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Journal articles

• Graves LP, Hughes LC, Irons SL, Possee RD, King LA, 'In cultured cells the baculovirus P10 protein forms two independent intracellular structures that play separate roles in occlusion body maturation and their release by nuclear disintegration'
  PLoS Pathogens (2019)
  ISSN: 1553-7366 eISSN: 1533-7374
  Abstract

  P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis.
  

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• Al-Mayah A, Bright S, Chapman K, Irons S, Luo P, Carter D, Goodwin E, Kadhim M., 'The non-targeted effects of radiation are perpetuated by exosomes'
  Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 772 (2014) pp.38-45
  ISSN: 0027-5107
  Abstract Exosomes contain cargo material from endosomes, cytosol, plasma membrane and microRNA molecules, they are released by a number of non-cancer and cancer cells into both the extracellular microenvironment and body fluids such as blood plasma.
   
  Recently we demonstrated radiation-induced non-targeted effects [NTE: genomic instability (GI) and bystander effects (BE)] are partially mediated by exosomes, particularly the RNA content. However the mechanistic role of exosomes in NTE is yet to be fully understood.
   
  The present study used MCF7 cells to characterise the longevity of exosome-induced activity in the progeny of irradiated and unirradiated bystander cells. Exosomes extracted from conditioned media of irradiated and bystander progeny were added to unirradiated cells. Analysis was carried out at 1 and 20/24 population doublings following medium/exosome transfer for DNA/chromosomal damage. Results confirmed exosomes play a significant role in mediating NTE of ionising radiation (IR). This effect was remarkably persistent, observed >20 doublings post-irradiation in the progeny of bystander cells. Additionally, cell progeny undergoing a BE were themselves capable of inducing BE in other cells via exosomes they released. Furthermore we investigated the role of exosome cargo. Culture media from cells exposed to 2 Gy X-rays was subjected to ultracentrifugation and four inoculants prepared, (a) supernatants with exosomes removed, and pellets with (b) exosome proteins denatured, (c) RNA degraded, and (d) a combination of protein–RNA inactivation. These were added to separate populations of unirradiated cells. The BE was partially inhibited when either exosome protein or exosome RNA were inactivated separately, whilst combined RNA–protein inhibition significantly reduced or eliminated the BE. These results demonstrate that exosomes are associated with long-lived signalling of the NTE of IR. Both RNA and protein molecules of exosomes work in a synergistic manner to initiate NTE, spread these effects to naïve cells, and perpetuate GI in the affected cells.
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• Beperet I, Irons SL, Simón O, King LA, Williams T, Possee RD, López-Ferber M, Caballero P, 'Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization'
  Journal of Virology 8 (6) (2014) pp.3548-3556
  ISSN: 0022-538X
  Abstract Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses—AcMNPV and SfMNPV—but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses.
   
  IMPORTANCE Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.
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• Al-Mayah AH, Irons SL, Pink RC, Carter DR, Kadhim MA, 'Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation'
  Radiation Research 177 (5) (2012) pp.539-545
  ISSN: 0033-7587 eISSN: 1938-5404
  Abstract

  Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation, a process known as the bystander effect. Bystander effects can also lead to chromosomal/genomic instability within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The factors that mediate this cellular communication can be transferred between cells via gap junctions or released into the extracellular media following irradiation, but their nature has not been fully characterized. In this study we tested the hypothesis that the bystander effect mediator contains an RNA molecule that may be carried by exosomes. MCF7 cells were irradiated with 2 Gy of X rays and the extracellular media was harvested. RNase treatment abrogated the ability of the media to induce early and late chromosomal damage in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the bystander effect, and genomic instability, are at least in part mediated by exosomes and implicate a role for RNA.

  Website 
• Irons SL, Serra V, Bowler D, Chapman K, Militi S, Lyng F, Kadhim M, 'The Effect of Genetic Background and Dose on Non-targeted Effects of Radiation'
  International Journal of Radiation Biology 88 (2012) pp.735-742
  ISSN: 0955-3002 eISSN: 1362-3095
  Abstract

  PURPOSE:

  This work investigates the hypothesis that genetic background plays a significant role in the signalling mechanisms underlying induction and perpetuation of genomic instability following radiation exposure.

