Biology subjects

Wilson, H.

Publications and source records attributed to Wilson, H..

At least 19 recordsLinked to original sources

bioRxiv2026

High-resolution mapping of RNA structural maturation during Cas9 assembly with ABEL-FRET

The structural flexibility of RNA is essential for forming ribonucleoprotein (RNP) complexes, which regulate diverse biological processes. This intrinsic property permits RNA to act as a dynamic scaffold along the assembly pathway as it folds into a specific structure for initial recognition by protein and undergoes conformational rearrangements for functional maturation as a complex. Yet, RNA flexibility and RNP multicomponent assembly create significant obstacles for traditional structural methods. To overcome these challenges, we applied recently developed ABEL-FRET spectroscopy to measure tether-free single-molecule Forster resonance energy transfer (smFRET) over extended observation times. Furthermore, ABEL-FRET enables the unique ability for simultaneous measurements of ultrahigh resolution smFRET and hydrodynamic size of individual complexes, which offers distinct advantages for studying dynamic RNA molecules that undergo assembly via sequential binding events. Using ABEL-FRET, we explored how the guide RNA (gRNA) of CRISPR genome editing system folds and modulates its structural flexibility to carry out the roles required for each assembly state from its unbound apo form to the functional Cas9 RNP state for target DNA cleavage. Multi-perspective view of gRNA structure gained by probing its two primary functional domains enabled to capture dramatic changes in gRNA flexibility that are highly dependent on its specific structural domains as well as assembly states. Collectively, our work with ABEL-FRET highlights the intrinsic link between the structural flexibility of RNA and its functionality in RNP assembly.

biophysics↗

bioRxiv2026

Cth1 is essential for gametogenesis and fertility in zebrafish

Infertility frequently arises from defects in germ cells and early embryonic transition. Successful fertilization depends on the developmental competence and molecular integrity of mature gametes from both parents, which are established through tightly coordinated programs of RNA regulation, metabolism, and genome maintenance. In our previous work, we identified Cth1 as a maternally regulated RNA decay factor essential for early embryonic development, acting through spatiotemporal control of maternal transcript clearance. Interestingly, Cth1 loss of function in adults also resulted in infertility, suggesting an additional and unexplored role during gametogenesis. Here, we extend these findings by defining the gametogenic function of Cth1 in zebrafish. Through detailed phenotypic, cytological and molecular characterization of Cth1 loss of function mutants, we show that Cth1 is highly enriched in germ cells and early embryos and is spatio-temporally localized across oogenesis and early development. Loss of Cth1 causes severe defects in early oogenesis and spermatogenesis, resulting in complete infertility in males and females. Mutant germ cells display transcriptomic changes consistent with metabolic and translational dysregulation, increased DNA damage, and striking abnormalities in gamete morphology. Together, these findings identify Cth1 as essential for gamete quality and fertility. Our study suggests a link between RNA decay mediated regulation of metabolism and genome integrity during germ cell development and reveals disruption of post-transcriptional control as a potential mechanism underlying infertility.

developmental biology↗

bioRxiv2026

Atomic modeling of radiation damage in cryoelectron microscopy datasets

Damage to biological specimens by the electron beam is the fundamental resolution-limiting factor in cryoelectron microscopy (cryo-EM) single particle analysis. There is, however, currently no method to accurately infer fluence-dependent changes to the specimen structure during electron irradiation. We develop a Bayesian framework to fit a sequence of atomic models to a series of cryo-EM reconstructions produced at increasing fluence. In particular, our algorithm is able to infer the ensemble average position and atomic displacement parameter of every atom in the macromolecule as a function of fluence. Application of the algorithm to cryo-EM datasets shows that the molecule expands during imaging and identifies environment-dependent variations in beam-induced damage. We use our results to propose a stochastic process model of this phenomenon. We envisage that our method will lead to a better mechanistic understanding of radiation damage to biological specimens and may contribute to efforts to mitigate its effects.

