Oligomerization and Cell Egress Controlled by Two Microdomains of Canine Distemper Virus Matrix Protein.

The multimeric matrix (M) protein of clinically relevant paramyxoviruses orchestrates assembly and budding activity of viral particles at the plasma membrane (PM). We identified within the canine distemper virus (CDV) M protein two microdomains, potentially assuming α-helix structures, which are essential for membrane budding activity. Remarkably, while two rationally designed microdomain M mutants (E89R, microdomain 1 and L239D, microdomain 2) preserved proper folding, dimerization, interaction with the nucleocapsid protein, localization at and deformation of the PM, the virus-like particle formation, as well as production of infectious virions (as monitored using a membrane budding-complementation system), were, in sharp contrast, strongly impaired. Of major importance, raster image correlation spectroscopy (RICS) revealed that both microdomains contributed to finely tune M protein mobility specifically at the PM. Collectively, our data highlighted the cornerstone membrane budding-priming activity of two spatially discrete M microdomains, potentially by coordinating the assembly of productive higher oligomers at the PM.IMPORTANCE Despite the availability of efficient vaccines, morbilliviruses (e.g., canine distemper virus [CDV] and measles virus [MeV]) still cause major health impairments. Although antivirals may support vaccination campaigns, approved inhibitors are to date still lacking. Targeting late stages of the viral life cycle (i.e., the cell exit system) represents a viable option to potentially counteract morbilliviral infections. The matrix (M) protein of morbillivirus is a major contributor to membrane budding activity and is assumed to assemble into dimers that further associate to form higher oligomers. Here, we rationally engineered M protein variants with modifications in two microdomains that potentially locate at dimer-dimer interfaces. Our results spotlight the cornerstone impact of both microdomains in membrane budding activity and further suggest a role of finely tuned high-order oligomer formation in regulating late stages of cell exit. Collectively, our findings highlight two microdomains in the morbilliviral M protein as novel attractive targets for drug design

Morbillivirus Glycoprotein Expression Induces ER Stress, Alters Ca2+ Homeostasis and Results in the Release of Vasostatin

Although the pathology of Morbillivirus in the central nervous system (CNS) is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV) that we inoculated into two different cell systems: a monkey cell line (Vero) and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H) markedly accumulated in the endoplasmic reticulum (ER). This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT), another ER resident chaperon critically involved in the response to misfolded proteins and in Ca2+ homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca2+ homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses

Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, “outrunning” the host’s immune response in demyelinating plaques, thus continuously eliciting new lesions

Highly potent bispecific sybodies neutralize SARS-CoV-2

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair (Sb#15 and Sb#68) that can bind simultaneously to the SARS-CoV-2 spike-RBD and efficiently neutralize pseudotyped and live-viruses by interfering with ACE2 interaction. Two spatially-discrete epitopes identified by cryo-EM translated into the rational design of bispecific and tri-bispecific fusions constructs, exhibiting up to 100- and 1000-fold increase in neutralization potency. Cryo-EM of the sybody-spike complex further revealed a novel up-out RBD conformation. While resistant viruses emerged rapidly in the presence of single binders, no escape variants were observed in presence of the bispecific sybody. The multivalent bispecific constructs further increased the neutralization potency against globally-circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the development of clinically relevant therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants

Reverse genetics as a tool to study molecular determinants of cell culture-adaptation and persistence in wild-type canine distemper virus

