. Red bars represent the residues in vUb that are significantly destabilized upon increasing temperature. The secondary structure elements of vUb are drawn on top of the plot.

Table 2.
Analysis of Guanidium chloride melting curves of Ub and vUb
ΔG0 (kcal mol−1)m (kcal mol−1)[GdnCl] (in M) at 50% unfolding
Ub 11.5 ± 1.2 3.2 ± 0.3 3.65
vUb 6.6 ± 0.5 3.4 ± 0.2 1.89
ΔG0 (kcal mol−1)m (kcal mol−1)[GdnCl] (in M) at 50% unfolding
Ub 11.5 ± 1.2 3.2 ± 0.3 3.65
vUb 6.6 ± 0.5 3.4 ± 0.2 1.89

The local stability of vUb and Ub were probed using NMR by measuring and comparing the temperature coefficients per residue. The 2D-HSQC experiment was recorded for 15N-vUb and 15N-Ub from 283 to 323 K at every 5 K increment (Figure 4C,D). The resonance shifts were fit linearly against temperatures to calculate the temperature coefficients (Figure 4E,F). The average temperature coefficient ΔδNHT values of almost all the secondary structures of vUb and Ub were nearly the same. The ΔΔδNHT (Ub–vUb) values are plotted in Figure 4G, where negative and positive values indicate residues in vUb with higher and lower stability than Ub residues. Intriguingly, the ΔΔδNHT values of non-conserved residues like V23, L26, and A31 in α1, T55, and M57 in α2, Q62, S65, and M69 in β5 strand were positive, indicating that these substitutions resulted in local instability in vUb (Figure 4G). Several residues had high positive values in α2, β5, and in the loop in between, implicating that vUb is relatively unstable in these loop regions (Figure 4G).

### vUb interacts with proteasome receptors Rpn10/S5a and Rpn13

The interaction of vUb with Ub binding domains in proteasome receptors was examined in vitro to assess if the differences in structure and stability are reflected in the interactions with cofactors. The Ub-26S proteasome pathway targets misfolded or tightly regulated proteins for degradation by conjugating them with a poly-Ub chain [40]. The ubiquitinated substrate interacts with non-ATPase subunits such as Rpn1, Rpn10/S5a, and Rpn13 of the proteasome via Ub [7,41–44]. Subsequently, the substrate enters the core particle for degradation. Since the insect proteasome subunits have a high degree of similarity to the mammalian system [45], and the proteins of mammalian proteasome receptors were available, the interaction between vUb and the ubiquitin-binding domains in proteasome receptors (Rpn10/S5a and Rpn13) were tested to examine if substrates conjugated with poly-vUb chains can be recognized by the proteasome receptors.

NMR spectroscopy and isothermal titration calorimetry (ITC) was used to determine if vUb binds to Rpn10/S5a. Uniformly labeled 15N-vUb was titrated with unlabeled S5a-UIMs, and 2D-HSQC experiments were recorded till saturation. The altered chemical environment at the interface upon binding changes the chemical shifts of backbone amide resonances (Figure 5A). The difference in chemical shifts between the apo and bound form of vUb was plotted as chemical shift perturbation (CSP) in Figure 5B. The hydrophobic surface patches of vUb consisting I44, V70, and L71 along with polar residues of β4 (K48 and Q49) had maximum CSPs, suggesting that these residues are present at the interface of S5a/vUb complex (Figure 5C). Additionally, the hydrophobic residues like A15, I30, L43, G47, and L67 and charged residues like D32, R42, and R72 are also present at the interface. The chemical shift values were fit against the ligand concentration in the NMR titration experiments to yield the dissociation constant (Kd) of 78 ± 12 µM (Supplementary Figure S2).

#### vUb binds to proteasome receptor S5a and Rpn13.

Figure 5.
vUb binds to proteasome receptor S5a and Rpn13.

