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The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific facilities. Pre-prints by Institute authors can be viewed on the Institute's bioRxiv channel. We believe that free and open access to the outputs of publicly‐funded research offers significant social and economic benefits, as well as aiding the development of new research. We are working to provide Open Access to as many publications as possible and these can be identified below by the padlock icon. Where this hasn't been possible, subscriptions may be required to view the full text.
 

Olsen KW, Castillo-Fernandez J, Zedeler A, Freiesleben NC, Bungum M, Chan AC, Cardona A, Perry JRB, Skouby SO, Borup R, Hoffmann ER, Kelsey G, Grøndahl ML Epigenetics

Does women's age affect the DNA methylation (DNAm) profile differently in mural granulosa cells (MGCs) from other somatic cells?

+view abstract Human reproduction , PMID: 32474592

Velanis CN, Perera P, Thomson B, de Leau E, Liang SC, Hartwig B, Förderer A, Thornton H, Arede P, Chen J, Webb KM, Gümüs S, De Jaeger G, Page CA, Hancock CN, Spanos C, Rappsilber J, Voigt P, Turck F, Wellmer F, Goodrich J Epigenetics

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

+view abstract PLoS genetics , PMID: 32463832

Senner CE, Chrysanthou S, Burge S, Lin HY, Branco MR, Hemberger M

The ten-eleven translocation factor TET1 and its conferred epigenetic modification 5-hydroxymethylcytosine (5hmC) have important roles in maintaining the pluripotent state of embryonic stem cells (ESCs). We previously showed that TET1 is also essential to maintain the stem cell state of trophoblast stem cells (TSCs). Here, we establish an integrated panel of absolute 5hmC levels, genome-wide DNA methylation and hydroxymethylation patterns, transcriptomes, and TET1 chromatin occupancy in TSCs and differentiated trophoblast cells. We show that the combined presence of 5-methylcytosine (5mC) and 5hmC correlates with transcriptional activity of associated genes. Hypoxia can slow down the global loss of 5hmC that occurs upon differentiation of TSCs. Notably, unlike in ESCs and epiblast cells, most TET1-bound regions overlap with active chromatin marks and TFAP2C binding sites and demarcate putative trophoblast enhancer regions. These chromatin modification and occupancy patterns are highly informative to identify novel candidate regulators of the TSC state.

+view abstract Stem cell reports , PMID: 32442533

Kwok A, Zvetkova I, Virtue S, Luijten I, Huang-Doran I, Tomlinson P, Bulger DA, West J, Murfitt S, Griffin J, Alam R, Hart D, Knox R, Voshol P, Vidal-Puig A, Jensen J, O'Rahilly S, Semple RK Immunology

Insulin signaling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, including lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidemia. We sought to investigate this discordance.

+view abstract Molecular metabolism , PMID: 32439336

Marshall JD, Courage ER, Elliott RF, Fitzpatrick MN, Kim AD, Lopez-Clavijo AF, Woolfrey BA, Ouimet M, Wakelam MJO, Brown RJ Signalling , Lipidomics

Lipoprotein lipase (LPL) is upregulated in atherosclerotic lesions and it may promote the progression of atherosclerosis, but the mechanisms behind this process are not completely understood. We previously showed that the phosphorylation of Akt within THP-1 macrophages is increased in response to the lipid hydrolysis products generated by LPL from total lipoproteins. Notably, the free fatty acid (FFA) component was responsible for this effect. In the present study, we aimed to reveal more detail as to how the FFA component may affect Akt signalling. We show that the phosphorylation of Akt within THP-1 macrophages increases with total FFA concentration and that phosphorylation is elevated up to 18 hours. We further show that specifically the palmitoleate component of the total FFA affects Akt phosphorylation. This is tied with changes to the levels of select molecular species of phosphoinositides. We further show that the total FFA component, and specifically palmitoleate, reduces apolipoprotein A-I-mediated cholesterol efflux, and that the reduction can be reversed in the presence of the Akt inhibitor MK-2206. Overall, our data support a negative role for the FFA component of lipoprotein hydrolysis products generated by LPL, by impairing macrophage cholesterol efflux via Akt activation.