  MATERIALS AND METHODS:

  Bone marrow from two strains of mice (CBA and C57) were exposed to a range of X-ray doses (0, 0.01, 0.1, 1 and 3 Gy). Different cellular signalling endpoints: Apoptosis, cytokine levels and calcium flux, were evaluated at 2 h, 24 h and 7 d post-irradiation to assess immediate and delayed effects.

  RESULTS:

  In CBA (radiosensitive) elevated apoptosis levels were observed at 24 h post X-irradiation, and transforming growth factor-β (TGF-β) levels which increased with time and dose. C57 showed a higher background level of apoptosis, and sustained apoptotic levels 7 days after radiation exposure. Levels of tumor necrosis factor-α (TNF-α were increased in C57 at day 7 for higher X-ray doses. TGF-β levels were higher in CBA, whilst C57 exhibited a greater TNF-α response. Calcium flux was induced in reporter cells on exposure to conditioned media from both strains.

  CONCLUSIONS:

  These results show genetic and dose specific differences in radiation-induced signalling in the initiation and perpetuation of the instability process, which have potential implications on evaluation of non-targeted effects in radiation risk assessment.

  Website 
• Bak A, Irons SL, Martiniere A, Blanc S, Drucker M, 'Host Cell Processes to Accomplish Mechanical and Non-circulative Virus Transmission'
  Protoplasma 249 (2012) pp.529-539
  ISSN: 0033-183X eISSN: 1615-6102
  Abstract

  Mechanical vector-less transmission of viruses, as well as vector-mediated non-circulative virus transmission, where the virus attaches only to the exterior of the vector during the passage to a new host, are apparently simple processes: the viruses are carried along with the wind, the food or by the vector to a new host. We discuss here, using the examples of the non-circulatively transmittedCauliflower mosaic virus that binds to its aphid vector's exterior mouthparts, and that of the mechanically (during feeding activity) transmitted Autographa californica multicapsid nucleopolyhedrovirus, that transmission of these viruses is not so simple as previously thought. Rather, these viruses prepare their transmission carefully and long before the actual acquisition event. Host–virus interactions play a pivotal and specialised role in the future encounter with the vector or the new host. This ensures optimal propagation and enlarges the tremendous bottleneck transmission presents for viruses and other pathogens.

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• Chapman KL, Al-Mayah AH, Bowler DA, Irons SL, Kadhim MA, 'No influence of serotonin levels in foetal bovine sera on radiation induced bystander effects and genomic instability'
  International Journal of Radiation Biology 88 (10) (2012) pp.781-785
  ISSN: 0955-3002 eISSN: 1362-3095
  Website 
• Barbi T, Irons SL, Pepponi I, Hawes C, Ma JKC, Drake PMW, 'Expression and plasma membrane localization of the mammalian B-cell receptor complex in transgenic Nicotiana tabacum'
  Plant Biotechnology Journal 9 (4) (2011) pp.455-465
  ISSN: 1467-7644
  Abstract

  P>The B-cell antigen receptor (BCR), displayed on the plasma membrane of mature B cells of the mammalian immune system, is a multimeric complex consisting of a membrane-bound immunoglobulin (mIg) noncovalently associated with the Ig alpha/Ig beta heterodimer. In this study, we engineered transgenic tobacco plants expressing all four chains of the BCR. ELISA, Western blotting and confocal microscopy demonstrated that the BCR was correctly assembled in plants, predominantly in the plasma membrane, and that the noncovalent link was detergent sensitive. This is the first example of a noncovalently assembled plasma membrane-retained heterologous receptor in plants. In B cells of the mammalian immune system, following antigen binding to mIg, BCR is internalized and tyrosine residues on Ig alpha and Ig beta are phosphorylated activating a signaling cascade through interaction with protein kinases that ultimately leads to the initiation of gene expression. Expression of the BCR may therefore be an important tool for the study of plant endocytosis and the identification of previously unknown plant tyrosine kinases. The specificity and diversity of the antibody repertoire, coupled to the signal transduction capability of the Ig alpha/Ig beta heterodimer, also indicates that plants expressing BCR may in future be developed as environmental biosensors.