(A) Overlay of 15N-edited HSQC spectra of 15N-vUb with different stoichiometric ratios of S5a. The resonances of residues I13 and I44 are zoomed for clarity. (B) The CSP observed in vUb upon titration with S5a is plotted against the vUb residue number. Mean and mean with standard deviation are highlighted in pink and purple, respectively. (C) The high CSPs are mapped on to the vUb structure. (D) Overlay of 15N-HSQC spectra of 15N-S5a with different stoichiometric ratios of vUb. Chemical shift assignments of 15N-S5a-UIMs were obtained from the BMRB server (id: 6233). T282 and E284 resonances are zoomed for clarity. (E) The CSP observed in S5a upon titration with vUb is plotted against the S5a residue number. Mean and mean with standard deviation are highlighted in blue and purple, respectively. (F) The residues with high CSPs are highlighted in the S5a structure. Best model structure of vUb (olive green) superimposed on proximal and distal Ub in S5a-K48 linked-diUb NMR structure (PDB: 2KDE). Zoomed images show the interaction of helix α1(1) and α3(2) of S5a with vUb. (G) The interaction between vUb and S5a is measured by ITC. The titration curve indicates a 1 : 2 stoichiometric of S5a:vUb complex. The fit of the ITC data yielded the dissociation constant (Kd) = 85 (± 9.9) μM, stoichiometry n = 2.0 (± 0.1), ΔH = −4.5 (± 0.3) kcal mol−1 and TΔS = 0.09 kcal mol−1. (H) The binding curve for Rpn13 Pru domain binding to vUb as determined by intrinsic tryptophan fluorescence, which yielded the dissociation constant of 40 (±15) nM. (I) Accumulation of Ub and vUb conjugates in HEK293T cells upon proteasomal complex inhibition. Cells were transfected with FLAG-Ub or FLAG-vUb, and post inhibition by the proteasomal inhibitor MG132, cell lysates were separated on SDS–PAGE and immunoblotted with anti-FLAG antibody. α-tubulin was used for loading control. The bottom panel of the bar graph shows the quantitative analysis of the accumulation of poly-Ub and poly-vUb conjugates (n = 3).

Figure 5.
vUb binds to proteasome receptor S5a and Rpn13.

(A) Overlay of 15N-edited HSQC spectra of 15N-vUb with different stoichiometric ratios of S5a. The resonances of residues I13 and I44 are zoomed for clarity. (B) The CSP observed in vUb upon titration with S5a is plotted against the vUb residue number. Mean and mean with standard deviation are highlighted in pink and purple, respectively. (C) The high CSPs are mapped on to the vUb structure. (D) Overlay of 15N-HSQC spectra of 15N-S5a with different stoichiometric ratios of vUb. Chemical shift assignments of 15N-S5a-UIMs were obtained from the BMRB server (id: 6233). T282 and E284 resonances are zoomed for clarity. (E) The CSP observed in S5a upon titration with vUb is plotted against the S5a residue number. Mean and mean with standard deviation are highlighted in blue and purple, respectively. (F) The residues with high CSPs are highlighted in the S5a structure. Best model structure of vUb (olive green) superimposed on proximal and distal Ub in S5a-K48 linked-diUb NMR structure (PDB: 2KDE). Zoomed images show the interaction of helix α1(1) and α3(2) of S5a with vUb. (G) The interaction between vUb and S5a is measured by ITC. The titration curve indicates a 1 : 2 stoichiometric of S5a:vUb complex. The fit of the ITC data yielded the dissociation constant (Kd) = 85 (± 9.9) μM, stoichiometry n = 2.0 (± 0.1), ΔH = −4.5 (± 0.3) kcal mol−1 and TΔS = 0.09 kcal mol−1. (H) The binding curve for Rpn13 Pru domain binding to vUb as determined by intrinsic tryptophan fluorescence, which yielded the dissociation constant of 40 (±15) nM. (I) Accumulation of Ub and vUb conjugates in HEK293T cells upon proteasomal complex inhibition. Cells were transfected with FLAG-Ub or FLAG-vUb, and post inhibition by the proteasomal inhibitor MG132, cell lysates were separated on SDS–PAGE and immunoblotted with anti-FLAG antibody. α-tubulin was used for loading control. The bottom panel of the bar graph shows the quantitative analysis of the accumulation of poly-Ub and poly-vUb conjugates (n = 3).

In the reverse titration, uniformly labeled 15N-S5a-UIMs (196–306 aa) was titrated with unlabeled vUb (Figure 5D). S5a includes two UIMs, UIM1: 206–230 aa and UIM2: 274–300 aa. The chemical shift assignment of S5a was obtained from the BMRB server (id: 6233) [46]. High CSPs were observed in both the UIMs, suggesting that both interacted with vUb (Figure 5E). The fit of chemical shifts against ligand concentration yielded different Kd values, 331 ± 25 µM for UIM1 and 21 ± 6 µM for UIM2 (Supplementary Figure S3). A similar mode of interaction was also observed in S5a/Ub complex, where UIM2 had a higher affinity for Ub than UIM1 [42,47]. Residues in the conserved hydrophobic patch (L216ALAL220) had high CSPs in UIM1 and interacted with vUb. Additionally, several polar and charged residues in UIM1 (R221, S223, E225, and E226) also interacted with vUb. Similarly, apart from the hydrophobic patch (I287AYAM291) in UIM2, several polar and charged residues (S279, S280, E284, E285, Q292, S294, Q296, and E299) also interacted with vUb (Figure 5F). The vUb/S5a interaction was further studied by ITC (Figure 5G). The binding isotherm in ITC was fitted to a 1 : 2 stoichiometry complex to obtain the overall Kd of 85 ± 9.9 µM, which agrees well with the values as calculated by 15N-vUb/S5a NMR titration. Overall, the affinity and the interface of the vUb/S5a complex is similar to the Ub/S5a complex, indicating that S5a can identify substrates conjugated with vUb.

