Neutralization, effector function and immune imprinting of Omicron variants.

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain1 (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting.

A short sequence in the tail of SARS-CoV-2 envelope protein controls accessibility of its PDZ-binding motif to the cytoplasm.

The carboxy-terminal tail of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope protein (E) contains a PDZ-binding motif (PBM) which is crucial for coronavirus pathogenicity. During SARS-CoV-2 infection, the viral E protein is expressed within the Golgi apparatus membrane of host cells with its PBM facing the cytoplasm. In this work, we study the molecular mechanisms controlling the presentation of the PBM to host PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins. We show that at the level of the Golgi apparatus, the PDZ-binding motif of the E protein is not detected by E C-terminal specific antibodies nor by the PDZ domain-containing protein-binding partner. Four alanine substitutions upstream of the PBM in the central region of the E protein tail is sufficient to generate immunodetection by anti-E antibodies and trigger robust recruitment of the PDZ domain-containing protein into the Golgi organelle. Overall, this work suggests that the presentation of the PBM to the cytoplasm is under conformational regulation mediated by the central region of the E protein tail and that PBM presentation probably does not occur at the surface of Golgi cisternae but likely at post-Golgi stages of the viral cycle.

Lyssavirus P-protein selectively targets STAT3-STAT1 heterodimers to modulate cytokine signalling.

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.

Regulation of NF-κB by the p105-ABIN2-TPL2 complex and RelAp43 during rabies virus infection.

At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBß-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNß, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling.

The shift in rabies epidemiology in France: time to adjust rabies post-exposure risk assessment.

The epidemiology of rabies in France and western Europe has changed during the past 22 years. In France, rabies in non-flying terrestrial mammals was declared to be eliminated in 2001, and the risk of rabies is now limited to contact with bats, rabid animals illegally imported from rabies-enzootic countries and traveller exposure in enzootic areas. We analysed the epidemiology of rabies in France from 1995 to 2016, describing and analysing data on human rabies surveillance as well as data on post-exposure prophylaxis (PEP) collected from the network of French antirabies clinics. Over the study period, seven individuals were diagnosed with rabies in France, all of whom were infected outside mainland France. PEP data analysis revealed an expected overall decrease in PEP administration for individuals exposed in mainland France, but there was still overuse of anti-rabies drugs, given the very low epidemiological risk. On the other hand, a significant increase in PEP delivered to individuals exposed abroad was evidenced. These epidemiological trends indicate that clear guidelines should be provided to support physicians' efforts to adjust rabies risk assessment to the evolution of the epidemiological situation.

Focal adhesion kinase is involved in rabies virus infection through its interaction with viral phosphoprotein P.

UNLABELLED: The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE: Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function.

Severe ketoalkalosis as initial presentation of imported human rabies in France.

We report a patient with an unusual initial metabolic presentation of imported human rabies who became symptomatic within 2 weeks of returning from Mali to France. This is the single case of imported human rabies identified in France within the past 11 years and the first report of viral RNA in bronchial secretions.

Interaction of rabies virus P-protein with STAT proteins is critical to lethal rabies disease.

BACKGROUND: Rabies virus (RABV) causes rabies disease resulting in >55,000 human deaths/year. The multifunctional RABV P-protein has essential roles in genome replication, and forms interactions with cellular STAT proteins that are thought to underlie viral antagonism of interferon-dependent immunity. However, the molecular details of P-protein-STAT interaction, and its importance to disease are unresolved. METHODS: Studies were performed using sequence/structure analysis, mutagenesis, immunoprecipitation, luciferase and qRT-PCR-based signaling assays, confocal microscopy and reverse genetics/in vivo infection. RESULTS: We identified a hydrophobic pocket of the P-protein C-terminal domain as critical to STAT-binding/antagonism. This interface was found to be functionally and spatially independent of the region responsible for N-protein interaction, which is critical to genome replication. Based on these findings, we generated the first mutant RABV lacking STAT-association. Growth of the virus in vitro was unimpaired, but it lacked STAT-antagonist function and was highly sensitive to interferon. Importantly, growth of the virus was strongly attenuated in brains of infected mice, producing no major neurological symptoms, compared with the invariably lethal wild-type virus. CONCLUSIONS: These data represent direct evidence that P-protein-STAT interaction is critical to rabies, and provide novel insights into the mechanism by which RABV coordinates distinct functions in interferon antagonism and replication.

Comparative analysis of rabies pathogenic and vaccine strains detection by RIG-I-like receptors.

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.

Mechanism of action of phthalazinone derivatives against rabies virus.