+view abstract PloS one , PMID: 32437392

Pikor NB, Mörbe U, Lütge M, Gil-Cruz C, Perez-Shibayama C, Novkovic M, Cheng HW, Nombela-Arrieta C, Nagasawa T, Linterman MA, Onder L, Ludewig B Immunology

Efficient generation of germinal center (GC) responses requires directed movement of B cells between distinct microenvironments underpinned by specialized B cell-interacting reticular cells (BRCs). How BRCs are reprogrammed to cater to the developing GC remains unclear, and studying this process is largely hindered by incomplete resolution of the cellular composition of the B cell follicle. Here we used genetic targeting of Cxcl13-expressing cells to define the molecular identity of the BRC landscape. Single-cell transcriptomic analysis revealed that BRC subset specification was predetermined in the primary B cell follicle. Further topological remodeling of light and dark zone follicular dendritic cells required CXCL12-dependent crosstalk with B cells and dictated GC output by retaining B cells in the follicle and steering their interaction with follicular helper T cells. Together, our results reveal that poised BRC-defined microenvironments establish a feed-forward system that determines the efficacy of the GC reaction.

+view abstract Nature immunology , PMID: 32424359

Ma CY, Marioni JC, Griffiths GM, Richard AC Immunology

Millions of naïve T cells with different TCRs may interact with a peptide-MHC ligand, but very few will activate. Remarkably, this fine control is orchestrated using a limited set of intracellular machinery. It remains unclear whether changes in stimulation strength alter the programme of signalling events leading to T cell activation. Using mass cytometry to simultaneously measure multiple signalling pathways during activation of murine CD8 T cells, we found a programme of distal signalling events that is shared, regardless of the strength of TCR stimulation. Moreover, the relationship between transcription of early response genes and and activation of the ribosomal protein S6 is also conserved across stimuli. Instead, we found that stimulation strength dictates the rate with which cells initiate signalling through this network. These data suggest that TCR-induced signalling results in a coordinated activation program, modulated in rate but not organization by stimulation strength.

+view abstract eLife , PMID: 32412411

Quinn KM, Linterman MA Immunology

In Covre et al. and Pereira et al., the authors demonstrate the parallels between senescent NK cells and senescent CD8 T cells, and formalise the mechanism by which senescent CD8 T cells become more NK cell-like, through the action of sestrins.

+view abstract Immunology and cell biology , PMID: 32406096

Meckelmann SW, Hawksworth JI, White D, Andrews R, Rodrigues P, O'Connor A, Alvarez-Jarreta J, Tyrrell VJ, Hinz C, Zhou Y, Williams J, Aldrovandi M, Watkins WJ, Engler AJ, Lo Sardo V, Slatter DA, Allen SM, Acharya J, Mitchell J, Cooper J, Aoki J, Kano K, Humphries SE, O'Donnell VB Signalling

Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA.

+view abstract Circulation. Genomic and precision medicine , PMID: 32396387

Borg M, Jacob Y, Susaki D, LeBlanc C, Buendía D, Axelsson E, Kawashima T, Voigt P, Boavida L, Becker J, Higashiyama T, Martienssen R, Berger F Epigenetics

Epigenetic marks are reprogrammed in the gametes to reset genomic potential in the next generation. In mammals, paternal chromatin is extensively reprogrammed through the global erasure of DNA methylation and the exchange of histones with protamines. Precisely how the paternal epigenome is reprogrammed in flowering plants has remained unclear since DNA is not demethylated and histones are retained in sperm. Here, we describe a multi-layered mechanism by which H3K27me3 is globally lost from histone-based sperm chromatin in Arabidopsis. This mechanism involves the silencing of H3K27me3 writers, activity of H3K27me3 erasers and deposition of a sperm-specific histone, H3.10 (ref. ), which we show is immune to lysine 27 methylation. The loss of H3K27me3 facilitates the transcription of genes essential for spermatogenesis and pre-configures sperm with a chromatin state that forecasts gene expression in the next generation. Thus, plants have evolved a specific mechanism to simultaneously differentiate male gametes and reprogram the paternal epigenome.

+view abstract Nature cell biology , PMID: 32393884

Ivanova E, Canovas S, Garcia-Martínez S, Romar R, Lopes JS, Rizos D, Sanchez-Calabuig MJ, Krueger F, Andrews S, Perez-Sanz F, Kelsey G, Coy P Epigenetics , Bioinformatics