The binding interaction between vUb and another proteasome receptor, Rpn13, was studied by NMR. The N-terminal domain of human Rpn13 (1–150 aa, Pru domain) interacts with Ub [43]. Uniformly labeled 15N-vUb was serially titrated with the Pru domain, and 15N-HSQC spectra were recorded at each titration point (Supplementary Figure S4A). The CSP plot is shown in Supplementary Figure S4B. The vUb CSPs were mapped on a model of the Rpn13/vUb complex obtained from the Rpn13/Ub structure (PDB: 2Z59, Supplementary Figure S4C). Amide resonances of residues R42, G47, K48, Q49, L50, and L71, disappeared during titration, suggesting peak broadening due to conformational exchange upon binding. Resonances of F4, F45, A46, H68, and V70 disappeared during the initial titration points, but reappeared in the later titration points, suggesting an intermediate exchange and high affinity of the interaction. A similar observation was made in the Rpn13/Ub complex [43]. The dissociation constant of the Rpn13/vUb complex was estimated using fluorescence spectroscopy. This titration experiment yielded the Kd 40 ± 15 nM (Figure 5H), which is similar to the affinity between Rpn13 and Ub. Overall, the interaction of vUb with proteasome receptors Rpn10/S5a and Rpn13 are similar to Ub (Table 3).

Table 3.
Ub binding with Rpn10 and Rpn13
UIMUIM2Rpn13
Ub1 350 µM2 73 µM2 0.3 µM3
vUb 331 µM2 21 µM2 0.04 µM3
UIMUIM2Rpn13
Ub1 350 µM2 73 µM2 0.3 µM3
vUb 331 µM2 21 µM2 0.04 µM3
1

Refer [43];

2

Calculated by NMR;

3

Calculated by Fluorescence.

The activation of vUb in cellular conditions was studied by the transfection of HEK293T cells with FLAG-vUb, followed by detection with an anti-FLAG antibody in the presence/absence of proteasome inhibitor MG132. In the absence of MG132, higher molecular mass signals were observed (Figure 5I). These could be pure FLAG-vUb chains or Ub chains that have incorporated FLAG-vUb during elongation. The chains accumulated upon proteasome inhibition, indicating that they are recognized and degraded by the proteasome (Figure 5I bottom graph). These results suggest that vUb can be activated to either form poly-vUb chains or be incorporated into poly-Ub chains in cells.

### Polyubiquitination activity of vUb is similar to Ub

The differences in structure and stability may affect the activation of vUb in the ubiquitination reaction. The in vitro polyubiquitination activity of vUb was measured using multiple E2 : E3 pairs. Initially, E2s that form specific Ub linkages were used, and later, an E2 that forms all Ub linkages was tested. E2-25K specifically forms K48-linked poly-Ub chains whereas, Ubc13 (along with its cofactor Mms2) specifically forms K63-linked poly-Ub chains. E2-25K and Ubc13 can synthesize poly-Ub chains without any E3 in vitro [5,49,50]. Since these E2s are 80–90% similar between insects and humans (Supplementary Figure S5), and the human E2 proteins were available, we have used the human E2s in the reactions. Ubiquitination reactions were carried out with Ube1, E2-25K, and either Ub or vUb in the reaction mix and probed with Ub antibody. The antibody detected Ub and vUb comparably (Supplementary Figure S7A). The intensity of polyubiquitin chains observed in the western blots was similar between Ub and vUb, indicating that vUb forms K48-linked chains with similar efficiency as Ub (Figure 6A, left panel). As expected, di-Ub or higher Ub chains did not form in the absence of the E2 (Supplementary Figure S7B and Figure 6C right panel). The amount of K63-linked Ub chains catalyzed by the Ubc13/Mms2 complex was less in vUb than Ub (Figure 6B, left panel).

