Viral OTU Deubiquitinases: A Structural and Functional Comparison

Recent studies have revealed that proteases encoded by three very diverse RNA virus groups share structural similarity with enzymes of the Ovarian Tumor (OTU) superfamily of deubiquitinases (DUBs). The publication of the latest of these reports in quick succession prevented proper recognition and discussion of the shared features of these viral enzymes. Here we provide a brief structural and functional comparison of these virus-encoded OTU DUBs. Interestingly, although their shared structural features and substrate specificity tentatively place them within the same protease superfamily, they also show interesting differences that trigger speculation as to their origins.

revealed that proteases encoded by three very diverse RNA virus groups share structural similarity with enzymes of the Ovarian Tumor (OTU) superfamily of deubiquitinases (DUBs). The publication of the latest of these reports in quick succession prevented proper recognition and discussion of the shared features of these viral enzymes. Here we provide a brief structural and functional comparison of these virus-encoded OTU DUBs. Interestingly, although their shared structural features and substrate specificity tentatively place them within the same protease superfamily, they also show interesting differences that trigger speculation as to their origins.
The proteasomal degradation pathway is an important cellular route to dispose of viral proteins, as exemplified by the turnover of the TYMV polymerase [45]. Moreover, the degradation of this protein is specifically counteracted by the deubiquitinase activity of TYMV PRO, which thus promotes virus replication [10]. The functional characterization of viral OTU DUBs remains incomplete and future studies will likely reveal additional roles in replication and virus-host interplay.
Polyubiquitin chains can adopt a number of different configurations, depending on the type of covalent linkage present within the polymer [1]. A distal Ub molecule can be linked via its C-terminus to one of seven internal lysine residues present in a proximal Ub molecule via an isopeptide bond. Alternatively, in the case of linear chains, the C-terminus of the distal Ub is covalently linked to the Nterminal methionine residue of the proximal Ub via a peptide bond. While human OTU proteases often show a distinct preference for one or two isopeptide linkage types [46], nairovirus OTUs and TYMV PRO appear to be more promiscuous in their substrate preference [22,25]. However, like most human OTU proteases, they seem unable to cleave linear polyubiquitin chains in vitro [22,25,46]. Arterivirus PLP2 has not been extensively studied in this respect.  [27,28], (E) yeast OTU1 (3BY4) [57], and (F) human OTUD3 (4BOU) [46]. The b-hairpin motifs of CCHFV OTU and DUGV OTU are indicated in boxes in panels A and B, respectively, and the zinc-finger motif of EAV PLP2 is boxed in panel C. Active sites are indicated with arrows. The CCHFV OTU, DUGV OTU, EAV PLP2, and yeast OTU1 domains were crystallized in complex with Ub, which has been removed for clarity. Structure images were generated using PyMol [60]. (G) Schematic representation of the CCHFV large (L) protein [61,62]. A similar organization is found in the DUGV L protein, but is not depicted. The OTU domain resides in the N-terminal region of this protein and is not involved in autoproteolytic cleavage events [48]. (H) Schematic representation of the EAV polyprotein 1ab [63]. PLP2 resides in nonstructural protein 2 (nsp2) and is responsible for the cleavage between nsp2 and nsp3 [51]. (I) Schematic representation of the TYMV ORF1 polyprotein [50]. PRO resides in the N-terminal product of this polyprotein and is responsible for two internal cleavages [49,50]. Key replicative enzymes are indicated in G, H, and I. It is important to note that many positive-strand RNA viruses, including arteriviruses and tymoviruses, encode polyproteins that are post-translationally cleaved by internal protease domains [47]. Thus, while CCHFV OTU is not involved in viral protein cleavage and its activity seems dispensable for replication ( Figure 1G) [48], both arterivirus PLP2 and tymovirus PRO are critically required for viral replication due to their primary role in polyprotein maturation ( Figure 1H, 1I) [49][50][51][52][53]. Interestingly, while both EAV PLP2 and TYMV PRO can process peptide bonds in cis and in trans [50,51], PRO does not cleave peptide bonds in linear polyubiquitin chains in vitro [25]. To date, activity of EAV PLP2 towards linear polyubiquitin chains has not been reported.
