Deubiquitinating activity of SARS-CoV-2 papain-like protease does not influence virus replication or innate immune responses in vivo.

The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.

Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis.

Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Biochemical Insights into Imipenem Collateral Susceptibility Driven by ampC Mutations Conferring Ceftolozane/Tazobactam Resistance in Pseudomonas aeruginosa.

Several Pseudomonas aeruginosa AmpC mutants have emerged that exhibit enhanced activity against ceftazidime and ceftolozane, while also evading inhibition by avibactam. Interestingly, P. aeruginosa strains harboring these AmpC mutations fortuitously exhibit enhanced carbapenem susceptibility. This acquired susceptibility was investigated by comparing the degradation of imipenem by wild-type and cephalosporin-resistant AmpC. We show that cephalosporin-resistant AmpC enzymes lose their efficacy for hydrolyzing imipenem and suggest that this may be due to their increased flexibility and dynamics relative to the wild type.

The endopeptidase of the maize-affecting Marafivirus type member maize rayado fino virus doubles as a deubiquitinase.

Marafiviruses are capable of persistent infection in a range of plants that have importance to the agriculture and biofuel industries. Although the genomes of a few of these viruses have been studied in-depth, the composition and processing of the polyproteins produced from their main ORFs have not. The Marafivirus polyprotein consists of essential proteins that form the viral replicase, as well as structural proteins for virus assembly. It has been proposed that Marafiviruses code for cysteine proteases within their polyproteins, which act as endopeptidases to autocatalytically cleave the polyprotein into functional domains. Furthermore, it has also been suggested that Marafivirus endopeptidases may have deubiquitinating activity, which has been shown to enhance viral replication by downregulating viral protein degradation by the ubiquitin (Ub) proteasomal pathway as well as tampering with cell signaling associated with innate antiviral responses in other positive-sense ssRNA viruses. Here, we provide the first evidence of cysteine proteases from six different Marafiviruses that harbor deubiquitinating activity and reveal intragenus differences toward Ub linkage types. We also examine the structural basis of the endopeptidase/deubiquitinase from the Marafivirus type member, maize rayado fino virus. Structures of the enzyme alone and bound to Ub reveal marked structural rearrangements that occur upon binding of Ub and provide insights into substrate specificity and differences that set it apart from other viral cysteine proteases.

Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1ß (nsp1ß) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1ß and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1ß and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1ß:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1ß within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1ß and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1ß and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

Frontrunners in the race to develop a SARS-CoV-2 vaccine.

Numerous studies continue to be published on the COVID-19 pandemic that is being caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Given the rapidly evolving global response to SARS-CoV-2, here we primarily review the leading COVID-19 vaccine strategies that are currently in Phase III clinical trials. Nonreplicating viral vector strategies, inactivated virus, recombinant protein subunit vaccines, and nucleic acid vaccine platforms are all being pursued in an effort to combat the infection. Preclinical and clinal trial results of these efforts are examined as well as the characteristics of each vaccine strategy from the humoral and cellular immune responses they stimulate, effects of any adjuvants used, and the potential risks associated with immunization such as antibody-dependent enhancement. A number of promising advancements have been made toward the development of multiple vaccine candidates. Preliminary data now emerging from phase III clinical trials show encouraging results for the protective efficacy and safety of at least 3 frontrunning candidates. There is hope that one or more will emerge as potent weapons to protect against SARS-CoV-2.

Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam.

Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and notorious for its broad-spectrum resistance to antibiotics. A key mechanism that provides extensive resistance to ß-lactam antibiotics is the inducible expression of AmpC ß-lactamase. Recently, a number of clinical isolates expressing mutated forms of AmpC have been found to be clinically resistant to the antipseudomonal ß-lactam-ß-lactamase inhibitor (BLI) combinations ceftolozane-tazobactam and ceftazidime-avibactam. Here, we compare the enzymatic activity of wild-type (WT) AmpC from PAO1 to those of four of these reported AmpC mutants, bearing mutations E247K (a change of E to K at position 247), G183D, T96I, and ΔG229-E247 (a deletion from position 229 to 247), to gain detailed insights into how these mutations allow the circumvention of these clinically vital antibiotic-inhibitor combinations. We found that these mutations exert a 2-fold effect on the catalytic cycle of AmpC. First, they reduce the stability of the enzyme, thereby increasing its flexibility. This appears to increase the rate of deacylation of the enzyme-bound ß-lactam, resulting in greater catalytic efficiencies toward ceftolozane and ceftazidime. Second, these mutations reduce the affinity of avibactam for AmpC by increasing the apparent activation barrier of the enzyme acylation step. This does not influence the catalytic turnover of ceftolozane and ceftazidime significantly, as deacylation is the rate-limiting step for the breakdown of these antibiotic substrates. It is remarkable that these mutations enhance the catalytic efficiency of AmpC toward ceftolozane and ceftazidime while simultaneously reducing susceptibility to inhibition by avibactam. Knowledge gained from the molecular analysis of these and other AmpC resistance mutants will, we believe, aid in the design of ß-lactams and BLIs with reduced susceptibility to mutational resistance.