#### A comparison of the polyubiquitination activity between Ub and vUb.

Figure 6.
A comparison of the polyubiquitination activity between Ub and vUb.

The polyubiquitination reaction with Ub/vUb by; (A) E2-25K with RNF38 and RNF4, (B) Ubc13/ Mms2 with RNF38 and RNF4, and (C) UbcH5b with RNF38 and RNF4. The reaction without E2 is shown in lane 1 and 2 in the right panel image. All the reactions were probed with the anti-Ub antibody. NC denotes the negative control, where the polyubiquitination reaction was carried out without ATP. (D) Depleting amounts of mono-Ub (top) and mono-vUb (down) against time were detected in a polyubiquitination assay using E2-25K and RNF38 as the E2 and E3, respectively, on SDS–PAGE gel by SYPRO stain. The amount of mono-Ub and mono-vUb are plotted at the bottom. (E,F) are the same as (D), where the E2 is Ubc13/ Mms2 and UbcH5b, respectively. (G) The rates of depletion of Ub and vUb were calculated by linear fitting of initial time points (n = 3). (H) Confirmation of di-vUb formation using UbcH5b and RNF38 by immuno-detection with an anti-Ub antibody. The same reaction was separated on an SDS–PAGE gel, and the di-vUb was excised for MS/MS analysis. (I) MS–MS spectrum of one of the tryptic peptides (Seq: QLEDSK54TMADYNIQK, MW: 1896.8887Da, 2+) that confirms the isopeptide formation by K54th residue.

Figure 6.
A comparison of the polyubiquitination activity between Ub and vUb.

The polyubiquitination reaction with Ub/vUb by; (A) E2-25K with RNF38 and RNF4, (B) Ubc13/ Mms2 with RNF38 and RNF4, and (C) UbcH5b with RNF38 and RNF4. The reaction without E2 is shown in lane 1 and 2 in the right panel image. All the reactions were probed with the anti-Ub antibody. NC denotes the negative control, where the polyubiquitination reaction was carried out without ATP. (D) Depleting amounts of mono-Ub (top) and mono-vUb (down) against time were detected in a polyubiquitination assay using E2-25K and RNF38 as the E2 and E3, respectively, on SDS–PAGE gel by SYPRO stain. The amount of mono-Ub and mono-vUb are plotted at the bottom. (E,F) are the same as (D), where the E2 is Ubc13/ Mms2 and UbcH5b, respectively. (G) The rates of depletion of Ub and vUb were calculated by linear fitting of initial time points (n = 3). (H) Confirmation of di-vUb formation using UbcH5b and RNF38 by immuno-detection with an anti-Ub antibody. The same reaction was separated on an SDS–PAGE gel, and the di-vUb was excised for MS/MS analysis. (I) MS–MS spectrum of one of the tryptic peptides (Seq: QLEDSK54TMADYNIQK, MW: 1896.8887Da, 2+) that confirms the isopeptide formation by K54th residue.

Even for the E2s that can synthesize poly-Ub chains without E3s, the addition of E3s in the reaction enhances the rate of ubiquitination [51], because E3s stabilize the closed conformation of E2 ∼ Ub, which leaves the active site prone to aminolysis by the substrate lysine [52]. Therefore, it was important to confirm if the synthesis of polymeric chains of vUb and Ub were also comparable in the presence of E3s. In vitro reactions were repeated in the presence of two different E3s: RNF38 [53,54] and RNF4 [55]. The synthesis of K48-linked polyubiquitin chains was indifferent between vUb and Ub in the presence of E3s (Figure 6A, middle and right panel). The synthesis of K63-linked vUb chains was slightly lower than Ub (Figure 6B, middle and right panel). Finally, UbcH5b was used as the E2 in the ubiquitination reaction, which synthesizes poly-Ub chains of various linkages [56,57]. Polyubiquitination by UbcH5b with E3s was similar between Ub and vUb (Figure 6C).

Analysis of the polyubiquitin chains may fail to detect minor differences in the activation of Ub and vUb. Since the free Ub is activated to form poly-Ub chains, the rate of free Ub depletion reflects the activity of Ub. Hence, the depleting free Ub and vUb was quantified over time in polyubiquitination reactions. These experiments were performed with the E2s E2-25K, UbcH5b, and Ubc13/Mms2, using RNF38 as the E3. Interestingly, minor differences were observed in the activation of Ub and vUb, depending on the E2 used in the reaction. The activation of vUb is slightly better than Ub in the case of E2-25K and UbcH5b (Figure 6D,F). However, in the presence of Ubc13/Mms2, the activation of vUb was slower than Ub (Figure 6E). Unlike the E2-25K and UbcH5b, activity of the Ubc13/Mms2 complex depends on the Mms2/acceptor-Ub interaction, which may account for the lower enzyme activity. Overall, the rate of depletion in free Ub was within 2-fold of free vUb, implying that they had similar activity (Figure 6G).

