Vaccinia virus (VACV) generates multiple distinct forms of infectious virus. Intracellular mature virus (IMV) particles are surrounded by a single lipid membrane and are mostly released upon lysis of the infected cell. Some IMVs are wrapped by an additional double membrane to form intracellular enveloped virus (IEV), also called wrapped virus (WV). IEVs are transported on microtubules to the cell surface where the outer membrane fuses with the plasma membrane (PM) to expose a double enveloped virion outside the cell. Extracellular enveloped virus (EEV) and CEV are surrounded by two membranes and are morphologically indistinguishable, but whereas EEV are released from the cell to mediate long range virus spread, CEV are retained on the cell surface and induce the formation of actin tails to propel virions to neighbouring cells. EEV and CEV are essential for efficient virus spread within the host. This thesis concerns viral and cellular factors that influence transport of IEV particles from the site of wrapping to the cell membrane.

For transport to the cell surface, IEVs rely on active microtubular transport, mediated by the cellular motor protein kinesin-I and the viral proteins E2, F12, which form a complex, and A36. While some of the players in this process are known, other aspects remain elusive, such as the mechanism by which E2/F12 are recruited to IEV, because they lack transmembrane regions. To study factors affecting IEV egress, a proteomic study was performed with A36, E2 and F12 as bait, and many potential interaction partners were identified. Amongst them, two cellular phosphoinositide (PI) phosphatases, myotubularin related protein (MTMR) 1 and su(var), enhancer of zeste, trithorax (SET)-binding factor (SBF) 1 were identified as major hits for the joint interactome of E2 and F12. This thesis has investigated the potential role of these and other cellular proteins in IEV egress.

MTMR1 and SBF1 both co-precipitated with E2 and F12 only when both viral proteins were present and required the full length E2 protein. The co-purification of the catalytically active myotubularin MTMR1 with the E2/F12 complex required the inactive SBF1. Knockout (KO) of SBF1 was found to reduce virus spread, EEV titres and CEV numbers, confirming its involvement in IEV morphogenesis or egress. Interestingly, KO of MTMR1 did not cause a phenotype, likely because of functional redundancy between different myotubularins (MTMs). Indeed, when MTMR1 and MTMR2 were both deleted, a similar phenotype to that of SBF1 KO cells was observed. While a level of redundancy seems to exist between the active forms, the KO of the inactive myotubularin SBF2 in SBF1 KO cells did not further the defect in spread. The defect in spread observed in both SBF1 KO and MTMR1/2 double KO cells was dependant on the presence of F12/E2 and resulted from an accumulation of IEV at the sites of wrapping, partially recapitulating the phenotype observed in infections with viruses lacking E2 or F12. Furthermore, the contribution of myotubularins to IEV egress depended on the presence of the CC domain of SBF1 and MTMR1, that allows them to heterodimerise, their membrane recruitment domains (RID) and the phosphoinositide phosphatase activity of MTMR1. All together, these data show VACV hijacks phosphoinositide signalling through the recruitment of myotubularins to promote IEV egress and supports endosomes as a source of membranes for wrapping of IEVs. Finally, we also identified a role for PI3P regulation in IMV morphogenesis.