The polymerization of actin beneath the virus particle provides a driving force that helps to enhance the cell-to-cell spread of the virus. In the absence of cell lysis, newly assembled vaccinia virions are largely only released from the Lifitegrast infected cell, when IEV fuse with the plasma membrane. However, to reach the plasma membrane the IEV first have to traverse the cortical actin cytoskeleton, which consists of an extremely dense arrangement of actin filaments beneath the plasma membrane. This dense actin cortex represents a significant physical barrier to IEV, given their large size. Vesicles and secretory granules also face a similar problem during exocytosis. A variety of studies have shown that the remodelling of cortical actin as well as active actin polymerization plays an essential role in facilitating exocytosis. It is therefore not surprising given their size, that the release of IEV from infected cells is also dependent on the assembly and organization of the cortical actin cytoskeleton. In addition, vaccinia appears to enhance its release from infected cells in culture by modulating the cortical actin by inhibiting RhoA signalling to mDia, a key regulator of actin polymerization. The virus achieves this by encoding F11, a protein that mimicks ROCK to interact with RhoA to inhibit its downstream signalling. F11-mediated inhibition of RhoA signalling is also responsible for stimulating virus-induced cell migration, which may also help to enhance the spread of infection. Our previous observations on the role of F11 in promoting viral release are based on the effects of over expressing dominant negative and activated RhoA and mDia clones coupled with pharmalogical approaches to modulate RhoA-mDia signalling and the actin cytoskeleton. To extend these observations and directly investigate whether that F11-mediated inhibition of RhoA signalling promotes viral release and spread we have generated recombinant viruses Perphenazine lacking F11 or expressing an F11 mutant, which is deficient in binding RhoA. We found that loss of F11 or its ability to bind RhoA significantly reduces the release of infectious virus from infected cells.