### Polyubiquitin chains formed by vUb includes a novel K54-linked chain

The substitution of R54 by K54 in vUb provides an interesting possibility, where a new linkage type (K54-linked) polyubiquitin chains can be formed in vUb. UbcH5b can synthesize all the varieties of linkage chains and may also form a K54-linked vUb chain. A reaction was carried out using UbcH5b, RNF38, and vUb. After the reaction, the products were separated on SDS–PAGE gel and simultaneously blotted with the Ub antibody (Figure 6H). The band corresponding to di-vUb was extracted from the SDS gel, digested by trypsin, and analyzed by MS/MS. The MS/MS spectra detected multiple types of isopeptide linkages viz. K6-, K11-, K48-, and K63- (Table 4 and Supplementary Figure S6A–D). Apart from these canonical isopeptide linkages, a peptide containing the novel K54-linkage in di-vUb was also detected (Figure 6I). The abundance of the K54-linked vUb chain was in the same order as the K6-, K11-, and K48-linked chains (Supplementary Table S2). The abundance of K63-linked vUb chains was greater than the rest by an order.

Table 4.
List of peptides of di-viralUb
Peptide seqCalculated mol weight; charge
QIFIK6TLTGK 1392.7799; 2+
LIFAGK48QLEDSK 1461.7827; 2+
Peptide seqCalculated mol weight; charge
QIFIK6TLTGK 1392.7799; 2+
LIFAGK48QLEDSK 1461.7827; 2+

#### Comparison of polyubiquitin conjugate formation between vUb and K54o-vUb mutant.

Figure 7.
Comparison of polyubiquitin conjugate formation between vUb and K54o-vUb mutant.

Polyubiquitination reaction with either wt-vUb or K54o-vUb mutant by (A) E2-25K without and with RNF4 and (B) by Ubc13/Mms2 without and with RNF4. The lane labeled as K54 represents the reaction where K54o-vUb was used as a Ub source. The negative control lane is labeled NC, where the polyubiquitination reaction was carried out without ATP. (C) Poly-vUb conjugate formation by K54o-vUb mutant with UbcH5b/RNF38. The reaction was separated on an SDS–PAGE gel, and the di-vUb band was excised for MS/MS analysis. (D) The reaction is the same as C, but the K0-vUb mutant is used in the second lane. (E) MS/MS spectrum of a tryptic peptide (Seq: QLEDSK54TMADYNIQR, MW: 1924.8949Da, 2+) confirmed K54-linked di-vUb in the reaction.

Figure 7.
Comparison of polyubiquitin conjugate formation between vUb and K54o-vUb mutant.

Polyubiquitination reaction with either wt-vUb or K54o-vUb mutant by (A) E2-25K without and with RNF4 and (B) by Ubc13/Mms2 without and with RNF4. The lane labeled as K54 represents the reaction where K54o-vUb was used as a Ub source. The negative control lane is labeled NC, where the polyubiquitination reaction was carried out without ATP. (C) Poly-vUb conjugate formation by K54o-vUb mutant with UbcH5b/RNF38. The reaction was separated on an SDS–PAGE gel, and the di-vUb band was excised for MS/MS analysis. (D) The reaction is the same as C, but the K0-vUb mutant is used in the second lane. (E) MS/MS spectrum of a tryptic peptide (Seq: QLEDSK54TMADYNIQR, MW: 1924.8949Da, 2+) confirmed K54-linked di-vUb in the reaction.

### K54-linked vUb chains are poorly cleaved by DUBs

#### Deubiquitination of vUb conjugates by DUBs.

Figure 8.
Deubiquitination of vUb conjugates by DUBs.

vUb conjugates were formed by ubiquitination reaction involving Ube1, UbcH5b, and RNF38 and quenched by EDTA. To observe the DUB effect, we have loaded 2× volume in the case of vUb and K54o-vUb mutant. (A) The deubiquitination reaction of these conjugates was carried out using DUBs in the table. DUB reaction by USP2CD (B), GST-AMSH (C), OTUBAIN1 (D), Cezanne, Trabid and OTUD3 (E), and YOD1 (F). The Ub controls for USP2CD DUBs have been shown next to (B). The lane labeled as K54 represents K54-linked poly-vUb chains formed by K54o-vUb.