biophysics↗

bioRxiv2026

Explaining the pathogenesis of African swine fever using knowledge-driven regulatory network modeling

A lethal DNA virus with significant economic impact on livestock farmers worldwide, the pathogenesis of African swine fever virus (ASFV) infection is complex and continues to challenge the development of effective vaccine candidates. The requirement for high-containment conditions further complicates its study, resulting in limitations in sample size and marker assessment that challenge conventional statistical analysis. In this work we demonstrate how prior knowledge of immune biology and pathogen-host proteome interactions can be leveraged and reconciled with sparse experimental data to deliver plausible mechanistically informed hypotheses describing ASFV illness progression. We apply large-scale automated mining of literature and pathway schema together with generative artificial intelligence (AI) to create closed-loop regulatory network models consisting of 133 pathogen and host proteins linked by 676 regulatory interactions. Immune regulatory tuning of these networks is reverse engineered to explain two distinct experimentally observed illness progression trajectories in only 5 markers measured every second day over a maximum of 8 days. Comparison of network model pools specific to each progression phenotype suggest that these significantly different outcomes may arise from altered regulatory tuning of genes coding for interleukin (IL)1{beta}, tumor necrosis factor (TNF) and Forkhead box protein (FOX)O4, potentially as a result of epigenetic adaptations. Simulated challenges with individual ASFV protein confirm broadly delayed interferon (IFN)-{gamma}I response in both phenotypes, with multigene family (MGF)505-3R offering the earliest induction and only in the more severe phenotype. Paradoxically, predictions suggest that this delay is preceded by an early IL-10 induction by this same viral protein. While added model granularity and validation is needed, we propose that this proof-of-concept knowledge driven approach offers an attractive solution to mechanistic hypothesis generation in data poor environments.

Systems Biology↗

bioRxiv2026

A versatile cryopreservation method for peri-gastrulation squamate embryos optimised using the veiled chameleon (C. calyptratus)

Stem cell technologies have become a vital component of conservation efforts around the globe. Biobanks and pluripotent stem cell lines help to ensure species and their genetic diversity are preserved. These efforts have however, focussed mostly on mammals and birds, and the cryopreservation protocols for embryos and cells were developed decades ago laying the basis for artificial reproductive techniques for species conservation. With over 20% of non-avian reptile species facing extinction, it is imperative to establish protocols for reptiles to ensure species preservation and also to facilitate the establishment of new reptile model organisms to match the standard of mammals. Here, we have generated a cryopreservation method for preserving early gastrulating veiled chameleon embryos as a representative squamate species. To this end, we first developed a tissue culture method for maintaining cells extracted from peri-gastrulation chameleon embryos and then tested different cryopreservation methods altering the concentration of the penetrating cryoprotectant DMSO and assessing the effect of the addition of non-penetrating cryoprotectants Trehalose and Sucrose. We then optimised a protocol for whole embryo vitrification in 20% DMSO with added Trehalose or Sucrose that can easily be adapted for fieldwork. Taken together, our method not only provides a protocol for conservation efforts but also lays the basis for mechanistic studies of early squamate embryo development by enabling cryopreservation of whole embryos in a fieldwork setting, which facilitates their live transport back to a laboratory for functional experiments or molecular analyses.

developmental biology↗

bioRxiv2025

Mesodermal-niche interactions direct specification and differentiation of pancreatic islet cells in human multilineage organoids

