Puck B. van Kasteren

Arterivirus and Nairovirus Ovarian Tumor Domain-Containing Deubiquitinases Target Activated RIG-I To Control Innate Immune Signaling

ABSTRACT The innate immune response constitutes the first line of defense against viral infection and is extensively regulated through ubiquitination. The removal of ubiquitin from innate immunity signaling factors by deubiquitinating enzymes (DUBs) therefore provides a potential opportunity for viruses to evade this host defense system. It was previously found that specific proteases encoded by the unrelated arteri- and nairoviruses resemble the ovarian tumor domain-containing (OTU) family of DUBs. In arteriviruses, this domain has been characterized before as a papain-like protease (PLP2) that is also involved in replicase polyprotein processing. In nairoviruses, the DUB resides in the polymerase protein but is not essential for RNA replication. Using both in vitro and cell-based assays, we now show that PLP2 DUB activity is conserved in all members of the arterivirus family and that both arteri- and nairovirus DUBs inhibit RIG-I-mediated innate immune signaling when overexpressed. The potential relevance of RIG-I-like receptor (RLR) signaling for the innate immune response against arterivirus infection is supported by our finding that in mouse embryonic fibroblasts, the production of beta interferon primarily depends on the recognition of arterivirus RNA by the pattern-recognition receptor MDA5. Interestingly, we also found that both arteri- and nairovirus DUBs inhibit RIG-I ubiquitination upon overexpression, suggesting that both MDA5 and RIG-I have a role in countering infection by arteriviruses. Taken together, our results support the hypothesis that arteri- and nairoviruses employ their deubiquitinating potential to inactivate cellular proteins involved in RLR-mediated innate immune signaling, as exemplified by the deubiquitination of RIG-I.

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

Significance Many viruses encode proteases that cleave both viral and host substrates. Arteriviruses encode such a dual-specificity protease (PLP2) that removes ubiquitin from cellular proteins involved in host immunity. Based on a 3D structure of PLP2, we engineered the protease to have diminished deubiquitinating activity without affecting its activity toward its viral substrate. Viruses expressing such engineered proteases displayed a significantly weakened ability to evade host immune responses. This result demonstrates a crucial role for PLP2 in arterivirus immune evasion and opens new possibilities for developing improved attenuated virus vaccines against economically important arteriviruses and other viruses encoding similar dual-specificity proteases. 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-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 dual-specificity proteases.

Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression.

Background: MERS-CoV papain-like protease (PLpro) processes viral polyproteins and has deubiquitinating activity. Results: A crystal structure of MERS-CoV PLpro bound to ubiquitin guided mutagenesis to disrupt PLpro deubiquitinating activity without affecting polyprotein cleavage. Conclusion: The deubiquitinating activity of MERS-CoV PLpro suppresses the induction of interferon-β expression. Significance: Our strategy to selectively disable PLpro deubiquitinating activity enables the study of its specific functions in infection. Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PLpro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PLpro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PLpro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PLpro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PLpro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PLpro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PLpro domain was found to suppress IFN-β promoter activation, PLpro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PLpro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PLpro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PLpro as a viral DUB during MERS-CoV infection.

Anti-Sclerostin antibody inhibits internalization of Sclerostin and Sclerostin-mediated antagonism of Wnt/LRP6 signaling.

Sclerosteosis is a rare high bone mass disease that is caused by inactivating mutations in the SOST gene. Its gene product, Sclerostin, is a key negative regulator of bone formation and might therefore serve as a target for the anabolic treatment of osteoporosis. The exact molecular mechanism by which Sclerostin exerts its antagonistic effects on Wnt signaling in bone forming osteoblasts remains unclear. Here we show that Wnt3a-induced transcriptional responses and induction of alkaline phosphatase activity, an early marker of osteoblast differentiation, require the Wnt co-receptors LRP5 and LRP6. Unlike Dickkopf1 (DKK1), Sclerostin does not inhibit Wnt-3a-induced phosphorylation of LRP5 at serine 1503 or LRP6 at serine 1490. Affinity labeling of cell surface proteins with [125I]Sclerostin identified LRP6 as the main specific Sclerostin receptor in multiple mesenchymal cell lines. When cells were challenged with Sclerostin fused to recombinant green fluorescent protein (GFP) this was internalized, likely via a Clathrin-dependent process, and subsequently degraded in a temperature and proteasome-dependent manner. Ectopic expression of LRP6 greatly enhanced binding and cellular uptake of Sclerostin-GFP, which was reduced by the addition of an excess of non-GFP-fused Sclerostin. Finally, an anti-Sclerostin antibody inhibited the internalization of Sclerostin-GFP and binding of Sclerostin to LRP6. Moreover, this antibody attenuated the antagonistic activity of Sclerostin on canonical Wnt-induced responses.