Figure 8.
Deubiquitination of vUb conjugates by DUBs.

vUb conjugates were formed by ubiquitination reaction involving Ube1, UbcH5b, and RNF38 and quenched by EDTA. To observe the DUB effect, we have loaded 2× volume in the case of vUb and K54o-vUb mutant. (A) The deubiquitination reaction of these conjugates was carried out using DUBs in the table. DUB reaction by USP2CD (B), GST-AMSH (C), OTUBAIN1 (D), Cezanne, Trabid and OTUD3 (E), and YOD1 (F). The Ub controls for USP2CD DUBs have been shown next to (B). The lane labeled as K54 represents K54-linked poly-vUb chains formed by K54o-vUb.

### K54-linked vUb chains are produced in cellular conditions

Whether K54-linked vUb chains can indeed participate in the Ubiquitin signaling in cells was tested by transfecting HEK293T with FLAG-vUb and FLAG-K54o-vUb. High molecular mass conjugates were detected with an anti-FLAG antibody, indicating that K54o-vUb can be incorporated into the poly-Ub chains (Figure 9A and Supplementary Figure S8D). We then investigated if exclusive vUb-linked polymeric chains could form in cellular conditions, using an S. cerevisiae model system. HA-tagged Ub and vUb were expressed under a constitutive TEF promoter in S. cerevisiae cells. We engineered yeast strains where the endogenous ubiquitin gene ubi4 was deleted (ubi4Δ). Ubi4 is a polyubiquitin gene, which encodes a fusion of 5 ubiquitin genes and is stress-inducible [70–72]. UBI4 is the major contributor to the cellular Ub pool, and the deletion of Ubi4 drastically decreases the cellular Ub in yeast cells [73]. The other three yeast ubiquitin genes ubi1, ubi2, and ubi3 encode Ubiquitin fused to ribosomal proteins, and therefore, the deletion of these genes is lethal [74]. The cells expressing HA-Ub and HA-vUb were treated with or without MG132 for 60 min. Higher molecular mass conjugates were observed in both HA-Ub and HA-vUb expressing cells (Figure 9B), suggesting that vUb-linked polymeric chains can be produced in cellular conditions. The amount of conjugates increased upon treatment with MG132, suggesting that vUb chains can signal proteasomal degradation (Figure 9C). Whether exclusive K54-linked polymeric vUb chains can be formed in cells was tested by expressing HA-K54o-vUb under the native ubiquitin promoter (ubi4 promoter), which is also induced during heat stress [71,73]. Similar to Ub and vUb, K54-linked vUb conjugates could also be observed upon induction of the K54o-vUb expression by heat stress (Figure 9D,E). Furthermore, upon treatment with MG132, higher molecular mass K54o-vUb conjugates accumulated, indicating that K54-linked vUb chains can signal proteasomal degradation (Figure 9F,G).

#### The detection of vUb and K54-linked vUb chains in cellular conditions.

Figure 9.
The detection of vUb and K54-linked vUb chains in cellular conditions.

(A) Accumulation of vUb and K54o-vUb conjugates in HEK293T cells upon proteasomal complex inhibition. Cells were transfected with FLAG-vUb or FLAG-K54o-vUb, and post inhibition by the proteasomal inhibitor MG132, cell lysates were separated on SDS–PAGE and immunoblotted with anti-FLAG antibody. α-tubulin was used as the loading control. The complete gel is shown in Supplementary Figure S8D. The lane labeled as FLAG-vUB-K54 is where the cells are transfected with FLAG-K54o-vUb. (B) Detection of vUb conjugates in ubi4Δ S. cerevisiae. Cells were transfected with HA-Ub and HA-vUb. Post the MG132 treatment, and proteins were extracted and separated on SDS–PAGE and immunoblotted with the anti-HA antibody. (C) The lanes in (B) are quantified and plotted. (D) HA-Ub, HA-vUb, and HA-K54o-vUb (labeled as HA-K54) are expressed under the ubi4 promoter. A subset of cells was heat stressed. The protocol for the detection of conjugates was similar to (B). (E) The lanes in (D) are quantified and plotted. (F) A subset of heat-stressed cells was treated with MG132 and detected using the protocol similar to (B). (G) The lanes in (F) are quantified and plotted. All the quantifications were done for n = 3 replicates.

Figure 9.
The detection of vUb and K54-linked vUb chains in cellular conditions.