Tissues develop and function within a highly complex microenvironment, where diverse cell types interact in tightly regulated spatial and temporal patterns1,2. Through distinct, relay-like waves of activity, these cells collectively shape tissue formation, ensuring that each component emerges in the right place at the right time1,2. Such coordinated cues establish the structural and biochemical framework that drives cell differentiation and tissue organization. Accurately modelling this process in humans requires the development of complex multicellular systems. Here, we pair spatial transcriptomics of the human foetal pancreas with an induced pluripotent stem cell (iPSC)-based multilineage model that faithfully recapitulate the complex, hierarchical processes underlying human pancreatic islet formation. We show that iPSC-derived pancreatic mesodermal lineages direct endocrine commitment from pancreatic progenitors, by suppressing off-target fates and orchestrating niche-mediated spatio-temporal cues that promote beta-cell differentiation. Our results identify a vascular-rich niche, featuring pancreatic pericytes, which is associated with a neural repulsion program and may contribute to shaping the islet microenvironment. We benchmark our in vitro multilineage organoid system against spatial transcriptomics of the human foetal pancreas. Together, these findings identify the key cellular actors and contact-dependent mechanisms that build the human endocrine pancreas, providing a critical model for studying human islet development and disease.

developmental biology↗

bioRxiv2025

Measurement of atomic scattering factors by cryo-electron microscopy

Determination of specimen structure from cryo-electron microscopy (cryo-EM) experiments relies on an accurate model of the electrostatic potential of the specimen. For biological macromolecules, the potential is strongly influenced by the presence of chemical bonds between atoms, a fact unaccounted for by models of electron scattering that are currently standard in the field. We propose a Bayesian approach to the estimation of atomic scattering factors which incorporates the effect of the molecular environment while remaining fast, interpretable and transferable between molecules. Our algorithm infers atomic scattering factors directly from maps of the electrostatic potential determined by cryo-EM single particle analysis, bypassing the need for computationally-intensive theoretical calculations. The algorithm is used to infer empirical scattering factors from high-resolution reconstructions of catalase enzymes. To illustrate its broad applicability, the algorithm is also applied to a training set of publicly-available cryo-EM data. The empirical scattering factors show improved agreement with a test set of cryo-EM reconstructions. The predictions are further validated by comparison with magnetic susceptibility values of organic compounds, as well as by application to the refinement of atomic models. Significance StatementUnderstanding the structure of biomolecules is key to explaining their function. Cryo-electron microscopy is a method for reconstructing the electrostatic potential distribution of a biological macro-molecule, a quantity which contains information about atomic positions and the redistribution of charge due to chemical bonding. These factors should be modelled when inferring the structure of the molecule from its electrostatic potential. We develop an improved model of the potential that takes into account chemical bonding while remaining computationally tractable. The parameters of the model are inferred from a selection of cryo-electron microscopy datasets using Bayesian methods.

biophysics↗

bioRxiv2025

Oogenesis and germinal bed morphology of the brown anole (A. sagrei)

BackgroundThe brown anole is a model species of the genus Anolis, a squamate (encompassing lizards and snakes) group widely studied in evolutionary, behavioral, and developmental biology. Full genome annotation, the establishment of gene editing techniques, and comprehensive description of reproductive tract morphology and embryogenesis in this species, has laid the foundation for functional studies. However, analysis of brown anole oogenesis is still required and vital to optimize genome modification, mutant line establishment, and analyses of the evolution of reproductive developmental mechanisms. ResultsHere, we characterize ovary morphology and gametogenesis in the female brown anole, A. sagrei using brightfield imaging, microCT, histology staining, electron microscopy, and confocal imaging. We define 10 stages of oocyte maturation which commences inside the oogonial nest within the germinal bed and concludes with the mature follicle ready to ovulate based on follicle size, yolk-acquisition, and follicular, cellular, and basement membrane architecture. ConclusionsWe describe the complete oogenesis of the brown anole in 10 stages and report that oogenesis is highly conserved within iguanids, a suborder of lizards. With our staging framework, we lay the foundation for functional studies of oogenesis and optimized gene-editing.

