Biology subjects

Fearns, R.

Publications and source records attributed to Fearns, R..

10 recordsLinked to original sources

bioRxiv2026

eIF4E3 drives translation of viral mRNAs with short 5' UTRs

Eukaryotic cells express three paralogs of the cap-binding protein eIF4E, yet the functions of the less-studied family members remain poorly understood. Here we demonstrate that eIF4E3, a paralog whose expression is tissue-restricted and whose activity is insensitive to inhibition by eIF4E-binding proteins (4EBPs), drives efficient translation of viral mRNAs with short 5 untranslated regions (UTRs). Many negative-strand RNA viruses (NSVs), including vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), and influenza A virus (IAV), produce mRNAs with extremely short 5UTRs that are incompatible with canonical cap-dependent scanning translation initiation. Using an auxin-inducible degron (AID) system to acutely deplete endogenous eukaryotic initiation factors during active viral infection, we demonstrate that translation of these short-UTR viral mRNAs occurs independently of eIF4E1, the canonical cap-binding protein, while remaining dependent on eIF4E3. In contrast, Ebola virus (EBOV), whose mRNAs bear long, structured 5UTRs, remains eIF4E1-dependent, implicating 5UTR length and structural complexity as cis determinants of eIF4E paralog selectivity. During VSV infection, 4EBP dephosphorylation sequesters eIF4E1 and broadly suppresses host cap-dependent translation. Because eIF4E3 escapes 4EBP-mediated regulation and preferentially engages short, unstructured 5UTRs, it is uniquely positioned to sustain viral protein synthesis under these conditions. These findings reveal that VSV exploits a cellular paralog-switching mechanism by co-opting eIF4E3 to maintain viral translation when canonical eIF4F activity is suppressed and establish eIF4E3 as a proviral factor whose tissue-restricted expression in the lung may influence susceptibility to clinically important respiratory pathogens.

molecular biology↗

bioRxiv2026

An immune-poised state of human bronchial epithelial cells mediates RSV resistance in adults

Respiratory syncytial virus (RSV) impairs STAT3 activation in infected ciliated cells in infants, but not adults, causing apoptosis to facilitate virus spread. How age regulates the STAT3 response to RSV in human bronchial epithelial cells (BEpiCs) is unknown. Here, we identify an age-associated immune-poised physiological state in human bronchial basal stem cells (BSCs) and progeny BEpiCs, rendering robust RIG-I to guard against the inactivation of RIG-I-STAT3 signaling by RSV nonstructural protein 2. In contrast, infant BEpiCs are vulnerable to such inactivation by RSV due to lower RIG-I levels. We show that acute respiratory viral infection in specific pathogen-free mice, rather than physical age, triggered sustained epigenetic and transcriptomic remodeling in BEpiCs that resembled age-associated changes in human BEpiCs. In addition, cytokine pretreatment of human neonatal BSCs induced an adult-like immune-poised state that was maintained in progeny BEpiCs to protect against RSV. Therefore, respiratory infection and inflammation during a lifetime imparts the effect of age to BSCs, which fortifies RIG-I in progeny cells to mediate RSV resistance.

microbiology↗

bioRxiv2025

Infant RSV infection desensitizes β2-adrenergic receptor via CXCL11-CXCR7 signaling in airway smooth muscle