Preimplantation embryos experience profound resetting of epigenetic information inherited from the gametes. Genome-wide analysis at single-base resolution has shown similarities but also species differences between human and mouse preimplantation embryos in DNA methylation patterns and reprogramming. Here, we have extended such analysis to two key livestock species, the pig and the cow. We generated genome-wide DNA methylation and whole-transcriptome datasets from gametes to blastocysts in both species. In oocytes from both species, a distinctive bimodal methylation landscape is present, with hypermethylated domains prevalent over hypomethylated domains, similar to human, while in the mouse the proportions are reversed.An oocyte-like pattern of methylation persists in the cleavage stages, albeit with some reduction in methylation level, persisting to blastocysts in cow, while pig blastocysts have a highly hypomethylated landscape. In the pig, there was evidence of transient de novo methylation at the 8-16 cell stages of domains unmethylated in oocytes, revealing a complex dynamic of methylation reprogramming. The methylation datasets were used to identify germline differentially methylated regions (gDMRs) of known imprinted genes and for the basis of detection of novel imprinted loci. Strikingly in the pig, we detected a consistent reduction in gDMR methylation at the 8-16 cell stages, followed by recovery to the blastocyst stage, suggesting an active period of imprint stabilization in preimplantation embryos. Transcriptome analysis revealed absence of expression in oocytes of both species of ZFP57, a key factor in the mouse for gDMR methylation maintenance, but presence of the alternative imprint regulator ZNF445. In conclusion, our study reveals species differences in DNA methylation reprogramming and suggests that porcine or bovine models may be closer to human in key aspects than in the mouse model.

+view abstract Clinical epigenetics , PMID: 32393379

Stammers M, Ivanova IM, Niewczas IS, Segonds-Pichon A, Streeter M, Spiegel DA, Clark J Biological Chemistry

Collagen is a structural protein whose internal cross-linking critically determines the properties and functions of connective tissue. Knowing how the cross-linking of collagen changes with age is key to understanding why the mechanical properties of tissues change over a lifetime. The current scientific consensus is that collagen cross-linking increases with age and that this increase leads to tendon stiffening. Here, we show that this view should be reconsidered. Using MS-based analyses, we demonstrate that during aging of healthy C57BL/6 mice, the overall levels of collagen cross-linking in tail tendon decrease with age. However, the levels of lysine glycation in collagen, which is not considered a cross-link, increased dramatically with age. We found that in 16-week-old diabetic db/db mice, glycation reaches levels similar to those observed in 98-week-old C57BL/6 mice, while the other cross-links typical of tendon collagen either decreased or remained the same as those observed in 20 week old WT mice. These results, combined with findings from mechanical testing of tendons from these mice, indicate that overall collagen cross-linking in mouse tendon decreases with age. Our findings also reveal that lysine glycation appears to be an important factor that contributes to tendon stiffening with age and in diabetes.

+view abstract The Journal of biological chemistry , PMID: 32381510

Wołodko K, Walewska E, Adamowski M, Castillo-Fernandez J, Kelsey G, Galvão A Epigenetics

Obesity is associated with infertility, decreased ovarian performance and lipotoxicity. However, little is known about the aetiology of these reproductive impairments. Here, we hypothesise that the majority of changes in ovarian physiology in diet-induced obesity (DIO) are a consequence of transcriptional changes downstream of altered leptin signalling. Therefore, we investigated the extent to which leptin signalling is altered in the ovary upon obesity with particular emphasis on effects on cumulus cells (CCs), the intimate functional companions of the oocyte. Furthermore, we used the pharmacological hyperleptinemic (LEPT) mouse model to compare transcriptional profiles to DIO.

+view abstract Cellular physiology and biochemistry , PMID: 32348667

Liston A, Whyte CE Immunology

The microbiome is increasingly recognized for its ability to modulate human health. Colonization with gut symbionts induces Foxp3‐expressing regulatory T cells (Tregs) and expands their local numbers, a critical step in the suppression of intestinal inflammation and maintaining gut homeostasis. The molecular mechanism by which the microbiome interacts with peripherally induced Treg (pTreg) is likely complex and multifactorial; however, part of the effect is mediated via the release of microbial fermentation products, such as butyrate and other short‐chain fatty acids.

+view abstract Immunology and cell biology , PMID: 32329090

Silva-Cayetano A, Linterman MA Immunology

The germinal centre (GC) is a specialized cellular structure that forms in response to antigenic stimulation. It generates long-term humoral immunity through the production of memory B cells and long-lived antibody-secreting plasma cells. Conventional GCs form within secondary lymphoid organs, where networks of specialised stromal cells that form during embryogenesis act as the stage upon which the various GC immune cell players are brought together, nurtured and co-ordinated to generate a productive response. In non-lymphoid organs, ectopic GCs can form in response to persistent antigenic and inflammatory stimuli. Unlike secondary lymphoid tissues, non-lymphoid organs do not have a developmentally programmed stromal cell network capable of supporting the germinal centre reaction; therefore, the local tissue stroma must be remodelled by inflammatory stimuli in order to host a GC reaction. These ectopic GCs produce memory B cells and plasma cells that form a critical component of the humoral immune response.