developmental biology↗

bioRxiv2025

Epiblast Lumenogenesis is not a mammalian-specific trait

Epiblast lumenogenesis, which leads to amniotic cavity formation, is a hallmark of mammalian embryogenesis, and required for crucial developmental processes such as anterior-posterior patterning and gastrulation. Based on avian model-organisms, the epiblast in reptiles is thought to form a monolayered flat disc that undergoes anterior-posterior patterning and gastrulation. Here, we report that the squamate, veiled chameleon (Chameleo calyptratus), exhibits epiblast lumenogenesis and amniogenesis prior to anterior-posterior patterning. Using SEM, immunofluorescence, and histology techniques, we demonstrate that chameleon epiblast lumenogenesis occurs via a purse-string-like mechanism involving the formation of supracellular actin cables in concentric rings around the epiblast followed by constriction that closes the epiblast lumen. Through expression analyses of Nodal1, Nodal2, Cerberus, Lefty, Brachyury, Wnt3A, and Bmp2, and immunostaining for Brachyury, we uncovered a Wnt3a- and Brachyury-positive ring at the edge of the epiblast concomitant with lumenogenesis, and preceding anterior-posterior patterning and gastrulation. Furthermore, we report anterior-posterior patterning in veiled chameleons occurs independently of Cerberus and Lefty. These processes that mediate epiblast lumenogenesis in chameleons, result in a morphology remarkably similar to human embryos, despite 300 million years of evolutionary separation. Taken together, we show that pre-gastrulation epiblast lumenogenesis is not mammalian-specific, but also occurs in some non-avian reptiles.

developmental biology↗

bioRxiv2024

Structural and functional characterization of the KHNYN extended-diKH domain for mediating ZAP antiviral activity

Zinc finger antiviral protein (ZAP) binds CpG dinucleotides in viral RNA and targets them for decay. ZAP interacts with several cofactors to form the ZAP antiviral system, including KHNYN, a multidomain endoribonuclease required for ZAP-mediated RNA decay. However, it is unclear how the individual domains in KHNYN contribute to its activity. Here, we demonstrate that the KHNYN amino terminal extended-diKH (ex-diKH) domain is required for antiviral activity and present its crystal structure. The structure belongs to a rare group of KH-containing domains, characterized by a non-canonical arrangement between two type-1 KH modules, with an additional helical bundle. N4BP1 is a KHNYN paralog with an ex-diKH domain that functionally complements the KHNYN ex-diKH domain. Interestingly, the ex-diKH domain structure is present in N4BP1-like proteins in lancelets, which are basal chordates, indicating that it is evolutionarily ancient. While many KH domains demonstrate RNA binding activity, biolayer interferometry and electrophoretic mobility shift assays indicate that the KHNYN ex-diKH domain does not bind RNA. Furthermore, residues required for canonical KH domains to bind RNA are not required for KHNYN antiviral activity. By contrast, an inter-KH domain cleft in KHNYN is a potential protein-protein interaction site and mutations that eliminate arginine salt bridges at the edge of this cleft decrease KHNYN antiviral activity. This suggests that this domain could be a binding site for an unknown KHNYN cofactor.

microbiology↗

bioRxiv2024

Pre-oviposition development of the brown anole (Anolis sagrei)

The brown anole, Anolis sagrei, has emerged as a representative squamate species during the past several decades. Novel functional tools have been established to manipulate embryogenesis through genome editing or the introduction of small molecule inhibitors, and their effective use requires a thorough understanding of early anole embryogenesis. To enable precise and reproducible staging of anole embryos, we need knowledge of the progression of anole embryogenesis and morphogenesis. While post-oviposition development has been described, the pre-oviposition period remains to be explored. Here, we provide the first staging series of pre-oviposition development for the brown anole. Analysing the follicles and embryos through brightfield imaging, SEM, STEM, histology, and DAPI staining, we define 26 distinct developmental stages. Our dataset reveals that peri-gastrulation morphogenesis up to the initiation of neurulation diverges significantly from chicken, the common representative model of reptile embryogenesis. Furthermore, we followed heart development, neural crest cell migration and central nervous system development through immunofluorescence analyses and provide new comparative insights into the morphogenesis of each of these organ systems over time. Our work establishes the brown anole as a squamate model organism for cross clade evolutionary studies of early embryogenesis.

