Timothy Newsome

Sub-viral imaging of vaccinia virus using super-resolution microscopy

Publisher: Elsevier BV

Date: 12-2012

DOI: 10.1016/J.JVIROMET.2012.07.003

Abstract: The study of host-pathogen interactions over past decades has benefited from advances in microscopy and fluorescent imaging techniques. A particularly powerful model in this field is vaccinia virus (VACV), which due to its amenability to genetic manipulation has been a productive model in advancing the understanding of the transport of subcellular cargoes. Conventional light microscopy imposes an upper limit of resolution of ~250nm, hence knowledge of events occurring at the sub-viral resolution is based predominantly on studies utilising electron microscopy. The development of super-resolution light microscopy presents the opportunity to bridge the gap between these two technologies. This report describes the analysis of VACV replication using fluorescent recombinant viruses, achieving sub-viral resolution with three-dimensional structured illumination microscopy. This is the first report of successfully resolving poxvirus particle morphologies at the scale of single virus particles using light microscopy.

The Release of Vaccinia Virus from Infected Cells Requires RhoA-mDia Modulation of Cortical Actin

Publisher: Elsevier BV

Date: 05-2007

DOI: 10.1016/J.CHOM.2007.04.006

Abstract: Prior to being released from the infected cell, intracellular enveloped vaccinia virus particles are transported from their perinuclear assembly site to the plasma membrane along microtubules by the motor kinesin-1. After fusion with the plasma membrane, stimulation of actin tails beneath extracellular virus particles acts to enhance cell-to-cell virus spread. However, we lack molecular understanding of events that occur at the cell periphery just before and during the liberation of virus particles. Using live cell imaging, we show that virus particles move in the cell cortex, independently of actin tail formation. These cortical movements and the subsequent release of virus particles, which are both actin dependent, require F11L-mediated inhibition of RhoA-mDia signaling. We suggest that the exit of vaccinia virus from infected cells has strong parallels to exocytosis, as it is dependent on the assembly and organization of actin in the cell cortex.

A putative WAVE regulatory complex (WRC) interacting receptor sequence (WIRS) in the cytoplasmic tail of HSV-1 gE does not function in WRC recruitment or neuronal transport.

Publisher: Microbiology Society

Date: 03-2021

DOI: 10.1099/ACMI.0.000206

Abstract: HSV-1 envelope glycoprotein E (gE) is important for viral egress and cell-to-cell spread but the host protein(s) involved in these functions have yet to be determined. We aimed to investigate a role for the Arp2/3 complex and actin regulation in viral egress based on the identification of a WAVE Regulatory Complex (WRC) Interacting Receptor Sequence (WIRS) in the cytoplasmic tail (CT) of gE. A WIRS-dependent interaction between the gE(CT) and subunits of the WRC was demonstrated by GST-pulldown assay and a role for the Arp2/3 complex in cell-to-cell spread was also observed by plaque assay. Subsequent study of a recombinant HSV-1 gE WIRS-mutant found no significant changes to viral production and release based on growth kinetics studies, or changes to plaque and comet size in various cell types, suggesting no function for the motif in cell-to-cell spread. GFP-Trap pulldown and proximity ligation assays were unable to confirm a WIRS-dependent interaction between gE and the WRC in human cell lines though the WIRS-independent interaction observed in situ warrants further study. Confocal microscopy of infected cells of neuronal origin identified no impairment of gE WIRS-mutant HSV-1 anterograde transport along axons. We propose that the identified gE WIRS motif does not function directly in recruitment of the WRC in human cells, in cell-to-cell spread of virus or in anterograde transport along axons. Further studies are needed to understand how HSV-1 manipulates and traverses the actin cytoskeleton and how gE may contribute to these processes in a WIRS-independent manner.

