LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus.

Wild-type canine distemper virus (CDV) is an important pathogen of dogs as well as wildlife that can infect immune and epithelial cells through two known receptors: the signaling lymphocytic activation molecule (SLAM) and nectin-4, respectively. Conversely, the ferret and egg-adapted CDV-Onderstepoort strain (CDV-OP) is employed as an effective vaccine for dogs. CDV-OP also exhibits promising oncolytic properties, such as its abilities to infect and kill multiple cancer cells in vitro. Interestingly, several cancer cells do not express SLAM or nectin-4, suggesting the presence of a yet unknown entry factor for CDV-OP. By conducting a genome-wide CRISPR/Cas9 knockout (KO) screen in CDV-OP-susceptible canine mammary carcinoma P114 cells, which neither express SLAM nor nectin-4, we identified low-density lipoprotein receptor-related protein 6 (LRP6) as a host factor that promotes CDV-OP infectivity. Whereas the genetic ablation of LRP6 rendered cells resistant to infection, ectopic expression in resistant LRP6KO cells restored susceptibility. Furthermore, multiple functional studies revealed that (i) the overexpression of LRP6 leads to increased cell-cell fusion, (ii) a soluble construct of the viral receptor-binding protein (solHOP) interacts with a soluble form of LRP6 (solLRP6), (iii) an H-OP point mutant that prevents interaction with solLRP6 abrogates cell entry in multiple cell lines once transferred into recombinant viral particles, and (iv) vesicular stomatitis virus (VSV) pseudotyped with CDV-OP envelope glycoproteins loses its infectivity in LRP6KO cells. Collectively, our study identified LRP6 as the long sought-after cell entry receptor of CDV-OP in multiple cell lines, which set the molecular bases to refine our understanding of viral-cell adaptation and to further investigate its oncolytic properties. IMPORTANCE Oncolytic viruses (OV) have gathered increasing interest in recent years as an alternative option to treat cancers. The Onderstepoort strain of canine distemper virus (CDV-OP), an enveloped RNA virus belonging to the genus Morbillivirus, is employed as a safe and efficient vaccine for dogs against distemper disease. Importantly, although CDV-OP can infect and kill multiple cancer cell lines, the basic mechanisms of entry remain to be elucidated, as most of those transformed cells do not express natural receptors (i.e., SLAM and nectin-4). In this study, using a genome-wide CRISPR/Cas9 knockout screen, we describe the discovery of LRP6 as a novel functional entry receptor for CDV-OP in various cancer cell lines and thereby uncover a basic mechanism of cell culture adaptation. Since LRP6 is upregulated in various cancer types, our data provide important insights in order to further investigate the oncolytic properties of CDV-OP.

Structure and supramolecular organization of the canine distemper virus attachment glycoprotein.

Canine distemper virus (CDV) is an enveloped RNA morbillivirus that triggers respiratory, enteric, and high incidence of severe neurological disorders. CDV induces devastating outbreaks in wild and endangered animals as well as in domestic dogs in countries associated with suboptimal vaccination programs. The receptor-binding tetrameric attachment (H)-protein is part of the morbilliviral cell entry machinery. Here, we present the cryo-electron microscopy (cryo-EM) structure and supramolecular organization of the tetrameric CDV H-protein ectodomain. The structure reveals that the morbilliviral H-protein is composed of three main domains: stalk, neck, and heads. The most unexpected feature was the inherent asymmetric architecture of the CDV H-tetramer being shaped by the neck, which folds into an almost 90° bent conformation with respect to the stalk. Consequently, two non-contacting receptor-binding H-head dimers, which are also tilted toward each other, are located on one side of an intertwined four helical bundle stalk domain. Positioning of the four protomer polypeptide chains within the neck domain is guided by a glycine residue (G158), which forms a hinge point exclusively in two protomer polypeptide chains. Molecular dynamics simulations validated the stability of the asymmetric structure under near physiological conditions and molecular docking showed that two receptor-binding sites are fully accessible. Thus, this spatial organization of the CDV H-tetramer would allow for concomitant protein interactions with the stalk and head domains without steric clashes. In summary, the structure of the CDV H-protein ectodomain provides new insights into the morbilliviral cell entry system and offers a blueprint for next-generation structure-based antiviral drug discovery.

A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets.

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged-Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

Highly Potent Host-Specific Small-Molecule Inhibitor of Paramyxovirus and Pneumovirus Replication with High Resistance Barrier.

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parainfluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application. IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae. The inability of viruses to mutate against the inhibitor mitigated the critical issue of generation of escape variants. Importantly, compound 1 was successfully optimized to a highly potent variant, compound 2 (ZHAWOC21026), with a promising profile for pharmacological intervention.

Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity.

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.

Cryo-EM structure of the prefusion state of canine distemper virus fusion protein ectodomain.

