Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first ...step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.
The olfactory mucosa, where the first step of odor detection occurs, is a privileged pathway for environmental toxicants and pathogens toward the central nervous system. Indeed, some pathogens can ...infect olfactory sensory neurons including their axons projecting to the olfactory bulb allowing them to bypass the blood–brain barrier and reach the central nervous system (CNS) through the so‐called olfactory pathway. The respiratory syncytial virus (RSV) is a major respiratory tract pathogen but there is growing evidence that RSV may lead to CNS impairments. However, the mechanisms involved in RSV entering into the CNS have been poorly described. In this study, we wanted to explore the capacity of RSV to reach the CNS via the olfactory pathway and to better characterize RSV cellular tropism in the nasal cavity. We first explored the distribution of RSV infectious sites in the nasal cavity by in vivo bioluminescence imaging and a tissue clearing protocol combined with deep‐tissue imaging and 3D image analyses. This whole tissue characterization was confirmed with immunohistochemistry and molecular biology approaches. Together, our results provide a novel 3D atlas of mouse nasal cavity anatomy and show that RSV can infect olfactory sensory neurons giving access to the central nervous system by entering the olfactory bulb.
Cover Image for this issue: doi: 10.1111/jnc.14765.
While the respiratory syncytium virus (RSV) is mainly known for its damaged in the respiratory tract, it could also impact the central nervous system. We explored globally the impact of RSV in the mouse nasal cavity and showed that RSV can infect olfactory sensory neurons giving access to the central nervous system by entering the olfactory bulb. Our study also provides a 3D atlas of the nasal cavity in mice giving tools to improve our understanding of virus tropism in the upper respiratory tract.
Cover Image for this issue: 10.1111/jnc.14765.
Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ...ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway.
Infection of cells by respiratory syncytial virus induces the formation of cytoplasmic inclusion bodies (IBs) where all the components of the viral RNA polymerase complex are concentrated. However, ...the exact organization and function of these IBs remain unclear. In this study, we use conventional and super-resolution imaging to dissect the internal structure of IBs. We observe that newly synthetized viral mRNA and the viral transcription anti-terminator M2-1 concentrate in IB sub-compartments, which we term "IB-associated granules" (IBAGs). In contrast, viral genomic RNA, the nucleoprotein, the L polymerase and its cofactor P are excluded from IBAGs. Live imaging reveals that IBAGs are highly dynamic structures. Our data show that IBs are the main site of viral RNA synthesis. They further suggest that shortly after synthesis in IBs, viral mRNAs and M2-1 transiently concentrate in IBAGs before reaching the cytosol and suggest a novel post-transcriptional function for M2-1.Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.
De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In ...vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo-designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.
Negative‐sense, single‐stranded RNA (‐ssRNA) viruses comprise some of the deadliest human pathogens (Ebola, rabies, influenza A viruses etc.). Developing therapeutic tools relies on a better ...understanding of their multiplication cycle. For these viruses, the genome replication and transcription activities most‐often segregate in membrane‐less environments called inclusion bodies (IBs) or viral factories. These “organelles” usually locate far from the cell surface from where new virions are released, and ‐ssRNA viruses do not encode for transport factors. The efficient trafficking of the genome progeny toward the cell surface is most often ensured by mechanisms co‐opting the cellular machineries.
In this review, for each ‐ssRNA viral family, we cover the methods employed to characterize these host‐virus interactions, the strategies used by the viruses to promote the virus genome transport, and the current gaps in the literature. Finally, we highlight how Rab11 has emerged as a target of choice for the intracellular transport of ‐ssRNA virus genomes.
The genome replication of Negative‐sense, single‐stranded RNA viruses most‐often segregate in membrane‐less environments called inclusion bodies (IBs). These “organelles” usually locate far from the cell surface from where new virions are released. Here, for each viral family, we discuss how the genome progeny is transported from the IBs to reach the cell surface. We further highlight how the cellular protein Rab11, which has been associated to this process for several viruses, has gained a major importance in the field
The minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus (RSV) is a complex made of two viral proteins, the polymerase large protein (L) and the ...phosphoprotein (P). Here we have investigated the domain on P that is responsible for this critical P-L interaction. By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site. The role of this molecular recognition element of about 30 amino acid residues in the L-P interaction and RNA polymerase activity was evaluated in cellula using an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no good antivirals against RSV are available, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. Like all negative-strand RNA viruses, RSV codes for its own machinery to replicate and transcribe its genome. The core of this machinery is composed of two proteins, the phosphoprotein (P) and the large protein (L). Here, using recombinant proteins, we have mapped and characterized the P domain responsible for this L-P interaction and the formation of an active L-P complex. These findings extend our understanding of the mechanism of action of RSV RNA polymerase and allow us to define a new target for the development of drugs against RSV.
Biomolecular condensates have emerged as an important subcellular organizing principle
. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced ...compartments called inclusion bodies (IBs) or viroplasm
. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation
. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers
.
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell ...compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.
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•Identified a neonatal-specific subset of regulatory B (nBreg) cells in the blood•Neonatal nBreg cells are infected by RSV via the BCR and CX3CR1•RSV-infected nBreg cells produce anti-inflammatory IL-10 that dowregulates Th1 cell responses•Blood nBreg cells are a biomarker of lung disease severity in RSV+ patients
RSV infection leads to respiratory distress in very young children whereas it is asymptomic later in life. Zhivaki et al. found a neonatal-specific regulatory B cell that is recruited in the respiratory tract, gets infected by the virus, and correlates with high viral load and disease severity.
Respiratory syncytial virus (RSV) RNA synthesis occurs in cytoplasmic inclusion bodies (IBs) in which all the components of the viral RNA polymerase are concentrated. In this work, we show that RSV P ...protein recruits the essential RSV transcription factor M2-1 to IBs independently of the phosphorylation state of M2-1. We also show that M2-1 dephosphorylation is achieved by a complex formed between P and the cellular phosphatase PP1. We identified the PP1 binding site of P, which is an RVxF-like motif located nearby and upstream of the M2-1 binding region. NMR confirmed both P-M2-1 and P-PP1 interaction regions in P. When the P-PP1 interaction was disrupted, M2-1 remained phosphorylated and viral transcription was impaired, showing that M2-1 dephosphorylation is required, in a cyclic manner, for efficient viral transcription. IBs contain substructures called inclusion bodies associated granules (IBAGs), where M2-1 and neo-synthesized viral mRNAs concentrate. Disruption of the P-PP1 interaction was correlated with M2-1 exclusion from IBAGs, indicating that only dephosphorylated M2-1 is competent for viral mRNA binding and hence for a previously proposed post-transcriptional function.
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