RationaleAirflow obstruction refractory to {beta}2 adrenergic receptor ({beta}2AR) agonists is an important clinical feature of infant respiratory syncytial virus (RSV) bronchiolitis, with limited treatment options. This resistance is often linked to poor drug delivery and potential viral infection of airway smooth muscle cells (ASMCs). Whether RSV inflammation causes {beta}2AR desensitization in infant ASMCs is unknown. ObjectivesTo investigate the interaction of RSV inflammation with the {beta}2AR signaling pathway in infant ASMCs MethodsInfant precision-cut lung slices (PCLSs) and mouse pup models of RSV infection were subjected to airway physiological assays. Virus-free, conditioned media from RSV-infected infant bronchial epithelial cells in air-liquid interface (ALI) culture and nasopharyngeal aspirates (NPA) from infants with severe RSV bronchiolitis were collected and applied to infant PCLSs and ASMCs. Cytokines in these samples were profiled and assessed for the effects on {beta}2AR expression, cell surface distribution, and relaxant function in ASMCs. Measurements and Main ResultsConditioned media and NPA induced similar resistance to {beta}2AR agonists in ASMCs as RSV infection. Cytokine profiling identified CXCL11 as one of the most elevated signals following RSV infection. CXCL11 activated its receptor CXCR7 in a complex with {beta}2AR in ASMCs to promote {beta}2AR phosphorylation, internalization, and degradation. Blockade of CXCR7 partially restored airway relaxation in response to {beta}2AR agonists in infant PCLSs and mouse pup models of RSV infection. ConclusionsThe CXCL11-CXCR7 pathway plays a critical role in {beta}2AR desensitization in ASMCs during RSV infection and represents a potential therapeutic target in alleviating airflow obstruction in infant RSV bronchiolitis.

cell biology↗

bioRxiv2024

Structural and functional analysis of the Nipah virus polymerase complex

Nipah virus (NiV) is a bat-borne, zoonotic RNA virus that is among the most pathogenic viruses known to humans. The NiV polymerase, which mediates viral genome replication and mRNA transcription, is a drug target. However, NiV polymerase structures were previously unavailable. We determined the cryo-EM structure of the NiV polymerase complex, comprising the large protein (L) and its associated co-factor (P), and performed structural, biophysical, and functional analyses of the NiV polymerase. The complex assembles with a long P tetrameric coiled-coil that is capped by a bundle of -helices that we show are likely dynamic in solution. Highly conserved zinc-binding modules in the capping domain and a large insert in the RdRp palm domain that is short or absent in most other non-segmented negative strand RNA viruses are critical for replication and transcription. Our findings have the potential to aid in the rational development of drugs to combat NiV infection.

microbiology↗

bioRxiv2023

Age-related STAT3 signaling regulates severity of respiratory syncytial viral infection in human bronchial epithelial cells

Respiratory syncytial virus (RSV) can cause severe disease especially in infants; however, mechanisms of age-associated disease severity remain elusive. Here, employing human bronchial epithelium models generated from tracheal aspirate-derived basal stem cells of neonates and adults, we investigated whether age regulates RSV-epithelium interaction to determine disease severity. We show that following RSV infection, only neonatal epithelium model exhibited cytopathy and mucus hyperplasia, and neonatal epithelium had more robust viral spread and inflammatory responses than adult epithelium. Mechanistically, RSV-infected neonatal ciliated cells displayed age-related impairment of STAT3 activation, rendering susceptibility to apoptosis, which facilitated viral spread. In contrast, SARS-CoV-2 infection of ciliated cells had no effect on STAT3 activation and was not affected by age. Taken together, our findings identify an age-related and RSV-specific interaction with neonatal bronchial epithelium that critically contributes to severity of infection, and STAT3 activation offers a potential strategy to battle severe RSV disease in infants.

cell biology↗

bioRxiv2022

Ultrastructural characterization of a viral RNA and G-protein containing, membranous organelle formed in respiratory syncytial virus infected cells

Withdrawal statementThe authors have withdrawn their manuscript because since deposition of this preprint, they have become aware of the possibility that the findings reported may be an artefact. Carter et al. (J Struct Biol 2018 Jan;201(1):15-25. doi: 10.1016/j.jsb.2017.10.009) have reported that multilamellar bodies exhibit autofluorescence under cryogenic conditions. Thus, there is a possibility that stress induced by virus infection may stimulate formation of the features reported in this article, and autofluorescence, rather than specific labelling led them to identify the puncta as compartments containing RSV proteins or genomes. They are working to address these issues. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

microbiology↗

bioRxiv2022

DISTINCTIVE FEATURES OF THE RESPIRATORY SYNCYTIAL VIRUS PRIMING LOOP COMPARED TO OTHER NON-SEGMENTED NEGATIVE STRAND RNA VIRUSES