  Website 
• Barbante A, Irons SL, Hawes C, Frigerio L, Vitale A, Pedrazzini E, 'Anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plants'
  Plant Biotechnology Journal 6 (6) (2008) pp.560-575
  ISSN: 1467-7644
  Abstract

  The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.

  Website 
• Irons SL, Nuttall J, Floss DM, Frigerio L, Kotzer AM, Hawes C, 'Fluorescent protein fusions to a human immunodeficiency virus monoclonal antibody reveal its intracellular transport through the plant endomembrane system'
  Plant Biotechnology Journal 6 (7) (2008) pp.649-662
  ISSN: 1467-7644
  Abstract

  In order to further understand the production and intracellular trafficking of pharmaceutical proteins in plants, the light and heavy chains (LC and HC) of the human immunodeficiency virus neutralizing monoclonal antibody 2G12 were fused to fluorescent proteins [Venus and monomeric red fluorescent protein (mRFP)] to enable the visualization of their passage through the plant cell. Co- expression of LC and HC with various markers of the endomembrane system demonstrated that LC fusions were found in mobile punctate structures, which are likely to be pre-vacuolar compartments (PVCs) as a proportion of the LC fusions were found to be located in the vacuole. In addition, apoplast labelling was also observed with a 2G12LC-RFP fusion. The HC fusion expressed alone was found only in the endoplasmic reticulum (ER). When the LC and HC fusions were expressed together, they were found to co-locate to larger punctate structures, which were morphologically distinct from any observed on expression of LC or HC alone. These structures appeared to be in close association with the ER and their labelling partially overlapped with PVC marker fluorescence, but no increase in apoplast labelling was observed. Co-immunoprecipitation data demonstrated that the presence of the fluorescent proteins did not affect the assembly of the antibody, and also showed the association of BiP with the antibody chains. The antigen-binding activity of the Venus- fused 2G12 antibody was confirmed by enzyme-linked immunosorbent assay.

  Website 
• Irons S, Graumann K, Runions J, Evans DE, 'Studies on the nuclear envelope targeting and retention of the N-terminus of the mammalian lamin B receptor expressed in plant cells'
  Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology 150 (3) (2008) pp.202-202
  ISSN: 1095-6433
  Abstract

  A GFP fusion to the N-terminal 238 amino acids of the mammalian lamin B receptor (LBR) localises to the nuclear envelope (NE) when expressed in Nicotiana tabacum plants, showing properties expected of a native plant NE protein. In this study, we have used this chimaeric construct to explore evidence for common mechanisms of NE targeting and retention between plants and animals, given there is no plant homologue of the mammalian LBR or of one of its binding partners, lamin B. Binding mutants of LBR-GFP were created and fluorescence recovery after photobleaching of mutant and wild type constructs employed to examine their retention in the plant NE. Unmutated LBR-GFP was significantly less mobile in the NE than the lamin binding domain deletion mutant, which was also localised to theER and punctate structures in some cells. Mutation of the chromatin binding domain resulted in localisation of the protein in nuclear inclusions, in which it was immobile. Our findings, that expression of truncated LBR-GFP in plant cells results in altered targeting and retention relative to wt LBR-GFP, suggest that plant cells can recognize the INM-targeting motif of LBR. Altered mobility of the truncated probe indicates that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.

  Website 
• Graumann K, Evans DE, Irons S, Runions J, 'Dynamics of the lamin B receptor in the plant nuclear envelope'
  Comparative Biochemistry and Physiology A-molecular and Integrative Physiology 146 (4, Supplement) (2007) pp.S193-S193
  ISSN: 1095-6433
  Abstract

