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Volume microscopy is an ideal method to investigate microbial infections. In this study, we used Oryctes rhinoceros nudivirus (OrNV) as a model to illustrate the power of this technology in deciphering morphological changes associated with viral replication. Nudiviruses are large dsDNA rod-shaped enveloped viruses infecting a wide range of hosts. The best characterized member of the family, OrNV, infects rhinoceros beetle, a devastating pest damaging coconut and oil palm trees in Southeast Asia and the Pacific islands. Although previous electron microscopy studies have described the cellular changes associated with OrNV infection, little is known regarding the mechanism of viral assembly and egress. Here we used focussed ion beam and scanning electron microscopy to characterize the cellular remodelling associated with OrNV infection.
Bruno M. Humbel; Sailakshmi Velamoor; Allan Mitchell; Mihnea Bostina. Volume Microscopy of Nudivirus Infected Cells. Applications of Microscopy in Materials and Life Sciences 2021, 251 -259.
AMA StyleBruno M. Humbel, Sailakshmi Velamoor, Allan Mitchell, Mihnea Bostina. Volume Microscopy of Nudivirus Infected Cells. Applications of Microscopy in Materials and Life Sciences. 2021; ():251-259.
Chicago/Turabian StyleBruno M. Humbel; Sailakshmi Velamoor; Allan Mitchell; Mihnea Bostina. 2021. "Volume Microscopy of Nudivirus Infected Cells." Applications of Microscopy in Materials and Life Sciences , no. : 251-259.
Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.
Nadishka Jayawardena; Linde Miles; Laura Burga; Charles Rudin; Matthias Wolf; John Poirier; Mihnea Bostina. N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection. Viruses 2021, 13, 769 .
AMA StyleNadishka Jayawardena, Linde Miles, Laura Burga, Charles Rudin, Matthias Wolf, John Poirier, Mihnea Bostina. N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection. Viruses. 2021; 13 (5):769.
Chicago/Turabian StyleNadishka Jayawardena; Linde Miles; Laura Burga; Charles Rudin; Matthias Wolf; John Poirier; Mihnea Bostina. 2021. "N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection." Viruses 13, no. 5: 769.
The dynamics of nuclear envelope has a critical role in multiple cellular processes. However, little is known regarding the structural changes occurring inside the nucleus or at the inner and outer nuclear membranes. For viruses assembling inside the nucleus, remodeling of the intranuclear membrane plays an important role in the process of virion assembly. Here, we monitored the changes associated with viral infection in the case of nudiviruses. Our data revealed dramatic membrane remodeling inside the nuclear compartment during infection with Oryctes rhinoceros nudivirus , an important biocontrol agent against coconut rhinoceros beetle, a devastating pest for coconut and oil palm trees. Based on these findings, we propose a model for nudivirus assembly in which nuclear packaging occurs in fully enveloped virions.
Sailakshmi Velamoor; Allan Mitchell; Bruno M. Humbel; Wonmo Kim; Charlotte Pushparajan; Gabriel Visnovsky; Laura N. Burga; Mihnea Bostina. Visualizing Nudivirus Assembly and Egress. mBio 2020, 11, 1 .
AMA StyleSailakshmi Velamoor, Allan Mitchell, Bruno M. Humbel, Wonmo Kim, Charlotte Pushparajan, Gabriel Visnovsky, Laura N. Burga, Mihnea Bostina. Visualizing Nudivirus Assembly and Egress. mBio. 2020; 11 (4):1.
Chicago/Turabian StyleSailakshmi Velamoor; Allan Mitchell; Bruno M. Humbel; Wonmo Kim; Charlotte Pushparajan; Gabriel Visnovsky; Laura N. Burga; Mihnea Bostina. 2020. "Visualizing Nudivirus Assembly and Egress." mBio 11, no. 4: 1.