The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility

Publisher: Springer Science and Business Media LLC

Date: 05-03-2009

DOI: 10.1038/NATURE07773

Abstract: Understanding cell motility will require detailed knowledge not only of the localization of signalling networks regulating actin polymerization, but also of their dynamics. Unfortunately, many signalling networks are not amenable to such analysis, as they are frequently transient and dispersed. By contrast, the signalling pathways used by pathogens undergoing actin-based motility are highly localized and operate in a constitutive fashion. Taking advantage of this, we have analysed the dynamics of neuronal Wiskott-Aldrich syndrome protein (N-WASP), WASP-interacting protein (WIP), GRB2 and NCK, which are required to stimulate actin-related protein (ARP)2/3-complex-dependent actin-based motility of vaccinia virus, using fluorescence recovery after photobleaching. Here we show that all four proteins are rapidly exchanging, albeit at different rates, and that the turnover of N-WASP depends on its ability to stimulate ARP2/3-complex-mediated actin polymerization. Conversely, disruption of the interaction of N-WASP with GRB2 and/or the barbed ends of actin filaments increases its exchange rate and results in a faster rate of virus movement. We suggest that the exchange rate of N-WASP controls the rate of ARP2/3-complex-dependent actin-based motility by regulating the extent of actin polymerization by antagonizing filament capping.

Viruses That Exploit Actin-Based Motility for Their Replication and Spread

Publisher: Springer International Publishing

Date: 2016

DOI: 10.1007/164_2016_41

Abstract: The actin cytoskeleton is a crucial part of the eukaryotic cell. Viruses depend on host cells for their replication, and, as a result, many have developed ways of manipulating the actin network to promote their spread. This chapter reviews the various ways in which viruses utilize the actin cytoskeleton at discrete steps in their life cycle, from entry into the host cell, replication, and assembly of new progeny to virus release. Various actin inhibitors that function in different ways to affect proper actin dynamics can be used to parse the role of actin at these steps.

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naïve Mice

Publisher: Public Library of Science (PLoS)

Date: 23-12-2015

DOI: 10.1371/JOURNAL.PPAT.1005342

Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection

Publisher: The Company of Biologists

Date: 30-09-2022

DOI: 10.1242/JCS.260175

Abstract: Intracellular mature viruses (IMVs) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMVs can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMVs, like intracellular enveloped viruses (IEVs), the second form of vaccinia that are wrapped in Golgi-derived membranes, recruit kinesin-1 and undergo anterograde transport. In vitro reconstitution of virion transport in infected cell extracts revealed that IMVs and IEVs move toward microtubule plus ends with respective velocities of 0.66 and 0.56 µm/s. Quantitative imaging established that IMVs and IEVs recruit an average of 139 and 320 kinesin-1 motor complexes, respectively. In the absence of kinesin-1, there was a near-complete loss of in vitro motility and reduction in the intracellular spread of both types of virions. Our observations demonstrate that kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to elucidate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.

Exhalation of respiratory viruses by breathing, coughing, and talking

Publisher: Wiley

Date: 09-2009

DOI: 10.1002/JMV.21556

Abstract: There is a lack of quantitative information about the generation of virus aerosols by infected subjects. The exhaled aerosols generated by coughing, talking, and breathing were s led in 50 subjects using a novel mask, and analyzed using PCR for nine respiratory viruses. The exhaled s les from a subset of 10 subjects who were PCR positive for rhinovirus were also examined by cell culture for this virus. Of the 50 subjects, among the 33 with symptoms of upper respiratory tract infections, 21 had at least one virus detected by PCR, while amongst the 17 asymptomatic subjects, 4 had a virus detected by PCR. Overall, rhinovirus was detected in 19 subjects, influenza in 4 subjects, parainfluenza in 2 subjects, and human metapneumovirus in 1 subject. Two subjects were co-infected. Of the 25 subjects who had virus-positive nasal mucus, the same virus type was detected in 12 breathing s les, 8 talking s les, and in 2 coughing s les. In the subset of exhaled s les from 10 subjects examined by culture, infective rhinovirus was detected in 2. These data provide further evidence that breathing may be a source of respirable particles carrying infectious virus.

