Elsevier

Virus Research

Volume 209, 2 November 2015, Pages 87-99
Virus Research

Review
The role of signalling and the cytoskeleton during Vaccinia Virus egress

https://doi.org/10.1016/j.virusres.2015.01.024Get rights and content

Highlights

  • Vaccinia virus recruits kinesin-1 to move on microtubules to the plasma membrane.

  • Vaccinia modulates the cortical actin cytoskeleton by inhibiting RhoA signalling.

  • Vaccinia activates Src and Abl family kinases.

  • Vaccinia recruits a signalling network to induce Arp2/3 driven actin polymerization.

  • Actin polymerization promotes the cell-to-cell spread of Vaccinia.

Abstract

Viruses are obligate intracellular parasites that are critically dependent on their hosts to replicate and generate new progeny. To achieve this goal, viruses have evolved numerous elegant strategies to subvert and utilise the different cellular machineries and processes of their unwilling hosts. Moreover, they often accomplish this feat with a surprisingly limited number of proteins. Among the different systems of the cell, the cytoskeleton is often one of the first to be hijacked as it provides a convenient transport system for viruses to reach their site of replication with relative ease. At the latter stages of their replication cycle, the cytoskeleton also provides an efficient means for newly assembled viral progeny to reach the plasma membrane and leave the infected cell. In this review we discuss how Vaccinia virus takes advantage of the microtubule and actin cytoskeletons of its host to promote the spread of infection into neighboring cells. In particular, we highlight how analysis of actin-based motility of Vaccinia has provided unprecedented insights into how a phosphotyrosine-based signalling network is assembled and functions to stimulate Arp2/3 complex-dependent actin polymerization. We also suggest that the formin FHOD1 promotes actin-based motility of the virus by capping the fast growing ends of actin filaments rather than directly promoting filament assembly. We have come a long way since 1976, when electron micrographs of vaccinia-infected cells implicated the actin cytoskeleton in promoting viral spread. Nevertheless, there are still many unanswered questions concerning the role of signalling and the host cytoskeleton in promoting viral spread and pathogenesis.

Introduction

The hijacking of the cytoskeleton is a common strategy employed by viruses infecting virtually all organisms including bacteria, plants and animals (Dodding and Way, 2011, Erb and Pogliano, 2013, Niehl et al., 2013, Taylor et al., 2011). However, one of the most striking examples of viral subversion of the host actin and microtubule cytoskeletons occurs during Vaccinia virus infection (Dodding and Way, 2011, Welch and Way, 2013). Vaccinia is a large double stranded DNA virus, which replicates exclusively in the cytoplasm of infected cells and is the most studied member of the Orthopoxviridae (Moss, 2007). Vaccinia is perhaps best known for its use as the vaccine to protect against smallpox, a deadly human disease caused by its close relative Variola virus (Jacobs et al., 2009, Walsh and Dolin, 2011). Smallpox was eradicated more than 30 years ago. Nevertheless, Vaccinia is increasingly being used as a vaccine vector for a wide range of different diseases as well as for oncolytic therapies (Jacobs et al., 2009, Kirn and Thorne, 2009, Thorne, 2011, Volz and Sutter, 2013, Walsh and Dolin, 2011). The vaccinia virus genome consists of ∼200 kbp encoding for some 260 proteins, only about 80 of which, end up in infectious intracellular mature virus (IMV) particles (Chung et al., 2006, Resch et al., 2007, Yoder et al., 2006). This large coding capacity, which allows Vaccinia to infect and replicate in many different cell types, is in part due to its complex replication cycle, which involves the assembly of two morphologically distinct types of cytoplasmic virus particles (Fig. 1). The large genome also reflects the prodigious number of viral proteins Vaccinia uses to inhibit or suppress the antiviral activity of its host at all stages of its replication cycle (Haller et al., 2014). This includes inhibiting apoptosis of infected cells before new viral progeny are assembled and minimizing detection by the host immune system (Bahar et al., 2011, Mohamed and McFadden, 2009, Postigo and Ferrer, 2009).