Oncolytic viruses (OVs) are replication competent agents that selectively target cancer cells. After penetrating the tumor cell, viruses replicate and eventually trigger cell lysis, releasing the new viral progeny, which at their turn will attack and kill neighbouring cells. The ability of OVs to self-amplify within the tumor while sparing normal cells can provide several advantages including the capacity to encode and locally produce therapeutic protein payloads, and to prime the host immune system. OVs targeting of cancer cells is mediated by host factors that are differentially expressed between normal tissue and tumors, including viral receptors and internalization factors. In this review article, we will discuss the evolution of oncolytic viruses that have reached the stage of clinical trials, their mechanisms of oncolysis, cellular receptors, strategies for targeting cancers, viral neutralization and developments to bypass virus neutralization.
Nadishka Jayawardena; John T Poirier; Laura N Burga; Mihnea Bostina. Virus–Receptor Interactions and Virus Neutralization: Insights for Oncolytic Virus Development. Oncolytic Virotherapy 2020, ume 9, 1 -15.
AMA StyleNadishka Jayawardena, John T Poirier, Laura N Burga, Mihnea Bostina. Virus–Receptor Interactions and Virus Neutralization: Insights for Oncolytic Virus Development. Oncolytic Virotherapy. 2020; ume 9 ():1-15.
Chicago/Turabian StyleNadishka Jayawardena; John T Poirier; Laura N Burga; Mihnea Bostina. 2020. "Virus–Receptor Interactions and Virus Neutralization: Insights for Oncolytic Virus Development." Oncolytic Virotherapy ume 9, no. : 1-15.
The authors wish to make the following corrections to this paper
Cormac McCarthy; Nadishka Jayawardena; Laura N. Burga; Mihnea Bostina. Correction: McCarthy, C.; et al. Developing Picornaviruses for Cancer Therapy. Cancers 2019, 11, 685. Cancers 2020, 12, 553 .
AMA StyleCormac McCarthy, Nadishka Jayawardena, Laura N. Burga, Mihnea Bostina. Correction: McCarthy, C.; et al. Developing Picornaviruses for Cancer Therapy. Cancers 2019, 11, 685. Cancers. 2020; 12 (3):553.
Chicago/Turabian StyleCormac McCarthy; Nadishka Jayawardena; Laura N. Burga; Mihnea Bostina. 2020. "Correction: McCarthy, C.; et al. Developing Picornaviruses for Cancer Therapy. Cancers 2019, 11, 685." Cancers 12, no. 3: 553.
Cryofixation by high pressure freezing (HPF) followed by freeze substitution (FS) is a preferred method to prepare biological specimens for ultrastructural studies. It has been shown to achieve uniform vitrification and ultrastructure preservation of complex structures in different cell types. One limitation of HPF is the small sample volume of < 200 μm thickness and about 2000 μm across. A wool follicle is a rare intact organ in a single sample about 200 μm thick. Within each follicle, specialized cells derived from multiple cell lineages assemble, mature and cornify to make a wool fibre, which contains 95% keratin and associated proteins. In addition to their complex structure, large density changes during wool fibre development, limited water movement and accessibility of fixatives are some other issues negatively affecting the preservation of the follicle ultrastructure via conventional chemical processing. Here we show for the first time that HPF-FS of wool follicle could yield high quality tissue preservation for ultrastructural studies of wool follicles using transmission electron microscopy. This article is protected by copyright. All rights reserved.
Sailakshmi Velamoor; Marina Richena; Allan Mitchell; Sharon Lequeux; Mihnea Bostina; Duane Harland. High‐pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle. Journal of Microscopy 2020, 278, 18 -28.
AMA StyleSailakshmi Velamoor, Marina Richena, Allan Mitchell, Sharon Lequeux, Mihnea Bostina, Duane Harland. High‐pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle. Journal of Microscopy. 2020; 278 (1):18-28.
