Fig 1.
Sequence and structural diversity of nairovirus vOTUs.
(A) Phylogenetic tree of CLUSTALW aligned nairovirus vOTUs. The tree was constructed utilizing the Jones-Thornton-Taylor model in the MEGA7 program [70]. Current species groupings are indicated by colored ovals, and the assigned species denoted. Previous serogroup classification, if applicable, is shown in parentheses. Virus vOTUs included in this study are denoted by red lettering. Inset is a structure-based phylogenetic tree of vOTUs, with the mammalian Cezanne, A20, and OTULIN OTUs included for comparison. The tree was constructed using PDB IDs 3PRP, 4HXD, 5JZE, 6DX1, 6DX2, 6DX3, 6DX5, 5LRV, 5LRX, and 3ZNZ in the MultiSeq module of VMD [71]. Sequence accession numbers are included in S1 Table. CCHFV, Crimean-Congo hemorrhagic fever virus; GANV, Ganjam virus; NSDV, Nairobi sheep disease virus; DUGV, Dugbe virus; KUPEV, Kupe virus; HAZV, Hazara virus; TFLV, Tofla virus; TAGV, Taggert virus; TILLV, Tillamook virus; SAKV, Sakhalin virus; PRMV, Paramushir virus; AVAV, Avalon virus; ARTSV, Artashat virus; TFAV, Thiafora virus; ERVEV, Erve virus; HUGV, Hughes virus; FARV, Farallon virus; RAZAV, Raza virus; PSV, Punta Salinas virus; ZIRV, Zirqa virus; SOLV, Soldado virus; GRSV, Great Saltee virus; CASV, Caspiy virus; AHV, Abu Hammad virus; DGKV, Dera Ghazi Khan virus; SAPV, Sapphire II virus; WzTV, Wēnzhōu tick virus; BURV, Burana virus; HpTV-1, Huángpí tick virus 1; TcTV-1, Tǎchéng tick virus 1; TDYV, Tamdy virus; YOGV, Yogue virus; LPHV, Leopards Hill virus; QYBV, Qalyub virus; GERV, Geran virus; CHIMV, Chim virus; GOSV, Gossas virus; ISKV, Issyk-kul virus; UZAV, Uzun-Agach virus; KTRV, Keterah virus. (B) Nairovirus vOTUs tested in this study aligned using the T-Coffee sequence alignment program [72]. Percentages show the sequence identity relative to CCHFV vOTU. Generic vOTU secondary structure based on Define Secondary Structure of Proteins (DSSP) algorithm calculations for the vOTUs is shown in reddish orange, with the α3 and α4 helices of FARV vOTU shown in teal. The catalytic triad is boxed in black and the selectivity pocket in orange. Mutation sites related to the selectivity pocket are shown by yellow stars, sites related to differences in how FARV vOTU engages mono-Ub by blue stars, and the DGKV vOTU catalytic triad mutant by a green star. Mutation sites for the second Ub binding site in FARV vOTU are denoted by red stars. The region deleted in the FARV vOTUΔ79–107 construct is indicated by a bracket.
Fig 2.
Diversity of vOTU specific activity.
Activity of vOTUs towards towards Ub-AMC (A) and human ISG15-AMC (B). Values shown are the mean ± standard deviation of two independent experiments.
Fig 3.
vOTU preferences for different di-Ub linkages.
(A) Activity towards K48, K63, and K11 linked di-Ub FRET-TAMRA substrates. Values shown are the mean ± standard deviation of two independent experiments. (B) Gel cleavage assay of unlabeled di-Ub, visualized by Commassie Blue staining. (C) Summary of currently investigated vOTU di-Ub activity (present study and [32]).
Table 1.
Data collection and refinement statistics for vOTU crystal structures.
Fig 4.
Structural comparison of new vOTU structures.
(A) Secondary structure overlay of the vOTUs from CCHFV, DGKV, QYBV, and TAGV. The red circles highlight areas of the binding interface that show large structural divergence. The region of the active site is denoted by a black box, with a closeup shown. The relative activities of the DGKV vOTU WT and E152D mutant for Ub-AMC and the peptide Z-RLRGG-AMC are shown, with error bars representing the standard deviation of two independent experiments. (B-C) Specific molecular interactions accounting for structural differences can be identified in distinct regions of the vOTUs. CCHFV vOTU is colored gray, DGKV vOTU in teal, QYBV vOTU in orange, and TAGV vOTU in magenta. Black dashed lines denote atom pairs that are within hydrogen bonding distance.
