Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zincdependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-β mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dualspecificity proteases.interferon-stimulated gene 15 | ISG15 | +RNA T he synthesis and posttranslational cleavage of polyproteins is a common genome expression strategy used by positivestranded (+)RNA viruses of eukaryotes. It is used to cope with the consequences of cytoplasmic replication and the limitations of the eukaryotic translation machinery, which essentially preclude the use of (nuclear) RNA splicing and polycistronic mRNAs, respectively (1). The critical cleavage of these viral polyproteins into their functional subunits is mediated by internal virusencoded proteases (2-5), many of which have been found to also target cellular substrates to promote virus replication or subvert host antiviral responses. Well-known examples of such dual-specificity proteases are the poliovirus 2A and hepatitis C virus NS3/4A enzymes that, in addition to the viral polyprotein, target host cell proteins involved in translation and innate immune signaling, respectively (6-10).Arteriviruses are +RNA viruses that, together with the coronaand roniviruses, belong to the order Nidovirales and include equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV). EAV is the family prototype and can cause abortion in pregnant mares...

The attachment of ubiquitin (Ub) and the Ub-like (Ubl) molecule interferon-stimulated gene 15 (ISG15) to cellular proteins mediates important innate antiviral responses. Ovarian tumor (OTU) domain proteases from nairoviruses and arteriviruses were recently found to remove these molecules from host proteins, which inhibits Ub and ISG15-dependent antiviral pathways. This contrasts with the Ub-specific activity of known eukaryotic OTU-domain proteases. Here we describe crystal structures of a viral OTU domain from the highly pathogenic Crimean-Congo haemorrhagic fever virus (CCHFV) bound to Ub and to ISG15 at 2.5-Å and 2.3-Å resolution, respectively. The complexes provide a unique structural example of ISG15 bound to another protein and reveal the molecular mechanism of an ISG15 cross-reactive deubiquitinase. To accommodate structural differences between Ub and ISG15, the viral protease binds the β-grasp folds of Ub and C-terminal Ub-like domain of ISG15 in an orientation that is rotated nearly 75°with respect to that observed for Ub bound to a representative eukaryotic OTU domain from yeast. Distinct structural determinants necessary for binding either substrate were identified and allowed the reengineering of the viral OTU protease into enzymes with increased substrate specificity, either for Ub or for ISG15. Our findings now provide the basis to determine in vivo the relative contributions of deubiquitination and deISGylation to viral immune evasion tactics, and a structural template of a promiscuous deubiquitinase from a haemorrhagic fever virus that can be targeted for inhibition using small-molecule-based strategies.innate immunity | viral deubiquitinase | bunyavirus | Crimean-Congo haemorrhagic fever virus | viral immune evasion T he posttranslational modification of proteins by Ub and Ub-like (Ubl) molecules is a regulatory process that controls numerous biological events (1, 2). Ub is conjugated to a lysine residue of target proteins through an isopeptide bond between the terminal carboxyl group of Ub and ϵ-amino group of the target lysine (3). Additional Ub molecules can be conjugated to lysines within Ub itself to form polyubiquitin chains. Lys 48-linked polyubiquitination is the canonical signal that targets proteins for proteasomal degradation, whereas Lys 63-linked polyubiquitination can initiate proteasome-independent events (4). Both Lys 48-and Lys 63-linked polyubiquitination have been established as key signaling events that activate innate and adaptive immune responses (5).A critical innate immune response to viral infection is the rapid production of type I interferon (IFN) and tumor necrosis factor alpha (TNFα). The induction and activity of these antiviral cytokines is controlled by, among other factors, Ub and Ubl conjugation (4). A hallmark of type I IFN stimulation is the rapid production of the Ubl molecule ISG15 (6). ISG15 is composed of two tandem Ub-like folds (7) and is known to exhibit potent antiviral activity against several important viruses (8). It conjugates to ϵ-amino groups of ...

NagZ is an exo-N-acetyl-b-glucosaminidase, found within Gram-negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N-acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC b-lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo-N-acetyl-b-glucosaminidases: O-GlcNAcase and the b-hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring-group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2-N-acyl derivatives of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram-negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N-valeryl-PUGNAc, the most selective known inhibitor of NagZ over both the human b-hexosaminidases and O-GlcNAcase. The selectivity stems from the five-carbon acyl chain of N-valeryl-PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O-GlcNAcase homologue bound to a related inhibitor N-butyryl-PUGNAc, which bears a four-carbon chain and is selective for both NagZ and O-GlcNAcase over the human b-hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O-GlcNAcase homologue. A comparison of these complexes, and with the human b-hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2-N-acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated b-lactam resistance.

No abstract

No abstract