Chicago/Turabian StyleSailakshmi Velamoor; Marina Richena; Allan Mitchell; Sharon Lequeux; Mihnea Bostina; Duane Harland. 2020. "High‐pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle." Journal of Microscopy 278, no. 1: 18-28.
Recent advancements in oncolytic virotherapy commend a special attention to developing new strategies for targeting cancer cells with oncolytic viruses (OVs). Modifications of the viral envelope or coat proteins serve as a logical mean of repurposing viruses for cancer treatment. In this review, we discuss how detailed structural knowledge of the interactions between OVs and their natural receptors provide valuable insights into tumor specificity of some viruses and re-targeting of alternate receptors for broad tumor tropism or improved tumor selectivity.
Nadishka Jayawardena; Laura N Burga; John T Poirier; Mihnea Bostina. Virus–Receptor Interactions: Structural Insights For Oncolytic Virus Development. Oncolytic Virotherapy 2019, ume 8, 39 -56.
AMA StyleNadishka Jayawardena, Laura N Burga, John T Poirier, Mihnea Bostina. Virus–Receptor Interactions: Structural Insights For Oncolytic Virus Development. Oncolytic Virotherapy. 2019; ume 8 ():39-56.
Chicago/Turabian StyleNadishka Jayawardena; Laura N Burga; John T Poirier; Mihnea Bostina. 2019. "Virus–Receptor Interactions: Structural Insights For Oncolytic Virus Development." Oncolytic Virotherapy ume 8, no. : 39-56.
Varroa destructor and its associated viruses, in particular deformed wing virus (DWV), have been identified as probable causes of honey bee (Apis mellif era L.) colony losses. Evidence suggests that elevated DWV titres in bees could compromise sensory and communication abilities resulting in negative consequences for hygienic behaviour. As antennae play a central role in this behaviour, we compared antennal ultrastructure in DWV-symptomatic and asymptomatic bees. The results show that virus capsids accumulate in the basal regions of the antennal epithelium, close to the haemolymph. No virus particles were detected at the level of sensory sensilla, such as pore plates, nor within the sensory cell dendrites associated with these sensilla. However, membranous structures appeared to be more prevalent in supporting cells surrounding the dendrites of DWV-symptomatic bees. Para-crystalline arrays containing large numbers of virus particles were detected in the antennae of DWV-symptomatic bees but not in asymptomatic bees.
Seo Hyun Kim; Alison Mercer; Allan Mitchell; Joachim Rodrigues de Miranda; Vernon Ward; Fanny Mondet; Mihnea Bostina. Viral infections alter antennal epithelium ultrastructure in honey bees. Journal of Invertebrate Pathology 2019, 168, 107252 .
AMA StyleSeo Hyun Kim, Alison Mercer, Allan Mitchell, Joachim Rodrigues de Miranda, Vernon Ward, Fanny Mondet, Mihnea Bostina. Viral infections alter antennal epithelium ultrastructure in honey bees. Journal of Invertebrate Pathology. 2019; 168 ():107252.
Chicago/Turabian StyleSeo Hyun Kim; Alison Mercer; Allan Mitchell; Joachim Rodrigues de Miranda; Vernon Ward; Fanny Mondet; Mihnea Bostina. 2019. "Viral infections alter antennal epithelium ultrastructure in honey bees." Journal of Invertebrate Pathology 168, no. : 107252.
Oncolytic viruses (OVs) form a group of novel anticancer therapeutic agents which selectively infect and lyse cancer cells. Members of several viral families, including Picornaviridae, have been shown to have anticancer activity. Picornaviruses are small icosahedral non-enveloped, positive-sense, single-stranded RNA viruses infecting a wide range of hosts. They possess several advantages for development for cancer therapy: Their genomes do not integrate into host chromosomes, do not encode oncogenes, and are easily manipulated as cDNA. This review focuses on the picornaviruses investigated for anticancer potential and the mechanisms that underpin this specificity.
