Cordeiro, J. (2009) Modulation of Rho GTPase signalling during vaccinia virus infection. Doctoral thesis, UCL (University College London).
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Infection by vaccinia virus leads to reorganization of the actin cytoskeleton, changes in cell adhesion, loss of contact inhibition and stimulation of cell motility. Previous work by the group has led to the identification of a viral gene, F11L, which is highly conserved among the orthopoxvirus genomes and is essential for this virus-induced cell mobility. F11L has been shown to interact with the RhoA but does not interact with the two other 'classical’ members of the family Rac1 and Cdc42. Understanding the molecular interaction of F11L and RhoA as well as investigating additional Rho binding partners was the basis of my thesis project with a view to obtain additional insights into the role of Rho GTPase manipulation during the vaccinia life cycle. Deletion of F11L from the virus genome results in reduced virus release and smaller plaque sizes. Characterization of the interaction between F11L and RhoA revealed that it occurs via a region of limited sequence homology to the RhoA effector ROCKI, in the C-terminal part of the molecule. Interestingly, generation of a recombinant mutant virus (F11L-VK) that is unable to bind RhoA leads to an intermediate phenotype between ΔF11L and WR in terms of viral plaque size. Further bioinformatic analysis revealed a region in the N-terminus of F11L with limited sequence homology to hDia2C, a RhoD effector. Biochemical analysis led to the identification of this site as the RhoD binding domain in F11L. Importantly, this study revealed that F11L interacts directly with different Rho GTPases via unique motifs to induce cell motility and facilitate enhanced virus cell-to-cell spread. Furthermore, work done in collaboration with the laboratory of Mariano Esteban in Madrid using a mouse model of infection shows that in contrast to wild-type vaccinia, infection with ΔF11L does not result in animal death. Additionally, the F11L-VK virus shows limited spread in vivo but still induces animal death albeit at a later stage when compared to wild-type vaccinia. These results suggest that F11L has additional functions besides interacting with RhoA, consistent with my observations that it can interact with other Rho GTPases. These studies can provide valuable information about the importance of Rho GTPase manipulation for the life cycle of vaccinia virus but also indicate the potential for the genetic manipulation of F11L in existing poxvirus vectors that may improve their therapeutic potential, safety, immunogenicity and/or oncolytic activity.
|Title:||Modulation of Rho GTPase signalling during vaccinia virus infection|
|Additional information:||Supplementary data is included on a CDROM. Authorisation for digitisation not received|
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