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. 2013 Apr;87(7):3815-27.
doi: 10.1128/JVI.03252-12. Epub 2013 Jan 23.

Diversity of ubiquitin and ISG15 specificity among nairoviruses' viral ovarian tumor domain proteases

Glenn C Capodagli  1 , Michelle K Deaton, Erica A Baker, Ryan J Lumpkin, Scott D Pegan
Affiliations

Diversity of ubiquitin and ISG15 specificity among nairoviruses' viral ovarian tumor domain proteases

Glenn C Capodagli et al. J Virol. 2013 Apr.

Abstract

Nairoviruses are responsible for numerous diseases that affect both humans and animal. Recent work has implicated the viral ovarian tumor domain (vOTU) as a possible nairovirus virulence factor due to its ability to edit ubiquitin (Ub) bound to cellular proteins and, at least in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), to cleave the Ub-like protein interferon-stimulated gene 15 (ISG15), a protein involved in the regulation of host immunity. The prospective roles of vOTUs in immune evasion have generated several questions concerning whether vOTUs act through a preserved specificity for Ub- and ISG15-conjugated proteins and where that specificity may originate. To gain insight into the substrate specificity of vOTUs, enzymological studies were conducted on vOTUs from Dugbe, CCHFV, and Erve nairoviruses. These studies revealed that vOTUs originating from different nairoviruses display a significant divergence in their preference toward Ub and ISG15. In addition, a recently identified vOTU from turnip yellow mosaic tymovirus was evaluated to elucidate any possible similarities between vOTUs originating from different viral families. Although possessing a similar preference for certain polymeric Ub moieties, its activity toward Ub in general was significantly less then those of nairoviruses. Lastly, the X-ray crystallographic structure of the vOTU from the Dugbe nairovirus was obtained in complex with Ub to reveal structural commonalities of vOTUs originating from nairoviruses. The structure suggests that divergences between nairovirus vOTUs specificity originate at the primary structural level. Comparison of this structure to that originating from CCHFV identified key residues that infer the substrate specificity of vOTUs.

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Figures

Fig 1
Fig 1
Sequence alignment of several viral OTU proteases. The vOTUs are from Dugbe virus (DUGV; GenBank accession no. AAB18834.1), Crimean-Congo hemorrhagic fever virus (CCHFV; GenBank accession no. AAQ98866.2), Nairobi sheep disease virus (NSDV; GenBank accession no. ACH99799.1), Erve virus (ERVEV; GenBank accession no. AFH89032), rice stripe virus (RSV; GenBank accession no. ABC68333), turnip yellow mosaic virus (TYMV; GenBank accession no. NP_663297.1), and porcine reproductive and respiratory virus (PRRSV; GenBank accession no. Q9WJB2). The secondary structure of DUG vOTU according to Defined Secondary Structure of Proteins (DSSP) is shown as gray cylinders (helical regions), pink arrows (β-sheets), and gray lines (loops). Hashed gray line represents residues for which electron density was not defined in the crystal structure. Breaks denote regions were DUG vOTU does not have residues. Asterisks represent amino acid residues chosen for site-directed mutation. Orange brackets indicate residues that are part of vOTUs' catalytic triad. A yellow bracket outlines residues involved in a backbone shift between CCHFV and DUGV.
Fig 2
Fig 2
vOTU cleavage of peptide, hUb, and hISG15 AMC conjugates. The cleavage activities of vOTUs from CCHFV, DUGV, and TYMV for 50 μM ZRLRGG-AMC (a), 1 μM hUb-AMC (b), and 1 μM hISG15-AMC (c) were determined. Error bars represent standard deviations from the average.
Fig 3
Fig 3
Gel shift assay of vOTU polyubiquitination linkage specificity. A 10 μM concentration of each di-Ub linkage was incubated with either 4 nM CCHFV, 4 nM DUGV, 100 nM ERVEV, or 6 μM TYMV at 37°C for an hour with samples taken at the indicated time points. The samples were heat inactivated at 95°C for 5 min and then run on a 10 to 20% Mini-Protean Tris-Tricine precast gels (Bio-Rad). The bands were visualized by staining with Coomassie blue.
Fig 4
Fig 4
vOTU preference for FRET poly-Ub linkage substrates, as well as human and mouse ISG15. (a to c) vOTU cleavage activity for K48-linked (a), K63-linked (b), or K11-linked di-Ub (c). Turnover values were determined based on the increase in emission upon cleavage of 1 μM di-Ub in the presence of the vOTU from CCHFV, DUGV, and TYMV. (d) Relative rates of cleavage activity of 4 nM CCHF vOTU and ERVE vOTU for 1 μM hISG15-AMC (white) in the presence of 120 μM unlabeled ISG15 from either mouse (light gray) or human (dark gray) sources. Error bars represent standard deviations from the average.
Fig 5
Fig 5
Poly-Ub linkage gel shift assay. A 20 μM concentration of each tri-Ub linkage was incubated with either 4 nM CCHF vOTU, 4 nM DUG vOTU, 100 nM ERVE vOTU, or 6 μM TYM vOTU at 37°C for an hour with samples taken at the indicated minute time points. The samples were heat inactivated at 95°C for 5 min and then run on a 10 to 20% Mini-Protean Tris-Tricine precast gels.
Fig 6
Fig 6
Diagram representation of DUG vOTU-Ub. (a) DUG vOTU monomer labeled and colored according to secondary structure: helices and loops are indicated in gray, and β-sheets are indicated in pink. (b) Complex of DUG vOTU rendered as in panel a, and hUb is rendered in yellow. (c) Overlay of DUG vOTU-Ub complex colored as in b, with hUb bound to CCHF vOTU (orange). (d) Overlay of DUG vOTU, CCHF vOTU bound to hUb (green, PDP code 3PRP), and CCHF vOTU bound to hISG15 (teal, PDB code 3PSE) DUG vOTU is as in panel a, with the α3 helix of each structure rendered as loops and boxed in red.
Fig 7
Fig 7
Locations of selective mutagenesis in vOTUs of CCHFV and DUGV. Wall-eyed stereo views of the interactions between DUG vOTU (gray/pink) and CCHF vOTU (teal) with hUb (yellow) and hISG15 (magenta) for the α3-chimera (a) and residues 128 (b), 100 to 102 (c), and 10 (d) are shown. Gray labels indicate CCHF vOTU residues, white labels indicate DUG vOTU residues, black labels indicate hUb and hISG15 residues, and red labels and dashed lines indicate distances. All of the distances are measured in angstroms.
Fig 8
Fig 8
Effects of vOTU mutagenesis on vOTU activity toward peptide, hUb, and hISG15 AMC. The cleavage activity of mutants of CCHF vOTU (light gray) or DUG vOTU (dark gray) were determined at 1 μM against 1 μM hUb-AMC (a and d), 1 μM hISG15-AMC (b and e), or 50 μM ZRLRLGG-AMC (c and f). Error bars represent standard deviations from the average.
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