(A) Accumulation of vUb and K54o-vUb conjugates in HEK293T cells upon proteasomal complex inhibition. Cells were transfected with FLAG-vUb or FLAG-K54o-vUb, and post inhibition by the proteasomal inhibitor MG132, cell lysates were separated on SDS–PAGE and immunoblotted with anti-FLAG antibody. α-tubulin was used as the loading control. The complete gel is shown in Supplementary Figure S8D. The lane labeled as FLAG-vUB-K54 is where the cells are transfected with FLAG-K54o-vUb. (B) Detection of vUb conjugates in ubi4Δ S. cerevisiae. Cells were transfected with HA-Ub and HA-vUb. Post the MG132 treatment, and proteins were extracted and separated on SDS–PAGE and immunoblotted with the anti-HA antibody. (C) The lanes in (B) are quantified and plotted. (D) HA-Ub, HA-vUb, and HA-K54o-vUb (labeled as HA-K54) are expressed under the ubi4 promoter. A subset of cells was heat stressed. The protocol for the detection of conjugates was similar to (B). (E) The lanes in (D) are quantified and plotted. (F) A subset of heat-stressed cells was treated with MG132 and detected using the protocol similar to (B). (G) The lanes in (F) are quantified and plotted. All the quantifications were done for n = 3 replicates.

## Discussion

Ubiquitin is the central component of the Ubiquitin signaling pathway. NPV and GV from the Baculoviridae family encode Ubiquitin in their genomes. An evolutionary analysis of viral Ubiquitin and eukaryotic Ubiquitin from different viruses and host insects suggested the possibility of horizontal gene transfer, and the phylogenetic analysis further confirmed the likelihood of one common ancient ancestral gene [17]. However, the retention of vUb within the viral genomes indicates that it might have unique properties different from the eukaryotic Ub. Here, we have compared the structure, dynamics, stability, and activity of the viral vUb with the host eukaryotic Ub. We report that the deviation from the Ub sequence has resulted in a cavity at the buried core of vUb and a reduced number of salt-bridges (Figure 2). As a result, the stability of vUb is reduced compared with Ub (Figure 4). Further experiments reveal that the region between β2 and α1, and between α2 and β5 are dynamic and unstable in vUb when compared with Ub. Typically, there is a trade-off between stability and newly acquired functions, where stability is likely to be compromised when proteins acquire new activities by evolutionary processes [75]. The lower stability in vUb may suggest an acquired function of vUb, which needs further investigation.

A major function of Ub signaling is to regulate optimal levels of proteins in the cell by the Ub-proteasome pathway. The proteasome receptors identify Ub chains on the substrate by the hydrophobic surface patch on Ub [42,43,47]. Our structural analysis suggests that the surface properties of Ub are primarily retained in vUb. In particular, the L8-I44-V70 hydrophobic surface patch is conserved in vUb. However, the structure of the buried core in a protein can regulate its conformational flexibility and, consequently, its interactions with cofactors. In SUMO, minor changes in the core aromatic interactions dictate the affinity of SUMO for SUMO-interacting Motifs [76]. Interestingly, this is not the case in the context of vUb, where we found that the interaction of mono-vUb with the two proteasomal receptors Rpn10 and Rpn13 is indistinguishable from Ub. However, recent studies have suggested that the dynamics between the two Ub units in the di-Ub can be crucial for interacting with proteasome receptors [77,78]. Further studies using polymeric vUb chains will reveal more mechanistic details of how substrates conjugated with vUb chains interact with the proteasome receptors.

Our activity assays suggested that the polyubiquitination activity of vUb and Ub are comparable. E1 ∼ Ub and E2 ∼ Ub structures show that majority of interactions of Ub with the enzymes are through the C-terminal tail and the hydrophobic surface patch. The similarity of the surface hydrophobic patch and C-terminal tail probably ensures the equivalent activity between vUb and Ub. Using a yeast system, we verified that vUb chains could be made in vivo, and these chains can target a substrate for proteasomal degradation. These results suggest that vUb can effectively compete with the eukaryotic Ub to hijack the host Ub machinery. Since proteasome receptors can recognize vUb, antiviral responses may be tagged by the vUb chain for proteasomal degradation. In the yeast experiments, we have deleted ubi4 to remove the major source of endogenous Ub. However, the possibility of a minute pool of endogenous Ub coming from the ubi1-3 gene remains. Alternate strategies for similar experiments would be to use the Sup328 Ub shutoff yeast strain, where the four natural ubiquitin genes are replaced with a synthetic ubiquitin gene [79–81].