SUMMARY The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system of dogs. A75/17-CDV, which infects cell lines very inefficiently, was successfully adapted to grow in Vero cells and designated A75/17-V. Both genomes were sequenced, and the comparison revealed 7 differences located in the M, P/V/C and L genes. The mutations resulted in amino acid changes in the P, V, M and L proteins. Interestingly, the phenotype of infection of A75/17-V in Vero cells is mainly characterized by a cell-to-cell dissemination with no obvious syncytia formation. Since both CDV strains contain identical glycoproteins but differ in their RNA polymerase genes, we first investigated whether the inability of A75/17-CDV to infect Vero cells was linked to a restricted RNA synthesis. Towards this end, the A75/17-V N, P and L genes, which have been demonstrated to be necessary and sufficient to promote paramyxovirus replication, were cloned into the eukaryotic expression vector pTM-1. Their functionality was then assessed by showing their ability to rescue a bi-cistronic minireplicon construct, containing the eGFP and CAT reporter genes flanked by the two A75/17-V promoter regions. The A75/17-V P and L genes were then mutated to generate the wild-type A75/17-CDV P and L proteins. Surprisingly, the polymerase of A75/17-V was found to be significantly less active in Vero cells than that of A75/17- CDV. This result suggested that restricted wild-type CDV infection in Vero cells was likely due to an impediment in cell entry, despite the fact that the wild-type and Vero- adapted virus harbor the same glycoprotein genes. To investigate the molecular determinants of A75/17-V involved in the persistent infection in Vero cells, a second rescue system was established based on a molecularly cloned full-length A75/17-V genome. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein was inserted at the 3' proximal position in the A75/17-V cDNA clone. In this system, we used stably expressing T7 RNA polymerase cells (Bsr-T7), which allowed us to avoid the use of recombinant vaccinia virus to deliver the polymerase. By transfecting the relevant cDNA clone and the N, P and L expression plasmids in Bsr-T7 cells, and followed by a subculture with Vero cells, we indeed succeeded in recovering a persistent, GFP-expressing CDV. The virus was then characterized in vitro, and our results showed that the cloned virus behaved similarly to the parental A75/17-V. The Onderstepoort CDV vaccine strain (OP-CDV), which is genetically more distant to wild-type isolate, induces a cytopathic effect characterized by extensive cell-to-cell fusion. To investigate the roles of wild-type F and H proteins in producing the persistent phenotype of infection, the surface glycoproteins of OP-CDV and A75/17-CDV were first compared in a transient-expression system using Vero cells. In addition, the role of the cellular receptor SLAM (CD150) in fusion efficiency was tested in a Vero cell line expressing the dog SLAM molecule. We demonstrate that not only the wild-type H protein, but also the wild-type F protein is major determinant of a low fusion activity. These results were confirmed by studying recombinant A75/17-V viruses bearing OP-CDV surface glycoproteins. Moreover, we showed that the presence of SLAM modulates the fusion activity, demonstrating a crucial role of host cellular factors in determining cytolytic versus persistent infection. Furthermore, detailed analysis of the F protein showed that the unusually long signal peptide plays a major role in modulating the fusogenic activity of the protein. Finally, in a natural course of infection of dogs with CDV, infection of canine footpads can result in the so-called hard pad disease, characterized by proliferation of footpad keratinocytes. Subsequently, primary dog keratinocytes (PDK) have been shown to be permissive to support A75/17-CDV infection, which were characterized by a cell-to-cell spread with no obvious syncytium formation. Preliminary results showed that by passaging the wild virus only three times in these cells, an efficient and rapid dissemination was observed. This phenotype is associated with an increase of infectious virus release, without modulating the persistent infection. By sequencing both genomes, three mutations were found in the P, M and H genes, which all result in amino acid changes, suggesting an important role of the wild-type M protein in the unefficient infectious virus production

Clustered Lysine Residues of the Canine Distemper Virus Matrix Protein Regulate Membrane Association and Budding Activity.

The canine distemper virus (CDV) matrix (M) protein is multifunctional; it orchestrates viral assembly and budding, drives the formation of virus-like particles (VLPs), regulates viral RNA synthesis, and may support additional functions. CDV M may assemble into dimers, where each protomer is constituted by N-terminal and C-terminal domains (NTD and CTD, respectively). Here, to investigate whether electrostatic interactions between CDV M and the plasma membrane (PM) may contribute to budding activity, selected surface-exposed positively charged lysine residues, which are located within a large basic patch of CTD, were replaced by amino acids with selected properties. We found that some M mutants harboring amino acids with neutral and positive charge (methionine and arginine, respectively) maintained full functionality, including proper interaction and localization with the PM as well as intact VLP and progeny virus production as demonstrated by employing a cell exit-complementation system. Conversely, while the overall structural integrity remained mostly unaltered, most of the nonconservative M variants (carrying a glutamic acid; negatively charged) exhibited a cytosolic phenotype secondary to the lack of interaction with the PM. Consequently, such M variants were entirely defective in VLP production and viral particle formation. Furthermore, the proteasome inhibitor bortezomib significantly reduced wild-type M-mediated VLP production. Nevertheless, in the absence of the compound, all engineered M lysine variants exhibited unaffected ubiquitination profiles, consistent with other residues likely involved in this functionally essential posttranslational modification. Altogether, our data identified multiple surface-exposed lysine residues located within a basic patch of CDV M-CTD, critically contributing to PM association and ensuing membrane budding activity.IMPORTANCE Although vaccines against some morbilliviruses exist, infections still occur, which can result in dramatic brain disease or fatal outcome. Postexposure prophylaxis with antivirals would support global vaccination campaigns. Unfortunately, there is no efficient antiviral drug currently approved. The matrix (M) protein of morbilliviruses coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, molecular mechanisms supporting these functions remain poorly understood, which preclude the rationale design of inhibitors. Here, to investigate potential interactions between canine distemper virus (CDV) M and the plasma membrane (PM), we combined structure-guided mutagenesis of selected surface-exposed lysine residues with biochemical, cellular, and virological assays. We identified several lysines clustering in a basic patch microdomain of the CDV M C-terminal domain, which contributed to PM association and budding activity. Our findings provide novel mechanistic information of how morbilliviruses assemble and egress from infected cells, thereby delivering bases for future antiviral drug development

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture

Canine Distemper Virus Infects Canine Keratinocytes and Immune Cells by Using Overlapping and Distinct Regions Located on One Side of the Attachment Protein▿