Based on mutagenesis of putative catalytic residues, arterivirus PLP2 and tymovirus PRO were initially generally classified as papain-like cysteine proteases [51,54,55]. Now that crystal structures of these proteases are available, it is possible to search the DALI server [56] in order to identify structurally similar domains. Using the 3-dimensional coordinates of TYMV PRO, the most recently solved structure of a viral OTU protease, such a query identifies structural similarity with eukaryotic OTU DUBs as well as the nairovirus OTU domains and EAV PLP2 (Table 1). A superposition of these viral protease structures with yeast OTU1 [57] further highlights their similarities (Figure 2A-2C), and these comparisons together clearly position them within the OTU DUB superfamily. Sequence comparisons alone were insufficient to demonstrate this conclusively, as the similarity of viral OTU domains to each other and to eukaryotic OTU proteases is very limited and mostly restricted to the areas surrounding the active site residues [29].
Structural characterization of nairovirus (CCHFV and DUGV) OTU domains and EAV PLP2 in complex with Ub revealed that while these viral proteases adopt a fold that is consistent with eukaryotic OTU DUBs, they possess additional structural motifs in their S1 binding site that rotate the distal Ub relative to the binding orientation observed in eukaryotic OTU enzymes ( Figure 2D, 2E) [22][23][24][25][26]. In the case of CCHFV OTU, this alternative binding mode was shown to expand its substrate repertoire by allowing the enzyme to also accommodate ISG15. Since TYMV PRO was crystallized in its apo form [27,28], it remains to be determined whether its S1 site binds Ub in an orientation similar to nairovirus OTU and EAV PLP2 or eukaryotic OTU DUBs.
A remarkable feature of EAV PLP2 is the incorporation within the OTU-fold of a zinc finger that is involved in the interaction with Ub ( Figures 1C, 2E). The absence of similar internal zinc-finger motifs in other OTU superfamily members prompted us to propose that PLP2 prototypes a novel subclass of zinc-dependent OTU DUBs [26].
Finally, an interesting structural difference between TYMV PRO and other OTU proteases of known structure is the absence of a loop that generally covers the active site ( Figure 2F, 2G). Because of this, TYMV PRO lacks a complete oxyanion hole. It also lacks a third catalytic residue that would otherwise form the catalytic triad that has been observed in other OTU proteases ( Figure 2G). Lombardi et al. suggested that the resulting solvent exposure of the active site may contribute to the broad substrate specificity of TYMV PRO [28]. Interestingly, EAV PLP2 also has broad substrate specificity, cleaving Ub, ISG15, and the viral polyprotein, even though it does possess an intact oxyanion hole and an active site that is not solvent exposed. Future work may uncover additional aspects relating to the unusual architecture of the TYMV PRO active site.
The presence of structurally similar proteases, each displaying unique features, in these highly diverse virus groups suggests that their ancestors have independently acquired their respective OTU enzymes. Although their origins remain elusive, one possible scenario is the scavenging of an OTU DUB-encoding gene that directly enabled the ancestral virus to interact with the cellular ubiquitin landscape [29]. The absence of an OTU homologue in other lineages of the bunyavirus family strongly suggests that a nairoviral ancestor acquired an OTU DUB through heterologous recombination. In this scenario, the current differences between the nairoviral and eukaryotic OTU domains would reflect divergent evolution. In the case of arteriviruses, however, it is also conceivable that a preexisting papain-like protease that was initially only involved in polyprotein maturation acquired OTU-like features through a process of convergent evolution. Although rare, such a scenario would  account for the limited structural similarity between eukaryotic OTU domains and EAV PLP2, which contrasts with that observed for nairovirus OTU (Figure 2A, 2B; Table 1). For tymoviruses, which encode one (OTU) protease, the existence of related viruses that do not encode a protease domain or that encode one (papain-like) or two (OTU combined with a second papain-like) protease domains complicates the development of a straightforward scenario describing PRO acquisition and evolution [58]. These and other intriguing unsolved questions should be addressed through structural and functional studies of additional OTU-like proteases, be they viral or cellular, the results of which may shed more light on the various scenarios explaining the evolution of viral OTU domains.