%0 Journal Article
%~ PubMed
%A Lynn, Helena
%A Horsington, Jacquelyn
%A Ter, Lee Kuan
%A Han, Shuyi
%A Chew, Yee Lian
%A Diefenbach, Russell J
%A Way, Michael
%A Chaudhri, Geeta
%A Karupiah, Gunasegaran
%A Newsome, Timothy P
%T Loss of Cytoskeletal Transport during Egress Critically Attenuates Ectromelia Virus Infection In Vivo.
%B Journal of Virology
%D 2012
%C United States
%I American Society for Microbiology
%V 86
%N 13
%P 7427-7443
%@ 1098-5514
%X 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 ??A36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ??A36R 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 ??A36R 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.
%Z FOR Codes: 110804


%0 Journal Article
%~ PubMed
%A Horsington, Jacquelyn
%A Turnbull, Lynne
%A Whitchurch, Cynthia B
%A Newsome, Timothy P
%T Sub-viral imaging of vaccinia virus using super-resolution microscopy.
%B Journal of Virological Methods
%D 2012
%C Netherlands
%I Elsevier BV
%V 186
%N 1-2
%P 132-136
%@ 1879-0984
%X 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 resolving successfully poxvirus particle morphologies at the scale of single virus particles using light microscopy.
%Z FOR Codes: 60506


%0 Book Section
%A Newsome, Tim
%A Marty, Allison Joy
%A Lynn, Helena
%A Procter, Dean Joseph
%T Navigating the subcellular space: Lessons from vaccinia virus
%B Viral Transport, Assembly and Egress
%D 2011
%C India
%I Research Signpost
%V 
%N 
%P 155-178
%@ 9788130804330
%E Cunningham, Anthony
%E Diefenbach, Russell
%X 
%Z FOR Codes: 110804


%0 Journal Article
%~ PubMed
%A Dodding, Mark P
%A Newsome, Timothy P
%A Collinson, Lucy M
%A Edwards, Ceri
%A Way, Michael
%T An E2-F12 complex is required for IEV morphogenesis during vaccinia infection.
%B Cellular microbiology
%D 2009
%C United Kingdom
%I Wiley-Blackwell Publishing Ltd.
%V 11
%N 5
%P 808-24
%@ 1462-5822
%X The vaccinia virus protein, F12, has been suggested to play an important role in microtubule-based transport of intracellular enveloped virus (IEV). We found that GFP-F12 is recruited to IEV moving on microtubules but is released from virus particles when they switch to actin-based motility. In the absence of F12, although the majority of IEV remain close to their peri-nuclear site of assembly, a small number of IEV still move with linear trajectories at speeds of 0.85 ??m s(-1) , consistent with microtubule transport. Using a recombinant virus expressing GST-F12, we found that the viral protein E2 interacts directly with F12. In infected cells, GFP-E2 is observed on moving IEV as well as in the Golgi region, but is not associated with actin tails. In the absence of E2L, IEV accumulate in the peri-nuclear region and F12 is not recruited. Conversely, GFP-E2 is not observed on IEV in the absence of F12. Ultra-structural analysis of ??E2L- and ??F12L-infected cells reveals that loss of either protein results in defects in membrane wrapping during IEV formation. We suggest that E2 and F12 function as a complex that is necessary for IEV morphogenesis prior to their microtubule-based transport towards the plasma membrane.
%Z FOR Codes: 110804


%0 Journal Article
%~ PubMed
%A Stelzer-Braid, Sacha
%A Oliver, Brian G
%A Blazey, Angus J
%A Argent, Elizabeth
%A Newsome, Timothy P
%A Rawlinson, William D
%A Tovey, Euan R
%T Exhalation of respiratory viruses by breathing, coughing, and talking.
%B Journal of medical virology
%D 2009
%C United States
%I John Wiley & Sons, Inc.
%V 81
%N 9
%P 1674-9
%@ 0146-6615
%X 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 sampled in 50 subjects using a novel mask, and analyzed using PCR for nine respiratory viruses. The exhaled samples 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 samples, 8 talking samples, and in 2 coughing samples. In the subset of exhaled samples 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.
%Z FOR Codes: 110203


%0 Journal Article
%~ PubMed
%A Weisswange, Ina
%A Newsome, Timothy P
%A Schleich, Sibylle
%A Way, Michael
%T The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility.
%B Nature
%D 2009
%C 175 Fifth Ave, New Y
%I Springer-Verlag
%V 458
%N 7234
%P 87-91
%@ 1476-4687
%X 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.
%Z FOR Codes: 110804


%0 Journal Article
%~ PubMed
%A Berger, Jürg
%A Senti, Kirsten-André
%A Senti, Gabriele
%A Newsome, Timothy P
%A Asling, Bengt
%A Dickson, Barry J
%A Suzuki, Takashi
%T Systematic identification of genes that regulate neuronal wiring in the Drosophila visual system.
%B PLoS genetics
%D 2008
%C United States
%I Public Library of Science
%V 4
%N 5
%P e1000085
%@ 1553-7404
%X Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring.
%Z FOR Codes: 111303 60412


%0 Journal Article
%~ PubMed
%A Bracale, Aurora
%A Cesca, Fabrizia
%A Neubrand, Veronika E
%A Newsome, Timothy P
%A Way, Michael
%A Schiavo, Giampietro
%T Kidins220/ARMS is transported by a kinesin-1-based mechanism likely to be involved in neuronal differentiation.
%B Molecular biology of the cell
%D 2007
%C United States
%I American Society for Cell Biology
%V 18
%N 1
%P 142-52
%@ 1059-1524
%X 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.
%Z FOR Codes: 110804


%0 Journal Article
%~ PubMed
%A Arakawa, Yoshiki
%A Cordeiro, João V
%A Schleich, Sibylle
%A Newsome, Timothy P
%A Way, Michael
%T The release of vaccinia virus from infected cells requires RhoA-mDia modulation of cortical actin.
%B Cell host & microbe
%D 2007
%C United States
%I Cell Press
%V 1
%N 3
%P 227-40
%@ 1934-6069
%X 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.
%Z FOR Codes: 110804


%0 Journal Article
%~ PubMed
%A Newsome, Timothy P
%A Weisswange, Ina
%A Frischknecht, Friedrich
%A Way, Michael
%T Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus.
%B Cellular microbiology
%D 2006
%C United Kingdom
%I Wiley-Blackwell
%V 8
%N 2
%P 233-241
%@ 1462-5814
%X 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.
%Z FOR Codes: 110804 110899