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... Show moreThe 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. Show less
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... Show moreThe 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.Author summaryIn the last 3 years, it has become clear that emerging coronaviruses still pose a significant threat to human health. Despite the vaccines that are now available in many countries against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccine scarcity or the inability to gain proper protection from immunization due to an immunocompromised status still leaves various people that will benefit from the administration of potent and safe antiviral agents. In this research, we have developed ubiquitin variants (UbVs), small proteins that resemble a natural component of human cells called ubiquitin, that are able to bind a part of the virus that is essential for its survival, with high affinity and selectivity. This way, these UbVs are able to inhibit the production of new viral particles after infection, thus preventing the virus from spreading from cell to cell and wreaking havoc on the body. Show less
Zande, H.J.P. van der; Gonzalez, M.A.; Ruiter, K. de; Wilbers, R.H.P.; Garcia-Tardon, N.; Huizen, M. van; ... ; Guigas, B. 2021
Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth... Show moreType 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (omega 1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant omega 1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of omega 1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that omega 1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein omega 1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism. Show less
It is well established that polyubiquitin chains, in particular those linked through K48 and K63, play a key role in the regulation of the antiviral innate immune response. However, the role of the... Show moreIt is well established that polyubiquitin chains, in particular those linked through K48 and K63, play a key role in the regulation of the antiviral innate immune response. However, the role of the atypical chains linked via any of the other lysine residues (K6, K11, K27, K29, and K33) and the M1-linked linear chains have not been investigated very well yet in this context. This is partially due to a lack of tools to study these linkages in their biological context. Interestingly though, recent findings underscore the importance of the atypical chains in the regulation of the antiviral immune response. This review will highlight the most important advances in the study of the role of atypical ubiquitin chains, particularly in the regulation of intracellular antiviral innate immune signaling pathways. We will also discuss the development of new tools and how these can increase our knowledge of the role of atypical ubiquitin chains. Show less
Freeman, L.; Middeldorp, J.; Akker, E. van den; Oudijk, M.; Bax, C.; Huizen, M. van; ... ; Akker-van Marle, E. van den 2018
Objective: To assess the length and angle of mediolateral episiotomies performed by midwives and resident gynecologists at 3 teaching hospitals in the Netherlands, and determine the incidence of... Show moreObjective: To assess the length and angle of mediolateral episiotomies performed by midwives and resident gynecologists at 3 teaching hospitals in the Netherlands, and determine the incidence of obstetric anal sphincter injury. Methods: In this prospective audit conducted between February and September 2008, all women delivered at the 3 hospitals were examined in the labor room for perineal injury. When an injury was assessed as being grade 2 or higher, it was re-evaluated. The incidence of anal sphincter injury was then compared with that reported in the preceding year. Results: Of 1979 women delivered, 420 (21.2%) were given an episiotomy and 58 (2.9%) sustained anal sphincter injury. The episiotomies formed a mean angle of 40 degrees with the perineal midline. There was no difference in length or angle between the episiotomies performed by resident gynecologists and those performed by midwives, and the angle of most episiotomies was sufficiently wide. Compared with the preceding year, the rate of anal sphincter injury was significantly higher. Conclusion: The quality of episiotomies did not differ when performed by midwives or resident gynecologists. To improve the recognition and classification of obstetric anal sphincter injuries, audits based on an internationally accredited classification could easily become a part of routine hospital practice. (C) 2009 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. Show less