Papain-Like Protease 1 from Transmissible Gastroenteritis Virus: Crystal Structure and Enzymatic Activity toward Viral and Cellular Substrates

ABSTRACT Coronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two precursor polyproteins of the viral replicase and are also thought to modulate host cell functions to facilitate infection. The papain-like protease 1 (PL1pro) domain is present in nonstructural protein 3 (nsp3) of alphacoronaviruses and subgroup 2a betacoronaviruses. It participates in the proteolytic processing of the N-terminal region of the replicase polyproteins in a manner that varies among different coronaviruses and remains poorly understood. Here we report the first structural and biochemical characterization of a purified coronavirus PL1pro domain, that of transmissible gastroenteritis virus (TGEV). Its tertiary structure is compared with that of severe acute respiratory syndrome (SARS) coronavirus PL2pro, a downstream paralog that is conserved in the nsp3s of all coronaviruses. We identify both conserved and unique structural features likely controlling the interaction of PL1pro with cofactors and substrates, including the tentative mapping of substrate pocket residues. The purified recombinant TGEV PL1pro was shown to cleave a peptide mimicking the cognate nsp2|nsp3 cleavage site. Like its PL2pro paralogs from several coronaviruses, TGEV PL1pro was also found to have deubiquitinating activity in an in vitro cleavage assay, implicating it in counteracting ubiquitin-regulated host cell pathways, likely including innate immune responses. In combination with the prior characterization of PL2pro from other alphacoronaviruses, e.g., human coronaviruses 229E and NL63, our results unequivocally establish that these viruses employ two PLpros with overlapping specificities toward both viral and cellular substrates.

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.

Mutagenesis of the transmembrane domain of the SARS coronavirus spike glycoprotein: refinement of the requirements for SARS coronavirus cell entry.

BackgroundThe spike protein (S) of SARS Coronavirus (SARS-CoV) mediates entry of the virus into target cells, including receptor binding and membrane fusion. Close to or in the viral membrane, the S protein contains three distinct motifs: a juxtamembrane aromatic part, a central highly hydrophobic stretch and a cysteine rich motif. Here, we investigate the role of aromatic and hydrophobic parts of S in the entry of SARS CoV and in cell-cell fusion. This was investigated using the previously described SARS pseudotyped particles system (SARSpp) and by fluorescence-based cell-cell fusion assays.ResultsMutagenesis showed that the aromatic domain was crucial for SARSpp entry into cells, with a likely role in pore enlargement.Introduction of lysine residues in the hydrophobic stretch of S also resulted in a block of entry, suggesting the borders of the actual transmembrane domain. Surprisingly, replacement of a glycine residue, situated close to the aromatic domain, with a lysine residue was tolerated, whereas the introduction of a lysine adjacent to the glycine, was not. In a model, we propose that during fusion, the lateral flexibility of the transmembrane domain plays a critical role, as do the tryptophans and the cysteines.ConclusionsThe aromatic domain plays a crucial role in the entry of SARS CoV into target cells. The positioning of the aromatic domain and the hydrophobic domain relative to each other is another essential characteristic of this membrane fusion process.