Cormac McCarthy; Nadishka Jayawardena; Laura N. Burga; Mihnea Bostina. Developing Picornaviruses for Cancer Therapy. Cancers 2019, 11, 685 .
AMA StyleCormac McCarthy, Nadishka Jayawardena, Laura N. Burga, Mihnea Bostina. Developing Picornaviruses for Cancer Therapy. Cancers. 2019; 11 (5):685.
Chicago/Turabian StyleCormac McCarthy; Nadishka Jayawardena; Laura N. Burga; Mihnea Bostina. 2019. "Developing Picornaviruses for Cancer Therapy." Cancers 11, no. 5: 685.
Picornaviruses are small, icosahedral, nonenveloped, positive-sense, single-stranded RNA viruses that form one of the largest and most important viral families. Numerous Picornaviridae members pose serious health or agricultural threats, causing diseases such as poliomyelitis, hepatitis A, or foot-and-mouth disease. The antigenic characterization of picornavirus capsids plays an important role in understanding the mechanism of viral neutralization and the conformational changes associated with genome release, and it can point to regions which can be targeted by small-molecule compounds to be developed as antiviral inhibitors. In a recent study, Cao and colleagues applied this strategy to hepatitis A virus (HAV) and used cryo-electron microscopy (cryo-EM) to characterize a well-conserved antigenic site recognized by several monoclonal antibodies. They further used computational approaches to identify a small-molecule drug with a strong inhibitory effect on cell attachment.
Mihnea Bostina. Monoclonal antibodies point to Achilles’ heel in picornavirus capsid. PLOS Biology 2019, 17, e3000232 .
AMA StyleMihnea Bostina. Monoclonal antibodies point to Achilles’ heel in picornavirus capsid. PLOS Biology. 2019; 17 (4):e3000232.
Chicago/Turabian StyleMihnea Bostina. 2019. "Monoclonal antibodies point to Achilles’ heel in picornavirus capsid." PLOS Biology 17, no. 4: e3000232.
Macrofibrils, the main structural features within the cortical cells of mammalian hair shafts, are long composite bundles of keratin intermediate filaments (KIFs) embedded in a matrix of keratin-associated proteins. The KIFs can be helically arranged around the macrofibril central axis, making a cylinder within which KIF helical angle relative to macrofibril axis increases approximately linearly from macrofibril centre to edge. Mesophase-based self-assembly has been implicated in the early formation of macrofibrils, which first appear as liquid-crystal tactoids in the bulb of hair follicles. Formation appears to be driven initially by interactions between pre-keratinized KIFs. Differences in the nature of these KIF-KIF interactions could result in all macrofibrils being internally twisted in a single handedness, or a 50:50 mixture of handedness within each cortical cell. We data-mined 41 electron tomograms containing three-dimensional macrofibril data from previously published studies of hair and wool. In all 644 macrofibrils examined we found that within each tomogram all macrofibrils had the same handedness. We concluded that earlier reports of left- and right-handed macrofibrils were due to artefacts of imaging or data processing. A handedness marker was used to confirm (using re-imaged sections from earlier studies) that, in both human and sheep, all macrofibrils are left-handed around the macrofibril axis. We conclude that this state is universal within mammalian hair. This also supports the conclusion that the origin of macrofibril twist is the expression of chiral twisting forces between adjacent KIFs, rather than mesophase splay and bending forces relaxing to twisting forces acting within a confined space.
Duane P. Harland; Veronika Novotna; Marina Richena; Sailakshmi Velamoor; Mihnea Bostina; A. John McKinnon. Helical twist direction in the macrofibrils of keratin fibres is left handed. Journal of Structural Biology 2019, 206, 345 -348.
AMA StyleDuane P. Harland, Veronika Novotna, Marina Richena, Sailakshmi Velamoor, Mihnea Bostina, A. John McKinnon. Helical twist direction in the macrofibrils of keratin fibres is left handed. Journal of Structural Biology. 2019; 206 (3):345-348.