Rabies, a viral zoonosis, is responsible for almost 59,000 deaths each year, despite the existence of an effective post-exposure prophylaxis. Indeed, rabies causes acute encephalomyelitis, with a case-fatality rate of 100 % after the onset of neurological clinical signs. Therefore, the development of therapies to inhibit the rabies virus (RABV) is crucial. Here, we identified, from a 30,000 compound library screening, phthalazinone derivative compounds as potent inhibitors of RABV infection and more broadly of Lyssavirus and even Mononegavirales infections. Combining in vitro experiments, structural modelling, in silico docking and in vivo assays, we demonstrated that phthalazinone derivatives display a strong inhibition of lyssaviruses infection by acting directly on the replication complex of the virus, and with noticeable effects in delaying the onset of the clinical signs in our mouse model.

Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants.

Anosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals develop a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS-CoV-2 variants are neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validate the olfactory pathway as a major entry point into the brain in vivo and demonstrate in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks.

Susceptibilities of CNS Cells towards Rabies Virus Infection Is Linked to Cellular Innate Immune Responses.

Rabies is caused by neurotropic rabies virus (RABV), contributing to 60,000 human deaths annually. Even though rabies leads to major public health concerns worldwide, we still do not fully understand factors determining RABV tropism and why glial cells are unable to clear RABV from the infected brain. Here, we compare susceptibilities and immune responses of CNS cell types to infection with two RABV strains, Tha and its attenuated variant Th2P-4M, mutated on phospho- (P-protein) and matrix protein (M-protein). We demonstrate that RABV replicates in human stem cell-derived neurons and astrocytes but fails to infect human iPSC-derived microglia. Additionally, we observed major differences in transcription profiles and quantification of intracellular protein levels between antiviral immune responses mediated by neurons, astrocytes (IFNB1, CCL5, CXCL10, IL1B, IL6, and LIF), and microglia (CCL5, CXCL10, ISG15, MX1, and IL6) upon Tha infection. We also show that P- and M-proteins of Tha mediate evasion of NF-κB- and JAK-STAT-controlled antiviral host responses in neuronal cell types in contrast to glial cells, potentially explaining the strong neuron-specific tropism of RABV. Further, Tha-infected astrocytes and microglia protect neurons from Tha infection via a filtrable and transferable agent. Overall, our study provides novel insights into RABV tropism, showing the interest in studying the interplay of CNS cell types during RABV infection.

Structure of the rabies virus glycoprotein trimer bound to a prefusion-specific neutralizing antibody.

Rabies infection is nearly 100% lethal if untreated and kills more than 50,000 people annually, many of them children. Existing rabies vaccines target the rabies virus glycoprotein (RABV-G) but generate short-lived immune responses, likely because the protein is heterogeneous under physiological conditions. Here, we report the 3.39 Å cryo-electron microscopy structure of trimeric, prefusion RABV-G complexed with RVA122, a potently neutralizing human antibody. RVA122 binds to a quaternary epitope at the top of RABV-G, bridging domains and stabilizing RABV-G protomers in a prefusion state. RABV-G trimerization involves side-to-side interactions between the central α helix and adjacent loops, rather than contacts between central helices, and interactions among the fusion loops at the glycoprotein base. These results provide a basis from which to develop improved rabies vaccines based on RABV-G stabilized in the prefusion conformation.

Two overlapping domains of a lyssavirus matrix protein that acts on different cell death pathways.

The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

Structure of the nucleoprotein binding domain of Mokola virus phosphoprotein.

Mokola virus (MOKV) is a nonsegmented, negative-sense RNA virus that belongs to the Lyssavirus genus and Rhabdoviridae family. MOKV phosphoprotein P is an essential component of the replication and transcription complex and acts as a cofactor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. Here we present a structure for this domain of MOKV P, obtained by expression of full-length P in Escherichia coli, which was subsequently truncated during crystallization. The structure has a high degree of homology with P of rabies virus, another member of Lyssavirus genus, and to a lesser degree with P of vesicular stomatitis virus (VSV), a member of the related Vesiculovirus genus. In addition, analysis of the crystal packing of this domain reveals a potential binding site for the nucleoprotein N. Using both site-directed mutagenesis and yeast two-hybrid experiments to measure P-N interaction, we have determined the relative roles of key amino acids involved in this interaction to map the region of P that binds N. This analysis also reveals a structural relationship between the N-RNA binding domain of the P proteins of the Rhabdoviridae and the Paramyxoviridae.

Application of broad-spectrum resequencing microarray for genotyping rhabdoviruses.

The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.

Early Transcriptional Changes in Rabies Virus-Infected Neurons and Their Impact on Neuronal Functions.