developmental biology↗

bioRxiv2024

Sex differences in the cardiopulmonary and neuromuscular response to high-intensity interval exercise

Sex differences exist in the integrative response to exercise, however, these are typically researched during constant-load exercise. Interval exercise involves high-intensity efforts interspersed with recovery periods to repeatedly stress physiological systems, and it is currently unknown whether the response to this form of exercise differs between sexes. Ten males and ten females (age: 25{+/-}3 years) completed two experimental visits. First, an incremental treadmill exercise test was performed to obtain submaximal (lactate threshold) and maximal ([Formula] O2peak) data. Thereafter, visit two involved 4 x 3-min running intervals at 90% of the final incremental test velocity (v[Formula] O2peak), with 90 secs rest between intervals. Before exercise and after each interval, maximal voluntary contraction (MVC), quadriceps potentiated twitch (Qtw.pot), and voluntary activation (VA) were recorded. The rates of oxygen uptake ([Formula] O2), carbon dioxide production ([Formula] CO2) and ventilation ([Formula] E) were continuously recorded throughout. There was no sex difference in relative [Formula] O2peak (males: 47.2{+/-}6.0 vs. females: 44.4{+/-}5.8 ml.kg- 1.min-1, p=0.292). When expressed relative to peak values, there were no sex differences in the [Formula] O2 or [Formula] CO2 response to the interval task (p[≥]0.781). Females had greater [Formula] E, [Formula] E/[Formula] O2, and [Formula] E/[Formula] CO2 values during the first two intervals (p[≤]0.046). There were no sex differences in the reductions in MVC, Qtw.pot, and VA during the interval task (p[≥]0.150), however females had lesser reductions in Qtw.pot values post-exercise (-24{+/-}9 vs. -15{+/-}8%, p=0.044). Sex differences exist in the physiological response to interval exercise. Compared to males, females experienced greater hyperpnoea during the initial stages, and had lesser decreases in contractile function post-exercise.

physiology↗

bioRxiv2024

Single-cell transcriptome and T cell receptor profiling of the tuberculin skin test

The tuberculin skin test (TST) is a cutaneous delayed hypersensitivity reaction to antigen from Mycobacterium tuberculosis (Mtb). We provide the first single cell sequencing characterisation of the human TST reaction, based on skin suction blisters induced at the site of the TST on day 2 in 31 individuals. Integrated single cell RNA and TCR sequencing showed the immune response to be dominated by T cells, with smaller populations of NK cells and myeloid cells. T cells comprised CD4, CD8, gamma/delta and NK T cells, with 50% of all T cells identified as cytotoxic and 14% as regulatory. Interferon gamma gene expression was strongest in CD8 T cells, and distinct CD4 T helper lineages could not unambiguously be identified at this time point. Amongst myeloid cells, 63% displayed antimicrobial gene expression and 28% were functionally polarised towards antigen presentation with higher levels of HLA class 2 expression. We derived and validated transcriptional signatures for cell types and cellular functions relevant to the immune landscape of the TST. These data help to improve our understanding of the immune response to Mtb and enable further exploration of bulk transcriptomic data through context-specific cellular deconvolution.

immunology↗

bioRxiv2024

Sanctuary: A Starship transposon facilitating the movement of the virulence factor ToxA in fungal wheat pathogens