The morbilliviruses measles virus (MeV) and canine distemper virus (CDV) both rely on two surface glycoproteins, the attachment (H) and fusion proteins, to promote fusion activity for viral cell entry. Growing evidence suggests that morbilliviruses infect multiple cell types by binding to distinct host cell surface receptors. Currently, the only known in vivo receptor used by morbilliviruses is CD150/SLAM, a molecule expressed in certain immune cells. Here we investigated the usage of multiple receptors by the highly virulent and demyelinating CDV strain A75/17. We based our study on the assumption that CDV-H may interact with receptors similar to those for MeV, and we conducted systematic alanine-scanning mutagenesis on CDV-H throughout one side of the β-propeller documented in MeV-H to contain multiple receptor-binding sites. Functional and biochemical assays performed with SLAM-expressing cells and primary canine epithelial keratinocytes identified 11 residues mutation of which selectively abrogated fusion in keratinocytes. Among these, four were identical to amino acids identified in MeV-H as residues contacting a putative receptor expressed in polarized epithelial cells. Strikingly, when mapped on a CDV-H structural model, all residues clustered in or around a recessed groove located on one side of CDV-H. In contrast, reported CDV-H mutants with SLAM-dependent fusion deficiencies were characterized by additional impairments to the promotion of fusion in keratinocytes. Furthermore, upon transfer of residues that selectively impaired fusion induction in keratinocytes into the CDV-H of the vaccine strain, fusion remained largely unaltered. Taken together, our results suggest that a restricted region on one side of CDV-H contains distinct and overlapping sites that control functional interaction with multiple receptors

N-(3-Cyanophenyl)-2-phenylacetamide, an effective inhibitor of morbillivirus-induced membrane fusion with low cytotoxicity

Based on the structural similarity of viral fusion proteins within the family Paramyxoviridae, we tested recently described and newly synthesized acetanilide derivatives for their capacity to inhibit measles virus (MV)-, canine distemper virus (CDV)- and Nipah virus (NiV)-induced membrane fusion. We found that N-(3-cyanophenyl)-2-phenylacetamide (compound 1) has a high capacity to inhibit MV- and CDV-induced (IC(50) muM), but not NiV-induced, membrane fusion. This compound is of outstanding interest because it can be easily synthesized and its cytotoxicity is low [50 % cytotoxic concentration (CC(50)) >/= 300 muM], leading to a CC(50)/IC(50) ratio of approximately 100. In addition, primary human peripheral blood lymphocytes and primary dog brain cell cultures (DBC) also tolerate high concentrations of compound 1. Infection of human PBMC with recombinant wild-type MV is inhibited by an IC(50) of approximately 20 muM. The cell-to-cell spread of recombinant wild-type CDV in persistently infected DBC can be nearly completely inhibited by compound 1 at 50 muM, indicating that the virus spread between brain cells is dependent on the activity of the viral fusion protein. Our findings demonstrate that this compound is a most applicable inhibitor of morbillivirus-induced membrane fusion in tissue culture experiments including highly sensitive primary cells

Non-Destructive Examination Development for the JHR Material Testing Reactor

The Jules Horowitz Reactor (JHR) is a European material testing reactor (MTR) under construction at the CEA Cadarache centre. It will be dedicated to material and fuel irradiation tests, as well as to the production of medical isotopes. Gamma and X-Ray benches will be implemented in the reactor pool (RER), the irradiated component storage pool (EPI) and in a shielded hot cell for measuring either the whole underwater test device still containing the experimental sample or just the experimental sample before its extraction in the hot cell. The CEA/Cadarache Nuclear Measurement Laboratory (LMN) has been working in collaboration with VTT (Technical Research Centre in Finland Ltd.) since 2008 under a Finnish in-kind contribution agreement. This agreement focuses on the development of NDE systems implementing gamma-ray spectroscopy and high-energy X-ray imaging of the sample and irradiation device with the highest definition possible (resolution of 100 μm). The CEA-VTT technical specifications led to a European call for tenders launched by VTT. The contract was awarded to the Spanish company IDOM for the design, manufacturing, assembly and commissioning of: - Underwater gamma and X-ray (UGXR) mechanical benches and their associated gamma and X-ray collimation systems for the RER and EPI pools - Hot cell gamma and X-ray (HGXR) bench in the JHR NDE hot cell. The Final Design Reviews (FDR) of the UGXR and HGXR systems were completed in 2016. The design phase has been an iterative process in order to manage interfacing specifications and constraints: - Challenging experimental requirements, mainly to cover the wide diversity of sample shapes, sample activity levels and measurement processes, but also to achieve a level of mechanical accuracy to reach the ambitious geometrical resolution target in X-ray imaging, - Environmental constraints (immersion, radiation, compactness, limited accessibility for maintenance), - Nuclear safety constraints (seism, radiation protection). The whole design process has produced a number of elaborate and innovative mechatronic systems, which is rather unusual in nuclear applications since the resulting solutions have benefited from IDOM’s technological expertise in designing and commissioning large telescopes for the astronomy sector. Once the manufacturing phase and assembly finalised, the site acceptance tests for the UGXR and HGXR mechanical systems will be performed in 2019-2020 in the TOTEM facility at the CEA Cadarache center. The underwater benches will be tested in the CESARINE pool to check their requirements