+view abstract Current opinion in immunology , PMID: 32325390

Van Nieuwenhove E, Barber JS, Neumann J, Smeets E, Willemsen M, Pasciuto E, Prezzemolo T, Lagou V, Seldeslachts L, Malengier-Devlies B, Metzemaekers M, Haßdenteufel S, Kerstens A, van der Kant R, Rousseau F, Schymkowitz J, Di Marino D, Lang S, Zimmermann R, Schlenner S, Munck S, Proost P, Matthys P, Devalck C, Boeckx N, Claessens F, Wouters C, Humblet-Baron S, Meyts I, Liston A Immunology

The molecular cause of severe congenital neutropenia (SCN) is unknown in 30-50% of patients. SEC61A1 encodes the α subunit of the SEC61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease.

+view abstract The Journal of allergy and clinical immunology , PMID: 32325141

Hanna CW Epigenetics

As the maternal-foetal interface, the placenta is essential for the establishment and progression of healthy pregnancy, regulating both foetal growth and maternal adaptation to pregnancy. The evolution and functional importance of genomic imprinting are inextricably linked to mammalian placentation. Recent technological advances in mapping and manipulating the epigenome in embryogenesis in mouse models have revealed novel mechanisms regulating genomic imprinting in placental trophoblast, the physiological implications of which are only just beginning to be explored. This review will highlight important recent discoveries and exciting new directions in the study of placental imprinting.

+view abstract PLoS genetics , PMID: 32324732

Dalle Pezze P, Karanasios E, Kandia V, Manifava M, Walker SA, Gambardella Le Novère N, Ktistakis NT Signalling , Imaging

During macroautophagy/autophagy, the ULK complex nucleates autophagic precursors, which give rise to autophagosomes. We analyzed, by live imaging and mathematical modeling, the translocation of ATG13 (part of the ULK complex) to the autophagic puncta in starvation-induced autophagy and ivermectin-induced mitophagy. In nonselective autophagy, the intensity and duration of ATG13 translocation approximated a normal distribution, whereas wortmannin reduced this effect and shifted to a log-normal distribution. During mitophagy, multiple translocations of ATG13 with increasing time between peaks were observed. We hypothesized that these multiple translocations arise because the engulfment of mitochondrial fragments required successive nucleation of phagophores on the same target, and a mathematical model based on this idea reproduced the oscillatory behavior. Significantly, model and experimental data were also in agreement that the number of ATG13 translocations is directly proportional to the diameter of the targeted mitochondrial fragments. Thus, our data provide novel insights into the early dynamics of selective and nonselective autophagy. ATG: autophagy related 13; CFP: cyan fluorescent protein; dsRED: red fluorescent protein; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IVM: ivermectin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: PtdIns-3-phosphate; ULK: unc-51 like autophagy activating kinase.

+view abstract Autophagy , PMID: 32320309

Wojdyla K, Collier AJ, Fabian C, Nisi PS, Biggins L, Oxley D, Rugg-Gunn PJ Epigenetics , Mass Spectrometry

Naive and primed human pluripotent stem cells (hPSC) provide valuable models to study cellular and molecular developmental processes. The lack of detailed information about cell-surface protein expression in these two pluripotent cell types prevents an understanding of how the cells communicate and interact with their microenvironments. Here, we used plasma membrane profiling to directly measure cell-surface protein expression in naive and primed hPSC. This unbiased approach quantified over 1,700 plasma membrane proteins, including those involved in cell adhesion, signaling, and cell interactions. Notably, multiple cytokine receptors upstream of JAK-STAT signaling were more abundant in naive hPSC. In addition, functional experiments showed that FOLR1 and SUSD2 proteins are highly expressed at the cell surface in naive hPSC but are not required to establish human naive pluripotency. This study provides a comprehensive stem cell proteomic resource that uncovers differences in signaling pathway activity and has identified new markers to define human pluripotent states.

+view abstract Stem cell reports , PMID: 32302559

Hull RM, Houseley J Epigenetics

Carefully maintained and precisely inherited chromosomal DNA provides long-term genetic stability, but eukaryotic cells facing environmental challenges can benefit from the accumulation of less stable DNA species. Circular DNA molecules lacking centromeres segregate randomly or asymmetrically during cell division, following non-Mendelian inheritance patterns that result in high copy number instability and massive heterogeneity across populations. Such circular DNA species, variously known as extrachromosomal circular DNA (eccDNA), microDNA, double minutes or extrachromosomal DNA (ecDNA), are becoming recognised as a major source of the genetic variation exploited by cancer cells and pathogenic eukaryotes to acquire drug resistance. In budding yeast, circular DNA molecules derived from the ribosomal DNA (ERCs) have been long known to accumulate with age, but it is now clear that aged yeast also accumulate other high-copy protein-coding circular DNAs acquired through both random and environmentally-stimulated recombination processes. Here, we argue that accumulation of circular DNA provides a reservoir of heterogeneous genetic material that can allow rapid adaptation of aged cells to environmental insults, but avoids the negative fitness impacts on normal growth of unsolicited gene amplification in the young population.