There is increasing evidence that mobile genetic elements can drive the emergence of pathogenic fungal species by moving virulence genes horizontally. The 14 kbp ToxhAT transposon has been shown to be moving the necrotrophic effector, ToxA, horizontally between fungal species that infect Triticum aestivum (wheat), namely Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana. All three species utilise the ToxA protein to infect wheat. Previous genomic evidence found ToxhAT in distinct chromosomal positions in two isolates of B. sorokiniana, indicating that the transposon is still active in this species. Here we confirm the movement of ToxhAT using long-read Nanopore MinION sequencing of eight novel and one previously published B. sorokiniana isolates. One event of independent transposition of ToxhAT was observed, and target site duplications of "TA" were identified, confirming this was an autonomous movement facilitated by a yet unidentified transposase. Whole genome analysis revealed that ToxhAT is a passenger embedded in a much larger, conserved 170-196 kbp mobile genetic element. This element, termed Sanctuary, belongs to the newly described Starship transposon superfamily. This classification is based on the presence of short direct repeats, empty insertion sites, a putative tyrosine recombinase gene and other features of Starship transposons. We also show that ToxhAT has been independently captured by two different Starships, Sanctuary and Horizon which share little to no sequenced identity, outside of ToxhAT. This classification makes Horizon and Sanctuary part of a growing number of Starships involved in the horizontal gene transfer of adaptive genetic material between fungal species. ImportanceThe work presented here expands our understanding of a novel group of mobile genetic elements called Starships that facilitate the horizontal exchange of virulence genes in fungal pathogens. Our analysis shows that Sanctuary and ToxhAT are likely active and autonomous transposons in the B. sorokiniana genome. We also show that the smaller ToxhAT transposon has been independently captured by two different Starships, viz. Sanctuary in B. sorokiniana and Horizon in P. tritici-repentis and P. nodorum. Outside of ToxhAT these two Starships share no sequence identity. The capture of ToxhAT by two different mobile elements in three different fungal wheat pathogens demonstrates how horizontal transposon transfer is driving the evolution of virulence in these important wheat pathogens.

genomics↗

bioRxiv2023

Heterozygous missense variant in GLI2 impairs human endocrine pancreas development

Missense variants are the most common type of coding genetic variants. Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development. Here, we applied CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in a paediatric hyperglycaemic patient and family members. GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic {beta}-cell development in mice. However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes. A set of isogenic iPSC lines harbouring the missense variant were studied for their ability to differentiate into pancreatic {beta}-like cells. Interestingly, iPSCs carrying the missense variant showed altered GLI2 transcriptional activity and impaired endocrine differentiation. Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a novel variant that predisposes to diabetes.

developmental biology↗

bioRxiv2022

Phase-separating pyrenoid proteins form complexes in the dilute phase

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play a significant role in the stability, dynamics, and regulation of condensates.

biophysics↗

bioRxiv2022

Single-nucleotide Differences and Cell Type Decide the Subcellular Localization of miRNA Isoforms (isomiRs), tRNA-derived Fragments (tRFs) and rRNA-derived Fragments (rRFs)

BackgroundMicroRNAs (miRNAs) and their isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent ~95% of all short RNAs found in cells. All three types modulate mRNA and protein abundance and are dysregulated in diseases. Experimental studies to date assumed that the subcellular localization of these molecules is well understood and constant across cell types. ResultsWe investigated the localization of isomiRs, tRFs, and rRFs in biological replicates from three frequently-used model cell lines. In each case, we analyzed the contents of the nucleus, cytoplasm, whole mitochondrion, mitoplast, and the whole cell. We used a rigorous mathematical model to account for cross-fraction contamination and technical errors and adjusted abundances accordingly. We found that isomiRs, tRFs, and rRFs exhibit complex and unexpected patterns of subcellular localization. These patterns depend on the type of the RNA molecule, its exact sequence, and the cell type. Even for "sibling" RNAs from the same parental RNA whose sequences differ by only a few nucleotides, their subcellular localization depends on each siblings exact sequence and the cell type. ConclusionsPrevious studies of isomiRs, tRFs, and rRFs that used ectopic expression without accounting for isoforms may need to be re-evaluated. Future experiments with these molecules will need to distinguish among the multiple isoforms and account for the fact that each isoforms abundance and destination depend on its exact sequence and cell type. The findings additionally suggest the existence of an intracellular trafficking program for isomiRs, tRFs, and rRFs and, by extension, expanded roles for these molecules - both dimensions await characterization. To help design future experiments, we compiled a first-of-its-kind Atlas to catalogue the subcellular localization and abundance of 5,898 isomiRs, tRFs, and rRFs across three model cell lines. Results SummaryO_LIWe analyzed the distribution of microRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) in the{whitebullet} nucleus {whitebullet}cytoplasm {whitebullet}mitochondrion, and {whitebullet}mitoplast of biological replicates from three cell lines from the same tissue. C_LIO_LIWe corrected the measured abundances by accounting for cross-fraction contamination and technical errors through a rigorous mathematical model. C_LIO_LIOur analysis revealed complex localization patterns involving numerous isomiRs, tRFs, and rRFs. C_LIO_LIThe subcellular localization of these RNAs depends on their exact sequence and differs even for molecules that arise from the same parental miRNA, tRNA, or rRNA. C_LIO_LIFor a given RNA, its subcellular localization additionally depends on cell type. C_LIO_LIThe findings have implications for previous and future molecular studies of the function of isomiRs, tRFs, and rRFs. C_LIO_LIThe findings suggest the existence of a complex subcellular trafficking program, and hint at expanded functions for these RNA molecules that differ by compartment. C_LIO_LITo assist with the design of future experiments, we created a first-of-its-kind Atlas that catalogues the subcellular distribution and abundance of 5,898 isomiRs, tRFs, and rRFs across three cell lines. C_LI