Fig 5.
Selectivity pocket of nairovirus vOTUs.
(A) The CCHFV vOTU Ub bound structure (PDB ID 3PRP) is shown with the location of the selectivity pocket indicated with a box. The selectivity pockets of the other vOTUs are shown, with the Ub (purple) modeled in from the CCHFV structure based on a secondary alignment of the vOTUs. DUGV vOTU (PDB ID 4HXD) is colored silver and ERVEV vOTU (PDB ID 5JZE) is colored brown. The other vOTUs are colored as in Fig 3. All models were generated by aligning the vOTUs with the CCHFV vOTU Ub bound structure in Coot. (B) The extra space (black arrow) existing in the ERVEV-mouse ISG15 structure (PDB ID 5JZE), with the CCHFV vOTU bound to Ub included for comparison (Panel I). QYBV vOTU with Ub and mouse ISG15 (green) modeled in based on vOTU secondary structure alignments, with an arginine also modeled into the space opened up by the mouse ISG15 conformation (Panel II). (C) Activities for Ub-AMC for mutant DGKV, QYBV, and TAGV vOTUs relative to WT. Error bars represent the standard deviation of two independent experiments.
Table 2.
Isothermal Titration Calorimetry of TAGV-Ub binding.
Fig 6.
(A) Overall structure of the FARV vOTU with the secondary structure denoted based on DSSP. The extended regions of the α3 and α4 helices are colored in teal. Intervening amino acids lacking electron density are represented by an orange dashed line. (B) Molecular features of the extended α3-α4 helices of FARV vOTU, with CCHFV vOTU included for comparison. Atom pairs within hydrogen bonding distance are denoted by black dashes.
Fig 7.
Model of molecular contributors to FARV mono- and poly-Ub activity.
(A) FARV vOTU (reddish orange) in a Coot-calculated secondary structure overlay with CCHFV vOTU (gray) bound to Ub (PDB ID 3PRP). The selectivity pocket of FARV vOTU is shown in Panel I, with other elements potentially diminishing FARV vOTU Ub activity in Panels II and III. Black dashes show hydrogen bond interactions. Inset shows the Ub-AMC activity relative to WT of FARV and TAGV vOTU mutants. Error bars represent the standard deviation of two independent experiments. (B) Enzymatic activity of FARV vOTUΔ79–107 compared to WT for Ub-AMC and K48/K63 FRET-TAMRA (Panel I), gel cleavage assays of K48/K63 di-Ub with FARV vOTUΔ79–107 (Panel II), and gel cleavage assays of K48/K63 tri-Ub with WT FARV vOTU and FARV vOTUΔ79–107 (Panel III). (C) Model of tri-Ub binding with FARV vOTU. The proximal Ub of K6 linked (PDB ID 5OHP), K11 linked (PDB ID 5LRV), K48 linked (PDB ID 5E6J), and K63 linked (PDB ID 2JF5) di-Ub was anchored to bound mono-Ub based on a secondary structure alignment in Coot. The filled circle indicates the common space that would likely be occupied by the Ub interacting with the second site of interaction of FARV vOTU.
Fig 8.
Second site of FARV vOTU interaction with di-Ub.
(A) Model of FARV vOTU (reddish orange) bound to K63-linked di-Ub (purple; PDB ID 2JF5). FARV vOTU was overlaid with CCHFV vOTU bound to Ub (PDB ID 3PRP; not rendered) based on secondary structure alignment of the vOTUs. The distal Ub was anchored to the bound mono-Ub by aligning the secondary structure in Coot, followed by manual bond rotations within Lys63 of the proximal Ub in PyMol to model a plausible fit with minimal clashes based on the CCHFV vOTU active site and protease surface. The predicted region of FARV vOTU engagement with the proximal Ub is indicated by a black box. (B) Closeup view of the predicted region, with the residues selected for mutation shown as sticks. Activity of the mutants relative to WT is shown for Ub-AMC, Ub-Rh110, K48 di-Ub FRET-TAMRA, and K63 di-Ub FRET-TAMRA (right).