GxxxG Motif of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Transmembrane Domain Is Not Involved in Trimerization and Is Not Important for Entry

ABSTRACT Recently, a paper was published in which it was proposed that the GxxxG motif of the severe acute respiratory syndrome (SARS) coronavirus spike (S) protein transmembrane domain plays a vital role in oligomerization of the protein (E. Arbely, Z. Granot, I. Kass, J. Orly, and I. T. Arkin, Biochemistry 45:11349-11356, 2006). Here, we show that the GxxxG motif is not involved in SARS S oligomerization by trimerization analysis of S GxxxG mutant proteins. In addition, the capability of S to mediate entry of SARS S-pseudotyped particles overall was affected moderately in the mutant proteins, also arguing for a nonvital role for the GxxxG motif in SARS coronavirus entry.

In vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease 2

Abstract Arteriviruses are a family of positive-stranded RNA viruses that includes the prototypic equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV). Although several vaccines against these viruses are commercially available there is room for improvement, especially in the case of PRRSV. The ability of arteriviruses to counteract the immune response is thought to decrease the efficacy of the current modified live virus vaccines. We have recently shown that the deubiquitinase (DUB) activity of EAV papain-like protease 2 (PLP2) is important for the inhibition of innate immune activation during infection. A vaccine virus lacking PLP2 DUB activity may therefore be more immunogenic and provide improved protection against subsequent challenge than its DUB-competent counterpart. To test this hypothesis, twenty Shetland mares were randomly assigned to one of three groups. Two groups were vaccinated, either with DUB-positive (n =9) or DUB-negative (n =9) recombinant EAV. The third group (n =2) was not vaccinated. All horses were subsequently challenged with the virulent KY84 strain of EAV. Both vaccine viruses proved to be replication competent in vivo. In addition, the DUB-negative virus provided a similar degree of protection against clinical disease as its DUB-positive parental counterpart. Owing to the already high level of protection provided by the parental virus, a possible improvement due to inactivation of PLP2 DUB activity could not be detected under these experimental conditions. Taken together, the data obtained in this study warrant further in vivo investigations into the potential of using DUB-mutant viruses for the improvement of arterivirus vaccines.

In Vitro Enhancement of Respiratory Syncytial Virus Infection by Maternal Antibodies Does Not Explain Disease Severity in Infants

ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of severe respiratory illness in infants. At this young age, infants typically depend on maternally transferred antibodies (matAbs) and their innate immune system for protection against infections. RSV-specific matAbs are thought to protect from severe illness, yet severe RSV disease occurs mainly below 6 months of age, when neutralizing matAb levels are present. To investigate this discrepancy, we asked if disease severity is related to antibody properties other than neutralization. Some antibody effector functions are mediated via their Fc binding region. However, it has been shown that this binding may lead to antibody-dependent enhancement (ADE) of infection or reduction of neutralization, both possibly leading to more disease. In this study, we first showed that high levels of ADE of RSV infection occur in monocytic THP-1 cells in the presence of RSV antibodies and that neutralization by these antibodies was reduced in Vero cells when they were transduced with Fc gamma receptors. We then demonstrated that antibodies from cotton rats with formalin-inactivated (FI)-RSV-induced pulmonary pathology were capable of causing ADE. Human matAbs also caused ADE and were less neutralizing in vitro in cells that carry Fc receptors. However, these effects were unrelated to disease severity because they were seen both in uninfected controls and in infants hospitalized with different levels of RSV disease severity. We conclude that ADE and reduction of neutralization are unlikely to be involved in RSV disease in infants with neutralizing matAbs. IMPORTANCE It is unclear why severity of RSV disease peaks at the age when infants have neutralizing levels of maternal antibodies. Additionally, the exact reason for FI-RSV-induced enhanced disease, as seen in the 1960s vaccine trials, is still unclear. We hypothesized that antibodies present under either of these conditions could contribute to disease severity. Antibodies can have effects that may lead to more disease instead of protection. We investigated two of those effects: antibody-dependent enhancement of infection (ADE) and neutralization reduction. We show that ADE occurs in vitro with antibodies from FI-RSV-immunized RSV-infected cotton rats. Moreover, passively acquired maternal antibodies from infants had the capacity to induce ADE and reduction of neutralization. However, no clear association with disease severity was seen, ruling out that these properties explain disease in the presence of maternal antibodies. Our data contribute to a better understanding of the impact of antibodies on RSV disease in infants.