Biology subjects

Snijder, E. J.

Publications and source records attributed to Snijder, E. J..

22 records · Page 2Linked to original sources

bioRxiv2019

The deubiquitinating activity of Middle East respiratory syndrome coronavirus papain-like protease delays the innate immune response and enhances virulence in a mouse model

AbstractMiddle East respiratory syndrome coronavirus (MERS-CoV) continues to cause zoonotic infections and serious disease, primarily in the Arabian Peninsula, due to repeated spill-over from dromedary camels and subsequent nosocomial transmission. Approved MERS vaccines for use in animals or humans are not currently available. MERS-CoV replication requires the virus-encoded papain-like protease (PLpro) to cleave multiple sites in the viral replicase polyproteins, thereby releasing functional non-structural proteins. Additionally, PLpro is a deubiquitinating enzyme (DUB) that can remove ubiquitin(-like) moieties from substrates, presumably to counteract host antiviral responses. In previous work, we determined the crystal structure of MERS-CoV PLpro in complex with ubiquitin, facilitating the design of PLpro mutations that impair DUB activity without affecting viral polyprotein cleavage. Here, we introduced these DUB-inactivating mutations into the viral genome and examined their impact on MERS-CoV infection both in cell culture and in a lethal mouse model. Although overall replication of DUB-negative and wild-type (wt) recombinant MERS-CoV was comparable in multiple cell lines, infection with DUB-negative virus markedly increased mRNA levels for interferon (IFN)-{beta} and IFN-stimulated genes. Moreover, compared to a wt virus infection, the survival rate was significantly increased when DUB-negative MERS-CoV was used to infect transgenic mice expressing a human MERS-CoV receptor. Interestingly, DUB-negative and wt MERS-CoV replicated to the same titers in lungs of infected mice, but the DUB-negative virus was cleared faster, likely due to the observed accelerated and better-regulated innate immune responses, in contrast to delayed and subsequently excessive responses in wt virus-infected mice. This study provides the first direct evidence that the DUB activity of a coronaviral protease contributes to innate immune evasion and can profoundly enhance virulence in an animal model. Thus, reduction or removal of the innate immune-suppressive DUB activity of PLpros is a promising strategy for coronavirus attenuation in the context of rational vaccine development.\n\nAuthor SummaryAlthough zoonotic coronaviruses such as Middle East respiratory coronavirus (MERS-CoV) have pandemic potential, therapeutics and vaccines that counteract this public health threat are not currently available. Coronaviruses typically employ multiple strategies to evade the hosts innate immune response, which may enhance clinical disease and/or reduce the efficacy of modified live vaccines. The MERS-CoV-encoded papain-like protease (PLpro) is not only crucial for the expression of functional replicase proteins, but has also been postulated to antagonize ubiquitination-dependent steps during the activation of the innate immune response. Here, we report the generation of engineered MERS-CoVs mutants in which PLpros deubiquitinating (DUB) activity was specifically disrupted without affecting virus viability. In this manner, we could demonstrate that the DUB activity of PLpro suppresses the interferon response in MERS-CoV-infected cells. Strikingly, in the lungs of mice infected with DUB-negative MERS-CoV, innate immune responses were induced at an earlier stage of infection than in wt virus-infected mice. This group also showed a clearly increased survival, indicating that the DUB activity is an important MERS-CoV virulence factor. This proof-of-concept study establishes that the engineering of DUB-negative coronaviruses, which elicit a more effective immune response in the host, is a viable strategy for vaccine development.

microbiology↗

bioRxiv2019

Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae

Cryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-lamella workflow is a low-throughput technique and can easily be obstructed by technical issues like the bending of the lamellae during the final cryo-FIB-milling steps. The severity of lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin lamella, leading to its bending and breakage. To protect the lamellae from these forces, we milled \"micro-expansion joints\" alongside the lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-lamella bending in any biological sample milled on EM grids.

microbiology↗

bioRxiv2019

Inhibition of arterivirus RNA synthesis by cyclophilin inhibitors is counteracted by mutations in replicase transmembrane subunits

Previously, the cyclophilin inhibitors cyclosporin A (CsA) and Alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales. Here we aimed to identify arterivirus proteins involved in the mode-of-action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to a [~]2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAVs CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA-resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely-related drug ALV CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.\n\nImportanceCurrently, no approved treatments are available to combat infections with nidoviruses, a group of plus-stranded RNA viruses including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporin A (CsA) and Alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and may thus represent a class of pan-nidovirus inhibitors. Here, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two trans-membrane subunits of the viral replication complex, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of nidoviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and other RNA viruses inhibited by these compounds.

microbiology↗

bioRxiv2018

The transcriptional and translational landscape of equine torovirus

The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates including cattle, sheep, goats, pigs and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV) we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyse the relative expression levels of the known torovirus proteins and transcripts, chimaeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle) and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilises a unique combination of discontinuous and non-discontinuous RNA synthesis to produce its subgenomic RNAs; indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated ORFs, located within the so-called 5 UTR. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.\n\nImportanceToroviruses infect cattle, goats, pigs and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens including severe acute respiratory syndrome (SARS) coronavirus and they share some common features, however the mechanism that they use to produce subgenomic RNA molecules differs. Here we performed deep sequencing to determine how equine torovirus produces subgenomic RNAs. In doing so, we also identified two previously unknown open reading frames \"hidden\" within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

microbiology↗