ReviewThe role of signalling and the cytoskeleton during Vaccinia Virus egress
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.
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