De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation. Author SummaryRSV has a significant impact on human health. It is the major cause of respiratory disease in infants and exerts a significant toll on the elderly and immunocompromised. RSV is a member of the Mononegavirales, the non-segmented, negative strand RNA viruses (nsNSVs). Like other viruses in this order, RSV encodes an RNA dependent RNA polymerase, which is responsible for transcribing and replicating the viral genome. Due to its essential role during the viral replication cycle, the polymerase is a promising candidate target for antiviral inhibitors and so a greater understanding of the mechanistic basis of its activities could aid antiviral drug development. In this study, we identified an amino acid residue within the RSV polymerase that appears to stabilize the RNA synthesis initiation complex and showed that it plays a role in both transcription and RNA replication. However, the corresponding residue in a different nsNSV polymerase does not appear to play a similar role. This work reveals a key feature of the RSV polymerase but identifies differences with the polymerases of other related viruses.

microbiology↗

bioRxiv2021

Direct RNA sequencing of Respiratory Syncytial Virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing on an Oxford nanopore device. This yielded over 150,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We were also able to examine frequencies with which polycistronic readthrough mRNAs were generated and to assess the length of the polyadenylated tails for each group of transcripts. We show that there is a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not consistent. We show that the polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host cells polyadenylation machinery and broadly declined in length for most transcripts as infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails is much less for N, SH and G transcripts compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates.

microbiology↗

bioRxiv2021

Structural architecture of a dimeric paramyxovirus polymerase complex

Human parainfluenza virus type 3 (hPIV3), a member of non-segmented, negative-strand RNA viruses (nsNSVs), is the second most common cause of severe respiratory disease in pediatrics. The transcription and replication processes of nsNSVs are catalyzed by a multi-functional RNA-dependent RNA polymerase (RdRp) complex composed of the large protein (L) and the phosphoprotein (P). Previous studies have shown that the polymerase can adopt a dimeric form, however, the structure of the dimer and how it functions are not understood. Here we determined the cryo-EM structure of hPIV3 L-P complex at 2.7 [A] with substantial structural details. A putative catalytic magnesium ion could be built in our structure, and structural comparison revealed atomic features conserved with other RNA viruses. Interactions identified between the two priming and intrusion loops and the connector domain potentially trigger the spatial movement of three C-terminal L domains for different steps of transcription and replication. Structural comparison with other nsNSV RdRps suggests common features of L-P binding. Furthermore, we report for the first time the structural basis of the L-L interaction in the partially modelled dimeric L-P structure, in which the connector domain of one L is positioned at the putative RNA template entry of the other L. Based on these findings, we propose a model by which L dimerization promotes efficient conversion of nascent RNA into a template.

molecular biology↗

bioRxiv2021

Helical Ordering of Envelope Associated Proteins and Glycoproteins in Respiratory Syncytial Virus Filamentous Virions

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Treatments for RSV disease are however limited and efforts to produce an effective vaccine have so far been unsuccessful. Understanding RSV virion structure is an important prerequisite for developing interventions to treat or prevent infection but has been challenging because of the fragility of virions propagated in cell culture. Here we show, using cryogenic electron microscopy (cryoEM) and cryogenic electron tomography (cryoET) of RSV particles cultivated directly on transmission electron microscopy (TEM) grids, that there is extensive helical symmetry in RSV filamentous virions. We have calculated a 16 [A] resolution three-dimensional reconstruction of the viral envelope, targeting the matrix protein (M) that forms an endoskeleton below the viral membrane. These data define a helical lattice of M proteins, showing how M is oriented relative to the viral envelope and that helical ordering of viral glycoproteins that stud the viral envelope is coordinated by the M layer. Moreover, the helically ordered viral glycoproteins in RSV filamentous virions cluster in pairs, which may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV filamentous virions. Overall, the structural data obtained provides molecular insight into the organization of the virion and the mechanism of its assembly.

microbiology↗