Over the years, we have identified the main players involved in vaccinia-induced actin polymerisation.
During vaccinia infection, a proportion of cell-associated enveloped viruses (CEV) attached to the plasma membrane induce Arp2/3 complex driven actin polymerisation to enhance their spread into neighbouring cells.
CEV achieve this feat by locally activating Src and Abl family kinases, which results in the phosphorylation of tyrosine 112 and 132 of A36, an integral viral membrane protein localised beneath the virus in the plasma membrane.
Phosphorylation of these two tyrosine residues leads to the recruitment of a signalling network, consisting of Grb2, Nck, WIP and N-WASP, the latter of which locally activates the Arp2/3 complex, stimulating actin polymerisation beneath the virus.
Recruitment of this signalling network is not unique to vaccinia, as it is also at the heart of a number of actin dependent cellular processes, including the formation of invadopodia during tumour cell invasion. Vaccinia therefore provides an excellent model to understand exactly how a co-operative phosphotyrosine-based Nck and N-WASP signalling network stimulates actin polymerisation.
Over the years, we have identified the main players involved in vaccinia-induced actin polymerisation (Welch and Way, 2013;Cell Host Microbe. 14: 242-55) and also uncovered an unexpected role for clathrin in enhancing this process (Humphries et al., 2012; Cell Host Microbe. 12: 346-59). Nck and N-WASP are essential, as they are required to recruit the WIP:N-WASP complex to the virus and activate the Arp2/3 complex respectively.
In contrast, the role of WIP in vaccinia actin tail formation has been more obscure. We have now found that WIP, or its homologue WIRE are required for N-WASP recruitment and actin-based motility of the virus (Donnelly et al., 2013; Current Biol. 23: 999-1006).
Using a combination of biochemical approaches and expression of mutants in cells lacking endogenous Nck, WIP or N-WASP we have obtained essential insights into the hierarchy and connections within this network.
WIP contains two Nck binding sites and is recruited to the virus, bound to N-WASP, by interacting with the second SH3 domain of Nck. N-WASP also contains two Nck binding sites, but its recruitment is dependent on its interaction with WIP rather than Nck. The first and third SH3 domains of Nck are not required to recruit the WIP:N-WASP complex but are involved in promoting actin assembly.
Our observations have established that WIP plays an essential role in linking Nck to N-WASP (Donnelly et al., 2013). Curiously, however, the interaction between Nck and N-WASP is not required for vaccinia actin tail formation, suggesting that an additional factor regulates the ability of N-WASP to activate the Arp2/3 complex beneath the virus.