Chicago/Turabian StyleDuane P. Harland; Veronika Novotna; Marina Richena; Sailakshmi Velamoor; Mihnea Bostina; A. John McKinnon. 2019. "Helical twist direction in the macrofibrils of keratin fibres is left handed." Journal of Structural Biology 206, no. 3: 345-348.
CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These ‘spacers’ provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
B. N. J. Watson; R. A. Easingwood; B. Tong; M. Wolf; G. P. C. Salmond; Raymond Staals; M. Bostina; P. C. Fineran. Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers. Philosophical Transactions of the Royal Society B: Biological Sciences 2019, 374, 20180090 .
AMA StyleB. N. J. Watson, R. A. Easingwood, B. Tong, M. Wolf, G. P. C. Salmond, Raymond Staals, M. Bostina, P. C. Fineran. Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers. Philosophical Transactions of the Royal Society B: Biological Sciences. 2019; 374 (1772):20180090.
Chicago/Turabian StyleB. N. J. Watson; R. A. Easingwood; B. Tong; M. Wolf; G. P. C. Salmond; Raymond Staals; M. Bostina; P. C. Fineran. 2019. "Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers." Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1772: 20180090.
Recently, the use of oncolytic viruses in cancer therapy has become a realistic therapeutic option. Seneca Valley Virus (SVV) is a newly discovered picornavirus, which has earned a significant reputation as a potent oncolytic agent. Anthrax toxin receptor 1 (ANTXR1), one of the cellular receptors for the protective antigen secreted by Bacillus anthracis, has been identified as the high-affinity cellular receptor for SVV. Here, we report the structure of the SVV-ANTXR1 complex determined by single-particle cryo-electron microscopy analysis at near-atomic resolution. This is an example of a shared receptor structure between a mammalian virus and a bacterial toxin. Our structure shows that ANTXR1 decorates the outer surface of the SVV capsid and interacts with the surface-exposed BC loop and loop II of VP1, “the puff” of VP2 and “the knob” of VP3. Comparison of the receptor-bound capsid structure with the native capsid structure reveals that receptor binding induces minor conformational changes in SVV capsid structure, suggesting the role of ANTXR1 as an attachment receptor. Furthermore, our results demonstrate that the capsid footprint on the receptor is not conserved in anthrax toxin receptor 2 (ANTXR2), thereby providing a molecular mechanism for explaining the exquisite selectivity of SVV for ANTXR1.
Nadishka Jayawardena; Laura N. Burga; Richard A. Easingwood; Yoshimasa Takizawa; Matthias Wolf; Mihnea Bostina. Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus. Proceedings of the National Academy of Sciences 2018, 115, E10934 -E10940.
AMA StyleNadishka Jayawardena, Laura N. Burga, Richard A. Easingwood, Yoshimasa Takizawa, Matthias Wolf, Mihnea Bostina. Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus. Proceedings of the National Academy of Sciences. 2018; 115 (46):E10934-E10940.
Chicago/Turabian StyleNadishka Jayawardena; Laura N. Burga; Richard A. Easingwood; Yoshimasa Takizawa; Matthias Wolf; Mihnea Bostina. 2018. "Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus." Proceedings of the National Academy of Sciences 115, no. 46: E10934-E10940.