molecular biology↗

bioRxiv2022

The P681H mutation in the Spike glycoprotein escapes IFITM restriction and is necessary for type I interferon resistance in the SARS-CoV-2 alpha variant

The appearance of new dominant variants of concern (VOCs) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) threatens the global response to the COVID-19 pandemic. Of these, the alpha variant (also known as B.1.1.7) that appeared initially in the UK became the dominant variant in much of Europe and North America in the first half of 2021. The Spike (S) glycoprotein of alpha acquired seven mutations and two deletions compared to the ancestral virus, including the P681H mutation in the polybasic cleavage site that has been suggested to enhance S cleavage. Here, we show that the alpha S protein confers a level of resistance to the effects of interferon-{beta} (IFN{beta}) in human lung epithelial cells. This correlates with resistance to an entry restriction mediated by interferon-induced transmembrane protein 2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is essential for resistance to IFN{beta} and context-dependent resistance to IFITMs in the alpha S. However, while this appears to confer changes in sensitivity to endosomal protease inhibition consistent with enhanced cell-surface entry, its reversion does not reduce cleaved S incorporation into particles, indicating a role downstream of furin cleavage. Overall, we suggest that, in addition to adaptive immune escape, mutations associated with VOCs may well also confer replication and/or transmission advantage through adaptation to resist innate immune mechanisms. IMPORTANCEThe emergence of Variants of Concern of SARS-CoV-2 has been a key challenge in the global response to the COVID-19 pandemic. Accumulating evidence suggests VOCs are being selected to evade the human immune response, with much interest focussed on mutations in the Spike protein that escape from neutralizing antibody responses. However, resistance to the innate immune response is essential for efficient viral replication and transmission. Here we show that the alpha (B.1.1.7) VOC of SARS-CoV-2 is substantially more resistant to type-1 interferons than the parental Wuhan-like virus. This correlates with resistance to the antiviral protein IFITM2, and enhancement by its paralogue IFITM3, that block virus entry into target cells. The key determinant of this is a proline to histidine change at position 681 in S adjacent to the furin-cleavage site that we have shown previously modulates IFITM2 sensitivity. Unlike other VOCs, in the context of the alpha spike, P681H modulates cell entry pathways of SARS-CoV-2, further reducing its dependence one endosomal proteases. Reversion of position 681 to a proline in viruses bearing the alpha spike is sufficient to restore interferon and IFITM2 sensitivity without reducing furin-mediated spike cleavage, suggesting post cleavage conformational changes in S are changing the viral entry pathway and therefore sensitivity to interferon. These data highlight the dynamic nature of the SARS CoV-2 S as it adapts to both innate and adaptive immunity in the human population.

microbiology↗