  The nuclear envelope (NE) is a double membrane system consisting of the inner nuclear envelope (INE), the outer nuclear envelope (ONE) and nuclear pore complexes (NPCs). Most of our knowledge about the NE proteome comes from studies in animal systems. Recent investigations in plant systems have shown that plants do not have homologues for the majority of animal NE proteins. In a previous study in our laboratory, a construct consisting of the N-terminus of the human lamin B receptor (LBR) fused to GFP was shown to target the plant INE. In mammalian cells, LBR is an intrinsic INE protein, whose targeting to the INE is facilitated by a nuclear localization signal and retention in the INE is achieved by LBR binding mainly to chromatin and lamins. In this study the targeting and retention of LBR–GFP in the plant NE has been investigated by introducing mutations in key domains of LBR and employing fluorescence recovery after photobleaching experiments. Mutation of the chromatin binding domain caused LBR to accumulate in nuclear inclusions in which it was immobile. Deletion of the lamin binding domain resulted in the construct being localized not only to the NE but also ER and to be significantly more mobile then the wild type LBR–GFP in the NE. In the case of both the lamin binding deletion and wild type LBR–GFP, mobility was found to be much greater than previously described in mammalian cells. (Abstracts of the Annual Main Meeting of the Society for Experimental Biology, Glasgow, Scotland, 31st March - 4th April, 2007)

  Website 
• Irons SL, Evans DE, Brandizzi F, 'The First 238 Amino Acids of the Human Lamin B Receptor Are Targeted to the Nuclear Envelope in Plants'
  Journal of Experimental Botany 54 (2003) pp.943-950
  ISSN: 0022-0957 eISSN: 1460-2431
  Abstract In plants, the nuclear envelope (NE) is one of the least characterized cellular structures. In particular, little is known about its dynamics during the cell cycle. This is due to the absence of specific markers for in vivostudies. To generate such an in vivo marker, the suitability of the human lamin B receptor (LBR) was tested. When the first 238 amino acids of the LBR, fused to the green fluorescent protein (GFP), were expressed in tobacco plants, fluorescence accumulated only at the NE of leaf epidermal cells. This was confirmed by electron microscopy. The protein was shown to be membrane‐integral by phase separation. Distribution of fluorescence was compared with two ER markers, GFP‐calnexin and GFP‐HDEL. While co‐localization of all three markers was noted at the NE, only LBR‐GFP was specific to the NE, while the other two also showed fluorescence of the cortical ER. These results suggest that common targeting mechanisms to those in animals and fungi exist in plants to direct and locate proteins to the NE. This chimaeric construct is the first available fluorescent integral membrane protein marker to be targeted exclusively to the plant NE and it provides a novel opportunity to investigate the dynamics of this membrane system in vivo. With it, the cell cycle was followed in tobacco BY‐2 cells stably expressing the fusion protein. The interphase labelling of the NE altered in metaphase into an ER‐like meshwork, suggesting the dispersal of the NE to ER as in animal cells. Finally, the meshwork of fluorescent membranes was lost and new fluorescent NE formed around the daughter nuclei.Website 

Book chapters

• Chambers A, Aksular M, Graves L, Irons SL, Possee RD, King LA, 'Overview of the baculovirus expression system' in Current Protocols in Protein Science, Wiley (2017)
  Website 

Conference papers

• Graumann K, Irons S, Runions J, Evans D, 'SUN domain proteins at the plant nuclear envelope'
  Comparative Biochemistry and Physiology A-molecular and Integrative Physiology 150 (3 (Supplement)) (2008) pp.S202-S202
  ISSN: 1095-6433
  Abstract

  The nuclear envelope (NE) is a double membrane system that forms a protective barrier around chromatin and organises intranuclear structures and activities. The outer nuclear membrane (ONM) is continuous with the ER and associates with cytoskeletal elements. The inner nuclear membrane (INM) interacts with chromatin and the nucleoskeleton and plays a fundamental role in orchestrating nuclear functions such as nucleic acid metabolism. Most of our knowledge of the NE proteome and its functions comes from studies in animal systems. Despite its importance, the plant NE remains poorly understood. Here we present the characterisation of two novel NE proteins, AtSUN1 and AtSUN2, plant homologues of a group of animal and yeast INM proteins containing a well conserved SUN (Sad1/UNC84 homology) domain important for nucleo-cytoskeletal linkage. Both proteins share a similar domain layout to their animal counterparts and appear to interact with each other as indicated by fluorescence resonance energy transfer. Confocal microscopy of fluorescent protein fusions and electron microscopy suggest localisation to the plant INM. Deletion of either the SUN domain or a nuclear localisation signal abolishes this localisation. These SUN domain proteins are the first true inner nuclear envelope proteins to be identified in plants and provide the first evidence for a plant Linker of Cytoskeleton and Nucleoskeleton Complex.

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