Seneca Valley virus (SVV), like some other members of the Picornaviridae , forms naturally occurring empty capsids, known as procapsids. Procapsids have the same antigenicity as full virions, so they present an interesting possibility for the formation of stable virus-like particles. Interestingly, although SVV is a livestock pathogen, it has also been found to preferentially infect tumor cells and is being explored for use as a therapeutic agent in the treatment of small-cell lung cancers. Here we used cryo-electron microscopy to investigate the procapsid structure and describe the transition of capsid protein VP0 to the cleaved forms of VP4 and VP2. We show that the SVV receptor binds the procapsid, as evidence of its native antigenicity. In comparing the procapsid structure to that of the full virion, we also show that a cage of RNA serves to stabilize the inside surface of the virus, thereby making it more acid stable. IMPORTANCE Viruses are extensively studied to help us understand infection and disease. One of the by-products of some virus infections are the naturally occurring empty virus capsids (containing no genome), termed procapsids, whose function remains unclear. Here we investigate the structure and formation of the procapsids of Seneca Valley virus, to better understand how they form, what causes them to form, how they behave, and how we can make use of them. One potential benefit of this work is the modification of the procapsid to develop it for targeted in vivo delivery of therapeutics or to make a stable vaccine against SVV, which could be of great interest to the agricultural industry.
Mike Strauss; Nadishka Jayawardena; Eileen Sun; Richard A. Easingwood; Laura N. Burga; Mihnea Bostina. Cryo-Electron Microscopy Structure of Seneca Valley Virus Procapsid. Journal of Virology 2018, 92, e01927-17 .
AMA StyleMike Strauss, Nadishka Jayawardena, Eileen Sun, Richard A. Easingwood, Laura N. Burga, Mihnea Bostina. Cryo-Electron Microscopy Structure of Seneca Valley Virus Procapsid. Journal of Virology. 2018; 92 (6):e01927-17.
Chicago/Turabian StyleMike Strauss; Nadishka Jayawardena; Eileen Sun; Richard A. Easingwood; Laura N. Burga; Mihnea Bostina. 2018. "Cryo-Electron Microscopy Structure of Seneca Valley Virus Procapsid." Journal of Virology 92, no. 6: e01927-17.
Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. It has shown promise as a cancer therapeutic in preclinical studies and early-phase clinical trials. Here, we have identified anthrax toxin receptor 1 (ANTXR1) as the receptor for SVV using genome-wide loss-of-function screens. ANTXR1 is necessary for permissivity in vitro and in vivo. However, robust SVV replication requires an additional innate immune defect. We found that SVV interacts directly and specifically with ANTXR1, that this interaction is required for SVV binding to permissive cells, and that ANTXR1 expression is necessary and sufficient for infection in cell lines with decreased expression of antiviral IFN genes at baseline. Finally, we identified the region of the SVV capsid that is responsible for receptor recognition using cryoelectron microscopy of the SVV-ANTXR1-Fc complex. These studies identify ANTXR1, a class of receptor that is shared by a mammalian virus and a bacterial toxin, as the cellular receptor for SVV.
Linde A. Miles; Laura N. Burga; Eric E. Gardner; Mihnea Bostina; John T. Poirier; Charles M. Rudin. Anthrax toxin receptor 1 is the cellular receptor for Seneca Valley virus. Journal of Clinical Investigation 2017, 127, 2957 -2967.
AMA StyleLinde A. Miles, Laura N. Burga, Eric E. Gardner, Mihnea Bostina, John T. Poirier, Charles M. Rudin. Anthrax toxin receptor 1 is the cellular receptor for Seneca Valley virus. Journal of Clinical Investigation. 2017; 127 (8):2957-2967.
Chicago/Turabian StyleLinde A. Miles; Laura N. Burga; Eric E. Gardner; Mihnea Bostina; John T. Poirier; Charles M. Rudin. 2017. "Anthrax toxin receptor 1 is the cellular receptor for Seneca Valley virus." Journal of Clinical Investigation 127, no. 8: 2957-2967.