Rabies is a zoonotic disease caused by rabies virus (RABV). As rabies advances, patients develop a variety of severe neurological symptoms that inevitably lead to coma and death. Unlike other neurotropic viruses that can induce symptoms of a similar range, RABV-infected post-mortem brains do not show significant signs of inflammation nor the structural damages on neurons. This suggests that the observed neurological symptoms possibly originate from dysfunctions of neurons. However, many aspects of neuronal dysfunctions in the context of RABV infection are only partially understood, and therefore require further investigation. In this study, we used differentiated neurons to characterize the RABV-induced transcriptomic changes at the early time-points of infection. We found that the genes modulated in response to the infection are particularly involved in cell cycle, gene expression, immune response, and neuronal function-associated processes. Comparing a wild-type RABV to a mutant virus harboring altered matrix proteins, we found that the RABV matrix protein plays an important role in the early down-regulation of host genes, of which a significant number is involved in neuronal functions. The kinetics of differentially expressed genes (DEGs) are also different between the wild type and mutant virus datasets. The number of modulated genes remained constant upon wild-type RABV infection up to 24 h post-infection, but dramatically increased in the mutant condition. This result suggests that the intact viral matrix protein is important to control the size of host gene modulation. We then examined the signaling pathways previously studied in relation to the innate immune responses against RABV, and found that these pathways contribute to the changes in neuronal function-associated processes. We further examined a set of regulated genes that could impact neuronal functions collectively, and demonstrated in calcium imaging that indeed the spontaneous activity of neurons is influenced by RABV infection. Overall, our findings suggest that neuronal function-associated genes are modulated by RABV early on, potentially through the viral matrix protein-interacting signaling molecules and their downstream pathways.

Attenuation of clinical and immunological outcomes during SARS-CoV-2 infection by ivermectin.

The devastating pandemic due to SARS-CoV-2 and the emergence of antigenic variants that jeopardize the efficacy of current vaccines create an urgent need for a comprehensive understanding of the pathophysiology of COVID-19, including the contribution of inflammation to disease. It also warrants for the search of immunomodulatory drugs that could improve disease outcome. Here, we show that standard doses of ivermectin (IVM), an anti-parasitic drug with potential immunomodulatory activities through the cholinergic anti-inflammatory pathway, prevent clinical deterioration, reduce olfactory deficit, and limit the inflammation of the upper and lower respiratory tracts in SARS-CoV-2-infected hamsters. Whereas it has no effect on viral load in the airways of infected animals, transcriptomic analyses of infected lungs reveal that IVM dampens type I interferon responses and modulates several other inflammatory pathways. In particular, IVM dramatically reduces the Il-6/Il-10 ratio in lung tissue and promotes macrophage M2 polarization, which might account for the more favorable clinical presentation of IVM-treated animals. Altogether, this study supports the use of immunomodulatory drugs such as IVM, to improve the clinical condition of SARS-CoV-2-infected patients.

COVID-19-related anosmia is associated with viral persistence and inflammation in human olfactory epithelium and brain infection in hamsters.

Whereas recent investigations have revealed viral, inflammatory, and vascular factors involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lung pathogenesis, the pathophysiology of neurological disorders in coronavirus disease 2019 (COVID-19) remains poorly understood. Olfactory and taste dysfunction are common in COVID-19, especially in mildly symptomatic patients. Here, we conducted a virologic, molecular, and cellular study of the olfactory neuroepithelium of seven patients with COVID-19 presenting with acute loss of smell. We report evidence that the olfactory neuroepithelium is a major site of SARS-CoV2 infection with multiple cell types, including olfactory sensory neurons, support cells, and immune cells, becoming infected. SARS-CoV-2 replication in the olfactory neuroepithelium was associated with local inflammation. Furthermore, we showed that SARS-CoV-2 induced acute anosmia and ageusia in golden Syrian hamsters, lasting as long as the virus remained in the olfactory epithelium and the olfactory bulb. Last, olfactory mucosa sampling from patients showing long-term persistence of COVID-19-associated anosmia revealed the presence of virus transcripts and of SARS-CoV-2-infected cells, together with protracted inflammation. SARS-CoV-2 persistence and associated inflammation in the olfactory neuroepithelium may account for prolonged or relapsing symptoms of COVID-19, such as loss of smell, which should be considered for optimal medical management of this disease.

A live measles-vectored COVID-19 vaccine induces strong immunity and protection from SARS-CoV-2 challenge in mice and hamsters.

Several COVID-19 vaccines have now been deployed to tackle the SARS-CoV-2 pandemic, most of them based on messenger RNA or adenovirus vectors.The duration of protection afforded by these vaccines is unknown, as well as their capacity to protect from emerging new variants. To provide sufficient coverage for the world population, additional strategies need to be tested. The live pediatric measles vaccine (MV) is an attractive approach, given its extensive safety and efficacy history, along with its established large-scale manufacturing capacity. We develop an MV-based SARS-CoV-2 vaccine expressing the prefusion-stabilized, membrane-anchored full-length S antigen, which proves to be efficient at eliciting strong Th1-dominant T-cell responses and high neutralizing antibody titers. In both mouse and golden Syrian hamster models, these responses protect the animals from intranasal infectious challenge. Additionally, the elicited antibodies efficiently neutralize in vitro the three currently circulating variants of SARS-CoV-2.