Detection of High-Explosive Materials within Fingerprints by Means of Optical-Photothermal Infrared Spectromicroscopy.

As we live under a constant threat of global terrorism, the effective detection of highly energetic materials is one of the critical procedures needed at a variety of locations, including airports, border checkpoints, and entrances to high-security buildings. In this work, the application of optical-photothermal infrared (O-PTIR) spectromicroscopy for the detection of highly explosive materials within fingerprints is described. High-explosive (HE) materials (e.g., PETN, RDX, C-4, or TNT) were used to prepare contaminated fingerprints. These were subsequently deposited on various objects, including microscopic glass slides, a table, a mug, etc. Samples deposited on glass slides were directly sent for analyses; for other samples, adhesive tapes were used to lift off fingermarks. In cases of difficulty in locating fingerprints, additional powders were used to enhance their visibility. Experiments were performed with a mIRage IR microscope working in a noncontact, far-field reflection mode, offering submicron IR spectroscopy and imaging. Fast imaging (several characteristic absorbances were selected for every substance of interest) was used to locate "suspicious" particles among various residues present in fingerprints. Subsequently, spectra were collected for those particles. Reflection mode O-PTIR spectra taken from powdered and nonenhanced fingerprints were of comparable quality to transmission mode FTIR spectra collected for pure HEs. On the basis of the performed experiments, we consider O-PTIR spectromicroscopy to open a new avenue for the nondestructive, efficient, and reliable analysis of exogenous substances deposited within fingerprints. The real significance of O-PTIR is in its ability to deliver high-quality, spatially resolved FTIR transmission-like spectra below the diffraction limit of infrared wavelengths, doing so in an easy-to-use reflection (far-field) mode. Collected spectra are also searchable and interpretable in both commercial and institutional IR databases without mathematical modeling.

Mutations in HYAL2, Encoding Hyaluronidase 2, Cause a Syndrome of Orofacial Clefting and Cor Triatriatum Sinister in Humans and Mice.

Orofacial clefting is amongst the most common of birth defects, with both genetic and environmental components. Although numerous studies have been undertaken to investigate the complexities of the genetic etiology of this heterogeneous condition, this factor remains incompletely understood. Here, we describe mutations in the HYAL2 gene as a cause of syndromic orofacial clefting. HYAL2, encoding hyaluronidase 2, degrades extracellular hyaluronan, a critical component of the developing heart and palatal shelf matrix. Transfection assays demonstrated that the gene mutations destabilize the molecule, dramatically reducing HYAL2 protein levels. Consistent with the clinical presentation in affected individuals, investigations of Hyal2-/- mice revealed craniofacial abnormalities, including submucosal cleft palate. In addition, cor triatriatum sinister and hearing loss, identified in a proportion of Hyal2-/- mice, were also found as incompletely penetrant features in affected humans. Taken together our findings identify a new genetic cause of orofacial clefting in humans and mice, and define the first molecular cause of human cor triatriatum sinister, illustrating the fundamental importance of HYAL2 and hyaluronan turnover for normal human and mouse development.

Structural and mechanistic analysis of a ß-glycoside phosphorylase identified by screening a metagenomic library.

Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl ß-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining ß-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to N-acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying ß-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications.

Molecular Basis for the Potent Inhibition of the Emerging Carbapenemase VCC-1 by Avibactam.

In 2016, we identified a new class A carbapenemase, VCC-1, in a nontoxigenic Vibrio cholerae strain that had been isolated from retail shrimp imported into Canada for human consumption. Shortly thereafter, seven additional VCC-1-producing V. cholerae isolates were recovered along the German coastline. These isolates appear to have acquired the VCC-1 gene (blaVCC-1) independently from the Canadian isolate, suggesting that blaVCC-1 is mobile and widely distributed. VCC-1 hydrolyzes penicillins, cephalothin, aztreonam, and carbapenems and, like the broadly disseminated class A carbapenemase KPC-2, is only weakly inhibited by clavulanic acid or tazobactam. Although VCC-1 has yet to be observed in the clinic, its encroachment into aquaculture and other areas with human activity suggests that the enzyme may be emerging as a public health threat. To preemptively address this threat, we examined the structural and functional biology of VCC-1 against the FDA-approved non-ß-lactam-based inhibitor avibactam. We found that avibactam restored the in vitro sensitivity of V. cholerae to meropenem, imipenem, and ertapenem. The acylation efficiency was lower for VCC-1 than for KPC-2 and akin to that of Pseudomonas aeruginosa PAO1 AmpC (k2/Ki = 3.0 × 103 M-1 s-1). The tertiary structure of VCC-1 is similar to that of KPC-2, and they bind avibactam similarly; however, our analyses suggest that VCC-1 may be unable to degrade avibactam, as has been found for KPC-2. Based on our prior genomics-based surveillance, we were able to target VCC-1 for detailed molecular studies to gain early insights that could be used to combat this carbapenemase in the future.

Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants.

The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses. A variety of viral DUBs (vDUBs), including the MERS-CoV papain-like protease, are responsible for cleaving the viral replicase polyproteins during replication, and are thereby critical components of the viral replication cycle. Together, this makes vDUBs highly attractive antiviral drug targets. However, structural similarity between the catalytic cores of vDUBs and human DUBs complicates the development of selective small molecule vDUB inhibitors. We have thus developed an alternative strategy to target the vDUB activity through a rational protein design approach. Here, we report the use of phage-displayed ubiquitin variant (UbV) libraries to rapidly identify potent and highly selective protein-based inhibitors targeting the DUB domains of MERS-CoV and CCHFV. UbVs bound the vDUBs with high affinity and specificity to inhibit deubiquitination, deISGylation and in the case of MERS-CoV also viral replicative polyprotein processing. Co-crystallization studies further revealed critical molecular interactions between UbVs and MERS-CoV or CCHFV vDUBs, accounting for the observed binding specificity and high affinity. Finally, expression of UbVs during MERS-CoV infection reduced infectious progeny titers by more than four orders of magnitude, demonstrating the remarkable potency of UbVs as antiviral agents. Our results thereby establish a strategy to produce protein-based inhibitors that could protect against a diverse range of viruses by providing UbVs via mRNA or protein delivery technologies or through transgenic techniques.

Synergistic activity of fosfomycin, ß-lactams and peptidoglycan recycling inhibition against Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the interconnection between peptidoglycan (PG) recycling, fosfomycin susceptibility and synergy between fosfomycin and ß-lactams in Pseudomonas aeruginosa METHODS: Fosfomycin MICs were determined by broth microdilution and Etest for a panel of 47 PAO1 mutants defective in several components of PG recycling and/or AmpC induction pathways. PAO1 fosfomycin MICs were also determined in the presence of a 5 mM concentration of the NagZ inhibitor PUGNAc. Population analysis of fosfomycin susceptibility and characterization of the resistant mutants that emerged was also performed for selected strains. Finally, fosfomycin, imipenem and fosfomycin + imipenem killing curves were assessed. RESULTS: Mutants defective in AmpG, NagZ or all three AmpD amidases showed a marked increase in fosfomycin susceptibility (at least two 2-fold dilutions with respect to WT PAO1). Moreover, PAO1 fosfomycin MICs were consistently reduced from 48 to 24 mg/L in the presence of a 5 mM concentration of PUGNAc. Fosfomycin hypersusceptibility of the ampG, nagZ and triple ampD mutants was also clearly confirmed in the performed population analysis, although the emergence of resistant mutants, through GlpT mutations, was not avoided. Synergy between fosfomycin and imipenem was evidenced for the WT strain, the AmpC-hyperproducing strain (triple AmpD mutant) and the NagZ and AmpG mutants in killing curves. Moreover, regrowth of resistant mutants was not evidenced for the combination. CONCLUSIONS: PG recycling inhibitors are envisaged as useful adjuvants in the treatment of P. aeruginosa infections with ß-lactams and fosfomycin and therefore further development of these molecules is encouraged.

Transactivation of programmed ribosomal frameshifting by a viral protein.

Programmed -1 ribosomal frameshifting (-1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes -1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual -2 frameshifting (-2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of -1 PRF, yielding a third, truncated nsp2 variant named "nsp2N." Remarkably, we now show that both -2 and -1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1ß). Embedded in nsp1ß's papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1ß with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.

Producing glucose 6-phosphate from cellulosic biomass: structural insights into levoglucosan bioconversion.

The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-ß-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.

The ß-lactamase gene regulator AmpR is a tetramer that recognizes and binds the D-Ala-D-Ala motif of its repressor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide.

Inducible expression of chromosomal AmpC ß-lactamase is a major cause of ß-lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to ß-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide. When exposed to ß-lactams, however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAc-pentapeptide from AmpR and convert it into an activator of ampC transcription. Here we describe the molecular interactions between AmpR (from Citrobacter freundii), its DNA operator, and repressor UDP-MurNAc-pentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N11-A LTTR binding motif and revealed that it can bind four repressor molecules in an apparently stepwise manner. A crystal structure of the AmpR effector-binding domain bound to UDP-MurNAc-pentapeptide revealed that the terminal D-Ala-D-Ala motif of the repressor forms the primary contacts with the protein. This observation suggests that 1,6-anhydroMurNAc-pentapeptide may convert AmpR into an activator of ampC transcription more effectively than 1,6-anhydroMurNAc-tripeptide (which lacks the D-Ala-D-Ala motif). Finally, small angle x-ray scattering demonstrates that the AmpR·DNA complex adopts a flat conformation similar to the LTTR protein AphB and undergoes only a slight conformational change when binding UDP-MurNAc-pentapeptide. Modeling the AmpR·DNA tetramer bound to UDP-MurNAc-pentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function.

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.

Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.

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 zinc-dependent 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-ß 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 dual-specificity proteases.