Mitochondria are dynamic organelles that continually adapt their morphology by fusion and fission events. An imbalance between fusion and fission has been linked to major neurodegenerative diseases, including Huntington’s, Alzheimer’s, and Parkinson’s diseases. A member of the Dynamin superfamily, dynamin-related protein 1 (DRP1), a dynamin-related GTPase, is required for mitochondrial membrane fission. Self-assembly of DRP1 into oligomers in a GTP-dependent manner likely drives the division process. We show here that DRP1 self-assembles in two ways: i) in the presence of the non-hydrolysable GTP analog GMP-PNP into spiral-like structures of ~36 nm diameter; and ii) in the presence of GTP into rings composed of 13−18 monomers. The most abundant rings were composed of 16 monomers and had an outer and inner ring diameter of ~30 nm and ~20 nm, respectively. Three-dimensional analysis was performed with rings containing 16 monomers. The single-particle cryo-electron microscopy map of the 16 monomer DRP1 rings suggests a side-by-side assembly of the monomer with the membrane in a parallel fashion. The inner ring diameter of 20 nm is insufficient to allow four membranes to exist as separate entities. Furthermore, we observed that mitochondria were tubulated upon incubation with DRP1 protein in vitro. The tubes had a diameter of ~ 30nm and were decorated with protein densities. These findings suggest DRP1 tubulates mitochondria, and that additional steps may be required for final mitochondrial fission.
Kaustuv Basu; Driss Lajoie; Tristan Aumentado-Armstrong; Jin Chen; Roman I. Koning; Blaise Bossy; Mihnea Bostina; Attila Sík; Ella Bossy-Wetzel; Isabelle Rouiller. Molecular mechanism of DRP1 assembly studied in vitro by cryo-electron microscopy. PLOS ONE 2017, 12, e0179397 .
AMA StyleKaustuv Basu, Driss Lajoie, Tristan Aumentado-Armstrong, Jin Chen, Roman I. Koning, Blaise Bossy, Mihnea Bostina, Attila Sík, Ella Bossy-Wetzel, Isabelle Rouiller. Molecular mechanism of DRP1 assembly studied in vitro by cryo-electron microscopy. PLOS ONE. 2017; 12 (6):e0179397.
Chicago/Turabian StyleKaustuv Basu; Driss Lajoie; Tristan Aumentado-Armstrong; Jin Chen; Roman I. Koning; Blaise Bossy; Mihnea Bostina; Attila Sík; Ella Bossy-Wetzel; Isabelle Rouiller. 2017. "Molecular mechanism of DRP1 assembly studied in vitro by cryo-electron microscopy." PLOS ONE 12, no. 6: e0179397.
CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1–Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas14–Cas2-32 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1–Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1–Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3′-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1–Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1–Cas2-3 adaptation complexes and their integration mechanism.
Robert Fagerlund; Max Wilkinson; Oleg Klykov; Arjan Barendregt; Frederick Pearce; Sebastian N. Kieper; Howard W. R. Maxwell; Angela Capolupo; Albert Heck; Kurt L. Krause; Mihnea Bostina; Richard Scheltema; Raymond Staals; Peter C. Fineran. Spacer capture and integration by a type I-F Cas1–Cas2-3 CRISPR adaptation complex. Proceedings of the National Academy of Sciences 2017, 114, 201618421 -E5128.
AMA StyleRobert Fagerlund, Max Wilkinson, Oleg Klykov, Arjan Barendregt, Frederick Pearce, Sebastian N. Kieper, Howard W. R. Maxwell, Angela Capolupo, Albert Heck, Kurt L. Krause, Mihnea Bostina, Richard Scheltema, Raymond Staals, Peter C. Fineran. Spacer capture and integration by a type I-F Cas1–Cas2-3 CRISPR adaptation complex. Proceedings of the National Academy of Sciences. 2017; 114 (26):201618421-E5128.
Chicago/Turabian StyleRobert Fagerlund; Max Wilkinson; Oleg Klykov; Arjan Barendregt; Frederick Pearce; Sebastian N. Kieper; Howard W. R. Maxwell; Angela Capolupo; Albert Heck; Kurt L. Krause; Mihnea Bostina; Richard Scheltema; Raymond Staals; Peter C. Fineran. 2017. "Spacer capture and integration by a type I-F Cas1–Cas2-3 CRISPR adaptation complex." Proceedings of the National Academy of Sciences 114, no. 26: 201618421-E5128.
SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators have been identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon.
Hannah G. Hampton; Matthew McNeil; Thomas J. Paterson; Blair Ney; Neil R. Williamson; Richard A. Easingwood; Mihnea Bostina; George P. C. Salmond; Peter C. Fineran. CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia. Microbiology 2016, 162, 1047 -1058.
AMA StyleHannah G. Hampton, Matthew McNeil, Thomas J. Paterson, Blair Ney, Neil R. Williamson, Richard A. Easingwood, Mihnea Bostina, George P. C. Salmond, Peter C. Fineran. CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia. Microbiology. 2016; 162 (6):1047-1058.
Chicago/Turabian StyleHannah G. Hampton; Matthew McNeil; Thomas J. Paterson; Blair Ney; Neil R. Williamson; Richard A. Easingwood; Mihnea Bostina; George P. C. Salmond; Peter C. Fineran. 2016. "CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia." Microbiology 162, no. 6: 1047-1058.
Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.
Rebekah A. Frampton; Elena Lopez Acedo; Vivienne L. Young; Danni Chen; Brian Tong; Corinda Taylor; Richard A. Easingwood; Andrew R. Pitman; Torsten Kleffmann; Mihnea Bostina; Peter C. Fineran. Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae. Viruses 2015, 7, 3361 -3379.
AMA StyleRebekah A. Frampton, Elena Lopez Acedo, Vivienne L. Young, Danni Chen, Brian Tong, Corinda Taylor, Richard A. Easingwood, Andrew R. Pitman, Torsten Kleffmann, Mihnea Bostina, Peter C. Fineran. Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae. Viruses. 2015; 7 (7):3361-3379.
Chicago/Turabian StyleRebekah A. Frampton; Elena Lopez Acedo; Vivienne L. Young; Danni Chen; Brian Tong; Corinda Taylor; Richard A. Easingwood; Andrew R. Pitman; Torsten Kleffmann; Mihnea Bostina; Peter C. Fineran. 2015. "Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae." Viruses 7, no. 7: 3361-3379.
Lentiviral vectors have proved an effective method to deliver transgenes into the brain; however, they are often hampered by a lack of spread from the site of injection. Modifying the viral envelope with a portion of a rabies envelope glycoprotein can enhance spread in the brain by using long-range axon projections to facilitate retrograde transport. In this study, we generated two chimeric envelopes containing the extra-virion and transmembrane domain of rabies SADB19 or CVS-N2c with the intra-virion domain of vesicular stomatitis virus. Viral particles were packaged containing a green fluorescent protein reporter construct under the control of the phosphoglycerokinase promoter. Both vectors produced high-titer particles with successful integration of the glycoproteins into the particle envelope and significant transduction of neurons in vitro. Injection of the SADB19 chimeric viral vector into the lumbar spinal cord of adult mice mediated a strong preference for gene transfer to local neurons and axonal terminals, with retrograde transport to neurons in the brainstem, hypothalamus and cerebral cortex. Development of this vector provides a useful means to reliably target select populations of neurons by retrograde targeting.
L Schoderboeck; S Riad; A M Bokor; H E Wicky; Mike Strauss; Mihnea Bostina; M J Oswald; Ruth Empson; Stephanie Hughes. Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord. Gene Therapy 2015, 22, 357 -364.
AMA StyleL Schoderboeck, S Riad, A M Bokor, H E Wicky, Mike Strauss, Mihnea Bostina, M J Oswald, Ruth Empson, Stephanie Hughes. Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord. Gene Therapy. 2015; 22 (5):357-364.
Chicago/Turabian StyleL Schoderboeck; S Riad; A M Bokor; H E Wicky; Mike Strauss; Mihnea Bostina; M J Oswald; Ruth Empson; Stephanie Hughes. 2015. "Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord." Gene Therapy 22, no. 5: 357-364.