Divergent roles of β‐ and γ‐actin isoforms during spread of vaccinia virus

Publisher: Wiley

Date: 17-03-2017

DOI: 10.1002/CM.21356

A tight squeeze: host control of Listeria invasion.

Publisher: Elsevier BV

Date: 02-2021

DOI: 10.1016/J.DEVCEL.2021.02.004

Cell-to-cell spread of vaccinia virus is promoted by TGF-β-independent Smad4 signalling

Publisher: Hindawi Limited

Date: 28-04-2020

DOI: 10.1111/CMI.13206

Systematic Identification of Genes that Regulate Neuronal Wiring in the Drosophila Visual System

Publisher: Public Library of Science (PLoS)

Date: 30-05-2008

DOI: 10.1371/JOURNAL.PGEN.1000085

Kidins220/ARMS Is Transported by a Kinesin-1–based Mechanism Likely to be Involved in Neuronal Differentiation

Publisher: American Society for Cell Biology (ASCB)

Date: 2007

DOI: 10.1091/MBC.E06-05-0453

Abstract: Kinase D-interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning (Kidins220/ARMS) is a conserved membrane protein mainly expressed in brain and neuroendocrine cells, which is a downstream target of the signaling cascades initiated by neurotrophins and ephrins. We identified kinesin light chain 1 (KLC1) as a binding partner for Kidins220/ARMS by a yeast two-hybrid screen. The interaction between Kidins220/ARMS and the kinesin-1 motor complex was confirmed by glutathione S-transferase-pull-down and coimmunoprecipitation experiments. In addition, Kidins220/ARMS and kinesin-1 were shown to colocalize in nerve growth factor (NGF)-differentiated PC12 cells. Using Kidins220/ARMS and KLC1 mutants, we mapped the regions responsible for the binding to a short sequence of Kidins220/ARMS, termed KLC-interacting motif (KIM), which is sufficient for the interaction with KLC1. Optimal binding of KIM requires a region of KLC1 spanning both the tetratricopeptide repeats and the heptad repeats, previously not involved in cargo recognition. Overexpression of KIM in differentiating PC12 cells impairs the formation and transport of EGFP-Kidins220/ARMS carriers to the tips of growing neurites, leaving other kinesin-1 dependent processes unaffected. Furthermore, KIM overexpression interferes with the activation of the mitogen-activated protein kinase signaling and neurite outgrowth in NGF-treated PC12 cells. Our results suggest that Kidins220/ARMS-positive carriers undergo a kinesin-1–dependent transport linked to neurotrophin action.

Vaccinia virus F5 is required for normal plaque morphology in multiple cell lines but not replication in culture or virulence in mice

Publisher: Elsevier BV

Date: 05-2014

DOI: 10.1016/J.VIROL.2014.03.020

Abstract: Vaccinia virus (VACV) gene F5L was recently identified as a determinant of plaque morphology that is truncated in Modified Vaccinia virus Ankara (MVA). Here we show that F5L also affects plaque morphology of the virulent VACV strain Western Reserve (WR) in some, but not all cell lines, and not via previously described mechanisms. Further, despite a reduction in plaque size for VACV WR lacking F5L there was no evidence of reduced virus replication or spread in vitro or in vivo. In vivo we examined two mouse models, each with more than one dose and measured signs of disease and virus burden. These data provide an initial characterization of VACV F5L in a virulent strain of VACV. Further they show the necessity of testing plaque phenotypes in more than one cell type and provide an ex le of a VACV gene required for normal plaque morphology but not replication and spread.

Loss of cytoskeletal transport during egress critically attenuates ectromelia virus infection in vivo

Publisher: American Society for Microbiology

Date: 07-2012

DOI: 10.1128/JVI.06636-11

Abstract: Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin comets, another phenotype dependent on A36 expression during VACV infection. ECTV ΔA36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.

Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus

Publisher: Hindawi Limited

Date: 02-2006

DOI: 10.1111/J.1462-5822.2005.00613.X

Abstract: Local activation of Src at the plasma membrane by extracellular vaccinia virus results in a signalling cascade that acts to stimulate actin polymerization beneath the virus to enhance its cell-to-cell spread. Initiation of this signalling cascade involves Src-mediated phosphorylation of tyrosine 112 and 132 of the viral membrane protein A36R. Here we show that recruitment of Src is dependent on its myristoylation and an interaction with A36R upstream of tyrosine 112 and 132. We further show that Src, Fyn and Yes have unique specificities towards these tyrosine residues. Using cell lines deficient in Src, Fyn and Yes, we demonstrate that multiple Src family members can stimulate vaccinia-induced actin polymerization and also uncover a role for Abl family kinases. Additionally, Abl and Arg are able to phosphorylate A36R in vitro and are recruited to vaccinia-induced actin tails. The ability of multiple families of tyrosine kinases to directly phosphorylate A36R ensures robust cell-to-cell spread of vaccinia virus will occur under a variety of cellular conditions.

SMAD proteins: Mediators of diverse outcomes during infection

Publisher: Elsevier BV

Date: 04-2022

DOI: 10.1016/J.EJCB.2022.151204

Abstract: Understanding the relationship between host and pathogen is key to combatting disease. SMAD transcription factors, which transmit TGF-β superfamily signalling, mediate an array of outcomes during embryogenesis, inflammation, cancer, and immunity. Surprisingly, these activities can sometimes be directly opposed for ex le, SMAD3 has been reported as tumour suppressor by arresting cell cycle progression but conversely promotes cancer metastasis. A growing body of literature has identified SMADs as prominent targets during viral and bacterial infection for modulating host signalling. During infection, the activity of SMAD-containing transcriptional complexes can be finely tuned by pathogens to enhance infectivity and spread. SMAD signalling can be modulated at many levels, such as upstream at the ligand and receptor, or by direct interactions with SMADs. These alterations can increase pathogen dissemination, induce fibrosis, over-activate, or attenuate the host immune response. Here, we summarise the erse mechanisms by which pathogens have evolved to sway SMAD signalling in their favour. Understanding the intricacies of host-pathogen interactions through this lens may elucidate aspects of SMAD function in cancer development, homoeostasis, and immune signalling previously overlooked. These insights are an opportunity to identify novel TGF-β or BMP-targeted therapeutics for applications to infectious disease contexts.

A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity

Publisher: Wiley

Date: 16-06-2003

DOI: 10.1093/EMBOJ/CDG287

Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection

Publisher: Cold Spring Harbor Laboratory

Date: 15-04-2022

DOI: 10.1101/2022.04.15.488437

Abstract: Intracellular mature virions (IMV) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMV can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMV, like intracellular enveloped virions (IEV), the second form of vaccinia, undergo anterograde transport and recruit kinesin-1. In vitro reconstitution of virion transport reveals that IMV and IEV move toward microtubule plus-ends with respective velocities of 0.66 and 0.56 μm/s. Quantitative imaging establishes IMV and IEV recruit an average of 65 and 115 kinesin-1 motor complexes respectively. In the absence of kinesin-1 there is a near-complete loss of in vitro motility and defects in the cellular spread of both virions. Our observations demonstrate kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to investigate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.

An E2-F12 complex is required for intracellular enveloped virus morphogenesis during vaccinia infection

Publisher: Hindawi Limited

Date: 05-2009

DOI: 10.1111/J.1462-5822.2009.01296.X

Cover Image, Volume 74, Issue 4

Publisher: Wiley

Date: 04-2017

DOI: 10.1002/CM.21365

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Publisher: MyJove Corporation

Date: 17-01-2014

DOI: 10.3791/51151

Trio combines with dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila

Publisher: Elsevier BV

Date: 04-2000

DOI: 10.1016/S0092-8674(00)80838-7

Abstract: Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. Here, we identify the guanine nucleotide exchange factor (GEF) Trio as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. We propose that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension.