Ub moieties can be conjugated through one of their seven lysine residues or the N-terminal methionine to form Ub chains [82]. Conventionally, there are eight possible types of homotypic Ub chains having a single linkage, and multiple possible heterotypic chains with mixed linkages. vUb contains an additional lysine K54, and our studies uncovered an atypical linkage formed by K54 in vUb. The topology of linkage may be different from the other Ub linkages studied before. The K54-linked vUb chains were also detected in vivo. Further studies are required to identify substrates conjugated to K54-linked vUb chains and probe the functional role of such Ub chains. The intestinal parasite Entamoeba histolytica encodes an extensive Ub-proteasome system. The Ub encoded by E. histolytica (EhUb) also harbors K54. Our studies suggest that EhUb may form atypical K54-chains via its conjugation cascade, whose functional relevance needs investigation [83]. Another Ubiquitin-like protein NEDD8 has high sequence identity with Ub but does not directly participate in the proteasome degradation. NEDD8 also includes K54 in its sequence, which can form NEDD8 conjugates. Unlike the vUb, NEDD8 conjugates are efficiently regulated by the NEDD8 specific protease, NEDP1 [84].

## Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

## Conflict of Interest

The authors declare that they have no conflict of interest with the content of this article.

## Funding

The NMR data were acquired at the NCBS-TIFR NMR Facility, supported by the Department of Atomic Energy, Government of India, under project no. 12-R&D-TFR-5.04-0900. The NMR facility is also partially supported by the Department of Biotechnology, B-Life grant under project no dbt/pr12422/med/31/287/2014. This work was also supported by intramural grants from the Tata Institute of Fundamental Research. This work was supported by CSIR-UGC fellowship Sr. No. 2121330479 to H.N., DST-INSPIRE Fellowship DST/INSPIRE/03/2016/001546 to V.V. from the Department of Science and Technology, Government of India, and Wellcome Trust-DBT India Alliance Intermediate Fellowship IA/I/14/2/501523 to S.L.

## Accession Numbers

The atomic co-ordinates and structure factors (PDB id: 6KNA) have been deposited in the Protein data bank (http://wwpdb.org/). The NMR chemical shift is deposited in the Biological Magnetic Resonance Bank under accession number 36278. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [88] (http://www.ebi.ac.uk/pride) partner repository with the dataset identifier PXD017215 and 10.6019/PXD017215.

## Author Contributions

H.N. carried out all cloning, protein purification, NMR experiments, NMR data analysis, stability experiments, binding assays, ubiquitination assays, and mammalian cell experiments. P.P.R. analyzed NMR data and helped with structure determination. V.V. performed cloning and the S. cerevisiae experiments. C.P. helped with MS data collection and analysis. S.L. conceptualized and supervised the S. cerevisiae experiments. R.D. developed the concept and directed the project. H.N. and R.D. wrote the initial draft of the manuscript. All authors contributed to the final draft of the manuscript.

## Acknowledgements

We acknowledge the support of the Department of Atomic Energy, Government of India, under project no. 12-R&D-TFR-5.04-0800. We thank Dipendra Basu for helping in making ML Tree. The NMR data were acquired at the NCBS-TIFR NMR Facility, supported by the Department of Atomic Energy, Government of India, under project no. 12-R&D-TFR-5.04-0900. This work was supported by intramural grants from the Tata Institute of Fundamental Research. This work was supported by CSIR-UGC fellowship Sr. No. 2121330479 to H.N., DST-INSPIRE Fellowship DST/INSPIRE/03/2016/001546 to V.V. from the Department of Science and Technology, Government of India, and Wellcome Trust-DBT India Alliance Intermediate Fellowship IA/I/14/2/501523 to S.L. All the NMR spectra were collected at NMR Facility. Mass spectrometry data were collected at the proteomics unit of Mass Spectrometry facility of the National Centre for Biological Sciences. The NMR facility is also partially supported by the Department of Biotechnology, B-Life grant under project no dbt/pr12422/med/31/287/2014.

## Abbreviations

• AcMNPV

Autographa californica multiple nucleo-polyhedrosis virus

•
• CSP

chemical shift perturbation

•
• DUBs

DeUbiquitinases

•
• GV

Granulovirus

•
• het-NOE

heteronuclear Overhauser enhancement

•
• ITC

isothermal titration calorimetry

•
• ML

maximum likelihood

•
• NPVs

nuclear polyhedrosis viruses

•
• PSM

peptide spectrum matches

•
• PTM

post-translation modification

•
• Ub

ubiquitin

•
• vUb

viral ubiquitin

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