Determining the molecular drivers of species-specific interferon-stimulated gene product 15 interactions with nairovirus ovarian tumor domain proteases

Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species’ ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species’ ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe virus and Ganjam virus OTUs bound to sheep ISG15 were solved and compared to complexes of Crimean-Congo hemorrhagic fever virus and Erve virus OTUs bound to human and mouse ISG15, respectively. Through mutational and structural analysis seven residues in ISG15 were identified that predominantly influence ISG15 species specificity among nairovirus OTUs. Additionally, OTU residues were identified that influence ISG15 preference, suggesting the potential for viral OTUs to adapt to different host ISG15s. These findings provide a foundation to further develop research methods to trace nairovirus-host relationships and delineate the full impact of ISG15 diversity on nairovirus infection.

1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 3.17 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based. The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density. The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5%

Metric
The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density. The numeric value is given above the bar.
Mol Chain Length Quality of chain 2 Entry composition i ○ There are 2 unique types of molecules in this entry. The entry contains 4920 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein called RNA-dependent RNA polymerase. 3 Residue-property plots i ○ These plots are drawn for all protein, RNA and DNA chains in the entry. The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometryand electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.

Mol Chain Residues
• Molecule 1: RNA-dependent RNA polymerase Chain A: • Molecule 2: Interferon stimulated gene 17 Chain D: There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively. The H(added) column lists the number of hydrogen atoms added and optimized by MolProbity. The Clashes column lists the number of clashes within the asymmetric unit, whereas Symm-Clashes lists symmetry related clashes. The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 8.

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
All (83)   There are no symmetry-related clashes.

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues. There are no Ramachandran outliers to report.

Protein sidechains i ○
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues. 5.4 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no carbohydrates in this entry.

Ligand geometry i ○
There are no ligands in this entry.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry. 6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9. 6.2 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no carbohydrates in this entry.

Ligands i ○
There are no ligands in this entry.

Other polymers i ○
There are no such residues in this entry.