+view abstract Current genetics , PMID: 32296868

Dembny P, Newman AG, Singh M, Hinz M, Szczepek M, Krüger C, Adalbert R, Dzaye O, Trimbuch T, Wallach T, Kleinau G, Derkow K, Richard BC, Schipke C, Scheidereit C, Stachelscheid H, Golenbock D, Peters O, Coleman M, Heppner FL, Scheerer P, Tarabykin V, Ruprecht K, Izsvák Z, Mayer J, Lehnardt S

Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs, such as HERV-K(HML-2), are reported to be involved in neurological disorders, little is known about their biological function. We report that RNA from an HERV-K(HML-2) envelope gene region binds to and activates human Toll-like receptor (TLR) 8, as well as murine Tlr7, expressed in neurons and microglia, thereby causing neurodegeneration. HERV-K(HML-2) RNA introduced into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimer's disease (AD), resulted in neurodegeneration and microglia accumulation. Tlr7-deficient mice were protected against neurodegenerative effects but were resensitized toward HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Transcriptome data sets of human AD brain samples revealed a distinct correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from individuals with AD compared with controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for species-specific TLRs 7/8 and imply a functional contribution of human endogenous retroviral transcripts to neurodegenerative processes, such as AD.

+view abstract JCI insight , PMID: 32271161

Zachari M, Ktistakis NT Signalling

Mitophagy, a conserved intracellular process by which mitochondria are eliminated via the autophagic machinery, is a quality control mechanism which facilitates maintenance of a functional mitochondrial network and cell homeostasis, making it a key process in development and longevity. Mitophagy has been linked to multiple human disorders, especially neurodegenerative diseases where the long-lived neurons are relying on clearance of old/damaged mitochondria to survive. During the past decade, the availability of novel tools to study mitophagy both and has significantly advanced our understanding of the molecular mechanisms governing this fundamental process in normal physiology and in various disease models. We here give an overview of the known mitophagy pathways and how they are induced, with a particular emphasis on the early events governing mitophagosome formation.

+view abstract Frontiers in cell and developmental biology , PMID: 32258042

Thompson O, von Meyenn F, Hewitt Z, Alexander J, Wood A, Weightman R, Gregory S, Krueger F, Andrews S, Barbaric I, Gokhale PJ, Moore HD, Reik W, Milo M, Nik-Zainal S, Yusa K, Andrews PW Epigenetics , Bioinformatics

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.

+view abstract Nature communications , PMID: 32251294

Ludwig-Begall LF, Lu J, Hosmillo M, de Oliveira-Filho EF, Mathijs E, Goodfellow I, Mauroy A, Thiry E Immunology

Noroviruses are recognized as the major cause of non-bacterial gastroenteritis in humans. Molecular mechanisms driving norovirus evolution are the accumulation of point mutations and recombination. Recombination can create considerable changes in a viral genome, potentially eliciting a fitness cost, which must be compensated via the adaptive capacity of a recombinant virus. We previously described replicative fitness reduction of the first generated WU20-CW1 recombinant murine norovirus, RecMNV. In this follow-up study, RecMNV's capability of replicative fitness recuperation and genetic characteristics of RecMNV progenies at early and late stages of an adaptation experiment were evaluated. Replicative fitness regain of the recombinant was demonstrated via growth kinetics and plaque size differences between viral progenies prior to and post serial passaging. Point mutations at consensus and sub-consensus population levels of early and late viral progenies were characterized via next-generation sequencing and putatively associated to fitness changes. To investigate the effect of genomic changes separately and in combination in the context of a lab-generated inter-MNV infectious virus, mutations were introduced into a recombinant WU20-CW1 cDNA for subsequent DNA-based reverse genetics recovery. We thus associated fitness loss of RecMNV to a C7245T mutation and functional VP2 (ORF3) truncation and demonstrated individual and cumulative compensatory effects of one synonymous OFR2 and two non-synonymous ORF1 consensus-level mutations acquired during successive rounds of replication. Our data provide evidence of viral adaptation in a controlled environment via genetic drift after genetic shift induced a fitness cost of an infectious recombinant norovirus.

+view abstract The Journal of general virology , PMID: 32242791