After binding to the cell membrane, virus entry occurs either by direct fusion with the plasma membrane (Carter et al., 2005, Law et al., 2006) or by low-pH endosomal entry pathway (Huang et al., 2008, Townsley et al., 2006). Moreover, Vaccinia actually promotes its uptake by stimulating actin-dependent macropinocytosis (Mercer and Helenius, 2008, Mercer et al., 2010, Schmidt et al., 2011). Having gained access to the cell cytoplasm, expression of early proteins is initiated allowing viral cores to uncoat and release their DNA (Kilcher et al., 2014, Mercer et al., 2012, Schmidt et al., 2013). This early protein expression is required for viral DNA replication, which occurs in viral factories located in a perinuclear region near the microtubule-organizing centre of the infected cell (Ploubidou et al., 2000, Roberts and Smith, 2008). Only after DNA replication does intermediate and late gene expression start, resulting in the assembly of intracellular mature virus (IMV) particles. IMV which represent the majority of viral progeny are infectious but are only released when the infected cell undergoes lysis (Roberts and Smith, 2008). Alternatively, some IMV can become intracellular enveloped virus (IEV) by being ‘wrapped’ by membrane cisternae derived from trans-Golgi or endosomal compartments containing a subset of viral proteins (Roberts and Smith, 2008, Smith et al., 2002). The molecular basis for this envelopment remains to be established, but it involves multiple integral viral membrane proteins as well as the Vaccinia E2 and F12 proteins (Dodding et al., 2009, Domi et al., 2008, Roper et al., 1998, Röttger et al., 1999, Sanderson et al., 1998, Smith et al., 2002, Wolffe et al., 1997, Wolffe et al., 1998). Once formed, IEV are transported to the cell periphery on microtubules by kinesin-1 before fusing with the plasma membrane (Fig. 1). Fusion of IEV with the plasma membrane results in two outcomes that have a different impact on the subsequent spread of infection. The extracellular enveloped virus (EEV), which are infectious and released from the cell, promote the long-range spread of vaccinia. Alternatively, after their fusion with the plasma membrane, some virions remain attached to the outside of the cell and are known as the cell-associated enveloped virus (CEV). It is the CEV that are responsible for the local actin-dependent cell-to-cell spread of vaccinia (Fig. 1). In this review, we will discuss our current understanding of how vaccinia uses and manipulates the cytoskeleton of the cell to enhance its spread.

Section snippets

IMV and IEV move on microtubules

Microtubule-based transport is the primary way in which cargoes are moved over micron distances in a directed fashion throughout the cell (Franker and Hoogenraad, 2013, Fu and Holzbaur, 2014, Stephens, 2012). It is perhaps not surprising then that viruses have developed numerous strategies to take advantage of this cellular transport system at all stages of their infection cycles (Dodding and Way, 2011, Greber and Way, 2006, Radtke et al., 2006). Moreover, the ability of large viruses, such as

F11 enhances viral spread by inhibiting RhoA signalling

When IEV reach the cell periphery, they cannot fuse with the plasma membrane until they traverse the cortical actin cytoskeleton, a dense mesh of actin filaments associated with the cytoplasmic face of the plasma membrane (Biro et al., 2013, Charras et al., 2006, Clark et al., 2013, Fritzsche et al., 2013). The actin cortex provides a cell with mechanical resilience (Clark et al., 2013, Fritzsche et al., 2013, Gauthier et al., 2012, Salbreux et al., 2012, Tinevez et al., 2009) and plays an

Src and Abl phosphorylation of A36 induces actin polymerization

Nearly 40 years ago, analysis of vaccinia infected cells in the electron microscope revealed the presence of virus particles on the tip of large microvilli projecting from the plasma membrane (Stokes, 1976). These projections appeared late in infection and contained actin, α-actinin, fimbrin and filamin but not tropomyosin or myosin (Hiller et al., 1981, Hiller et al., 1979, Krempien et al., 1981). These studies were essentially forgotten until 1995, when it was demonstrated that vaccinia is

Acknowledgements

We thank Drs. David Barry and Jasmine Abella in the Way lab, London Research Institute, Cancer Research UK for comments on the MS. F.L. and M.W are supported by funding from Cancer Research UK.

References (137)

  • N.C. Gauthier et al.

    Mechanical feedback between membrane tension and dynamics

    Trends Cell Biol.

    (2012)
  • U.F. Greber et al.

    A superhighway to virus infection

    Cell

    (2006)
  • S.L. Haller et al.

    Poxviruses and the evolution of host range and virulence

    Infect. Genet. Evol.

    (2014)
  • Y. Handa et al.

    Vaccinia virus F11 promotes viral spread by acting as a PDZ-containing scaffolding protein to bind myosin-9A and inhibit RhoA signaling

    Cell Host Microbe

    (2013)
  • G. Hiller et al.

    Fluorescence microscopical analysis of the life cycle of vaccinia virus in the chick embryo fibroblasts

    Exp. Cell Res.

    (1981)
  • G. Hiller et al.

    Interaction of assembled progeny pox viruses with the cellular cytoskeleton

    Virology

    (1979)
  • A.C. Humphries et al.