Measles virus (MeV) and canine distemper virus (CDV), two members of the Morbillivirus genus, are still causing important global diseases of humans and animals, respectively. To enter target cells, morbilliviruses rely on an envelope-anchored machinery, which is composed of two interacting glycoproteins: a tetrameric receptor binding (H) protein and a trimeric fusion (F) protein. To execute membrane fusion, the F protein initially adopts a metastable, prefusion state that refolds into a highly stable postfusion conformation as the result of a finely coordinated activation process mediated by the H protein. Here, we employed cryo-electron microscopy (cryo-EM) and single particle reconstruction to elucidate the structure of the prefusion state of the CDV F protein ectodomain (solF) at 4.3 Å resolution. Stabilization of the prefusion solF trimer was achieved by fusing the GCNt trimerization sequence at the C-terminal protein region, and expressing and purifying the recombinant protein in the presence of a morbilliviral fusion inhibitor class compound. The three-dimensional cryo-EM map of prefusion CDV solF in complex with the inhibitor clearly shows density for the ligand at the protein binding site suggesting common mechanisms of membrane fusion activation and inhibition employed by different morbillivirus members.

Ovine Herpesvirus 2 Encodes a Previously Unrecognized Protein, pOv8.25, That Targets Mitochondria and Triggers Apoptotic Cell Death.

Malignant catarrhal fever (MCF) is a rare but frequently lethal disease of certain cloven-hoofed animals. At least 10 different viruses, all members of the Macavirus genus in the subfamily Gammaherpesvirinae, are known as causative agents of MCF. Among these, ovine herpesvirus 2 (OvHV-2) is the most frequent and economically most important MCF agent. Phenotypically, MCF is characterized by severe lymphocytic arteritis-periarteritis, which leads to the accumulation of activated lymphocytes accompanied by apoptosis and necrosis in a broad range of tissues. However, a viral factor that might be responsible for tissue damage has not yet been identified. We have studied a seemingly intergenic locus on the OvHV-2 genome, which was previously shown to be transcriptionally highly active in MCF-affected tissue. We identified by 5' and 3' rapid amplification of cDNA ends (RACE) a conserved, double-spliced transcript that encoded a 9.9-kDa hydrophobic protein. The newly detected gene, Ov8.25, and its splicing pattern were conserved among OvHV-2 strains of different origins. Upon transient expression of synthetic variants of this gene in various cell types, including bovine lymphocytes, the protein (pOv8.25) was shown to target mitochondria, followed by caspase-dependent apoptosis and necrosis. Notably, a deletion mutant of the same protein lost these abilities. Finally, we detected pOv8.25 in brain-infiltrating lymphocytes of cattle with MCF. Thus, the cell death-causing properties of pOv8.25 in affected cells may be involved in the emergence of typical MCF-associated apoptosis and necrosis. Thus, we have identified a novel OvHV-2 protein, which might contribute to the phenotype of MCF-related lesions.IMPORTANCE Ovine herpesvirus 2 (OvHV-2) circulates among sheep without causing disease. However, upon transmission to cattle, the same virus instigates a frequently lethal disease, malignant catarrhal fever (MCF). While the cause of death and pathogenesis of tissue lesions are still poorly understood, MCF is characterized by the accumulation of lymphocytes in various tissues, associated with vasculitis and cell death. As infectious virus is hardly present in these lesions, the cause of cell death cannot be explained simply by viral replication. The significance of our research is in identifying and characterizing a previously overlooked gene of OvHV-2 (Ov8.25), which is highly expressed in animals with MCF. Its encoded protein targets mitochondria, causing apoptosis and necrosis, thus contributing to an understanding of the source and nature of cell death. As the corresponding genetic locus is also active in the context of MCF due to a different macavirus, we may have detected a common denominator of the disease phenotype.

Identification of Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion.

Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of "druggable" targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses.IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.

Primary resistance mechanism of the canine distemper virus fusion protein against a small-molecule membrane fusion inhibitor.

Morbilliviruses (e.g. measles virus [MeV] or canine distemper virus [CDV]) employ the attachment (H) and fusion (F) envelope glycoproteins for cell entry. H protein engagement to a cognate receptor eventually leads to F-triggering. Upon activation, F proteins transit from a prefusion to a postfusion conformation; a refolding process that is associated with membrane merging. Small-molecule morbilliviral fusion inhibitors such as the compound 3G (a chemical analog in the AS-48 class) were previously generated and mechanistic studies revealed a stabilizing effect on morbilliviral prefusion F trimers. Here, we aimed at designing 3G-resistant CDV F mutants by introducing single cysteine residues at hydrophobic core positions of the helical stalk region. Covalently-linked F dimers were generated, which highlighted substantial conformational flexibility within the stalk to achieve those irregular F conformations. Our findings demonstrate that "top-stalk" CDV F cysteine mutants (F-V571C and F-L575C) remained functional and gained resistance to 3G. Conversely, although not all "bottom-stalk" F cysteine variants preserved proper bioactivity, those that remained functional exhibited 3G-sensitivity. According to the recently determined prefusion MeV F trimer/AS-48 co-crystal structure, CDV residues F-V571 and F-L575 may directly interact with 3G. A combination of conformation-specific anti-F antibodies and low-resolution electron microscopy structural analyses confirmed that 3G lost its stabilizing effect on "top-stalk" F cysteine mutants thus suggesting a primary resistance mechanism. Overall, our data suggest that the fusion inhibitor 3G stabilizes prefusion CDV F trimers by docking at the top of the stalk domain.

Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering.

Morbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerization per se was not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCE MeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.