Mobilization of HIV Spread by Diaphanous 2 Dependent Filopodia in Infected Dendritic Cells

Publisher: Public Library of Science (PLoS)

Date: 07-06-2012

DOI: 10.1371/JOURNAL.PPAT.1002762

Phototracking Vaccinia Virus Transport Reveals Dynamics of Cytoplasmic Dispersal and a Requirement for A36R and F12L for Exit from the Site of Wrapping

Publisher: MDPI AG

Date: 24-07-2018

DOI: 10.3390/V10080390

Abstract: The microtubule cytoskeleton is a primary organizer of viral infections for delivering virus particles to their sites of replication, establishing and maintaining subcellular compartments where distinct steps of viral morphogenesis take place, and ultimately dispersing viral progeny. One of the best characterized ex les of virus motility is the anterograde transport of the wrapped virus form of vaccinia virus (VACV) from the trans-Golgi network (TGN) to the cell periphery by kinesin-1. Yet many aspects of this transport event are elusive due to the speed of motility and the challenges of imaging this stage at high resolution over extended time periods. We have established a novel imaging technology to track virus transport that uses photoconvertible fluorescent recombinant viruses to track subsets of virus particles from their site of origin and determine their destination. Here we image virus exit from the TGN and their rate of egress to the cell periphery. We demonstrate a role for kinesin-1 engagement in regulating virus exit from the TGN by removing A36 and F12 function, critical viral mediators of kinesin-1 recruitment to virus particles. Phototracking viral particles and components during infection is a powerful new imaging approach to elucidate mechanisms of virus replication.

Viruses that ride on the coat-tails of actin nucleation

Publisher: Elsevier BV

Date: 10-2015

DOI: 10.1016/J.SEMCDB.2015.10.008

Abstract: Actin nucleation drives a ersity of critical cellular processes and the motility of a select group of viral pathogens. Vaccinia virus and baculovirus, Autographa californica multiple nucleopolyhedrovirus, recruit and activate the cellular actin nucleator, the Arp2/3 complex, at the surface of virus particles thereby instigating highly localized actin nucleation. The extension of these filaments provides a mechanical force that bestows the ability to navigate the intracellular environment and promote their infectious cycles. This review outlines the viral and cellular proteins that initiate and regulate the signalling networks leading to viral modification of the actin cytoskeleton and summarizes recent insights into the role of actin-based virus transport.

TNF deficiency dysregulates inflammatory cytokine production, leading to lung pathology and death during respiratory poxvirus infection

Publisher: Proceedings of the National Academy of Sciences

Date: 22-06-2020

DOI: 10.1073/PNAS.2004615117

Abstract: Excessive tumor necrosis factor (TNF) production during some respiratory viral infections is associated with lung pathology and death. We show here that deficiency in TNF also causes significant pathology during respiratory poxvirus infection of mice but has no effect on viral load. TNF deficiency causes increased production of interleukin (IL)-6, IL-10, transforming growth factor beta, and interferon gamma and overactivation of STAT3 signaling. Cytokine blockade, or STAT3 inactivation, ameliorates lung pathology in TNF-deficient mice. The membrane form of TNF alone is necessary and sufficient for regulating inflammation and the prevention of lung pathology. Targeting specific cytokines or cytokine signaling pathways to can ameliorate lung inflammation during respiratory viral infections but the timing and duration of the interventive measures are critical.

A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

Publisher: Public Library of Science (PLoS)

Date: 21-03-2013

DOI: 10.1371/JOURNAL.PPAT.1003239

Loss of actin-based motility impairs ectromelia virus release in vitro but is not critical to spread in vivo

Publisher: MDPI AG

Date: 05-03-2018

DOI: 10.3390/V10030111

Universität Zürich

Location: Switzerland

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University Of Melbourne

Location: Australia

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Discovery Projects - Grant ID: DP1096623

Start Date: 02-2010

End Date: 02-2013

Amount: $315,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100010

Start Date: 12-2010

End Date: 12-2010

Amount: $720,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989920

Start Date: 2009

End Date: 12-2009

Amount: $750,000.00

Funder: Australian Research Council