    Clathrin potentiates vaccinia-induced actin polymerization to facilitate viral spread

    Cell Host Microbe

    (2012)
  • Y. Ivarsson

    Plasticity of PDZ domains in ligand recognition and signaling

    FEBS Lett.

    (2012)
  • B.L. Jacobs et al.

    Vaccinia virus vaccines: past, present and future

    Antiviral Res.

    (2009)
  • S. Kilcher et al.

    siRNA screen of early poxvirus genes identifies the AAA+ ATPase D5 as the virus genome-uncoating factor

    Cell Host Microbe

    (2014)
  • U. Krempien et al.

    Conditions for pox virus-specific microvilli formation studied during synchronized virus assembly

    Virology

    (1981)
  • K. Luck et al.

    The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains

    FEBS Lett.

    (2012)
  • H.K. Matthews et al.

    Changes in Ect2 localization couple actomyosin-dependent cell shape changes to mitotic progression

    Dev. Cell

    (2012)
  • J. Mercer et al.

    RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection

    Cell Rep.

    (2012)
  • T. Omelchenko et al.

    Myosin-IXA regulates collective epithelial cell migration by targeting RhoGAP activity to cell-cell junctions

    Curr. Biol.

    (2012)
  • A. Postigo et al.

    Viral inhibitors reveal overlapping themes in regulation of cell death and innate immunity

    Microbes Infect.

    (2009)
  • W. Resch et al.

    Protein composition of the vaccinia virus mature virion

    Virology

    (2007)
  • K.L. Roberts et al.

    Vaccinia virus morphogenesis and dissemination

    Trends Microbiol.

    (2008)
  • G. Salbreux et al.

    Actin cortex mechanics and cellular morphogenesis

    Trends Cell Biol.

    (2012)
  • N. Scaplehorn et al.

    Grb2 and nck act cooperatively to promote actin-based motility of vaccinia virus

    Curr. Biol.

    (2002)
  • D.E. Alvarez et al.

    The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin-based motility

    J. Cell. Biol.

    (2013)
  • T. Aoyama et al.

    Cayman ataxia protein caytaxin is transported by kinesin along neurites through binding to kinesin light chains

    J. Cell. Sci.

    (2009)
  • Y. Araki et al.

    The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport

    Embo J.

    (2007)
  • M. Biro et al.

    Cell cortex composition and homeostasis resolved by integrating proteomics and quantitative imaging

    Cytoskeleton (Hoboken)

    (2013)
  • E.A. Burton et al.

    Abl kinases regulate actin comet tail elongation via an N-WASP-dependent pathway

    Mol. Cell. Biol.

    (2005)
  • G.C. Carter et al.

    Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans

    J. Gen. Virol.

    (2005)
  • G.T. Charras et al.

    Reassembly of contractile actin cortex in cell blebs

    J. Cell. Biol.

    (2006)
  • E. Chieregatti et al.

    Myr 7 is a novel myosin IX-RhoGAP expressed in rat brain

    J. Cell. Sci.

    (1998)
  • C.S. Chung et al.

    Vaccinia virus proteome: identification of proteins in vaccinia virus intracellular mature virion particles

    J. Virol.

    (2006)
  • J.V. Cordeiro et al.

    F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection

    PLoS One

    (2009)
  • S. Cudmore et al.

    Actin-based motility of vaccinia virus

    Nature

    (1995)
  • S. Cudmore et al.

    Vaccinia virus: a model system for actin-membrane interactions

    J. Cell Sci.

    (1996)
  • V. Doceul et al.

    Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions

    J. Gen. Virol.

    (2012)
  • V. Doceul et al.

    Repulsion of superinfecting virions: a mechanism for rapid virus spread

    Science

    (2010)
  • M.P. Dodding et al.

    A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome

    Embo J.

    (2011)
  • M.P. Dodding et al.

    An E2-F12 complex is required for IEV morphogenesis during vaccinia infection

    Cell. Microbiol.

    (2009)
  • M.P. Dodding et al.

    Coupling viruses to dynein and kinesin-1

    Embo J.

    (2011)
  • A. Domi et al.

    Vaccinia virus E2L null mutants exhibit a major reduction in extracellular virion formation and virus spread

    J. Virol.

    (2008)
  • J.L. Duteyrat et al.

    Ultrastructural study of myxoma virus morphogenesis

    Arch. Virol.

    (2006)
  • M.A. Franker et al.

    Microtubule-based transport - basic mechanisms, traffic rules and role in neurological pathogenesis

    J. Cell Sci.

    (2013)
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