Investigating the structure and function of the Ebola virus transcription factor VP30 in order to develop anti-viral therapeutics

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Ebola virus (EBOV) is one of the most serious human pathogens in existence, responsible fordevastating hemorrhagic fevers with fatality rates of up to 70%. The recent outbreak of EBOV in WestAfrica has resulted in over 10,000 human deaths, as well as widespread collapse of essentialinfrastructure and economies of affected countries. The extent and duration of this outbreak, as wellas the massive impact on human well-being, have highlighted the urgent need for effective pre- andpost exposure therapies for EBOV infection. Particularly concerning is the long term healthimplications of EBOV and its ability to be harboured in immune privileged sites such as the eye, thetestes and the central nervous system. Most of the sustained re-emergence of the virus has beencaused by transmission from asymptomatic carriers. Anti-viral therapy is needed to access anddestroy the virus in these immune privileged sites. EBOV is a negative strand RNA virus, whichexpresses 6 proteins. This studentship will investigate the structure and function of one of these,namely VP30. This work will increase our understanding of the basic molecular processes of theEBOV life cycle as well as provide essential information for development of anti-viral treatments.VP30 is an essential building block of the large virus-encoded polymerase complex, which is amachine needed to decipher the information stored within EBOV genes and thus cause disease. Todo its job, VP30 must interact with other EBOV proteins, and also RNA that makes up the EBOVgenome. Our aims are to determine the three-dimensional structure of VP30 using crystallography,and determine how it fits with these other building blocks using X-ray crystallography and EM. Whenwe identify these interaction interfaces, we can design inhibitory compounds that will prevent VP30from making these interactions and thus prevent it from working. To investigate the association ofthese components within cells, we will make use of an EBOV replicon system developed by ourcollaborator Professor Julian Hiscox at The University of Liverpool. This system allows thereconstitution of active EBOV replication complexes able to perform authentic replication andtranscription, but does not require specialized biological containment. We will use confocalmicroscopy to first locate components of the EBOV replication complex in cells, progressing towardssuper resolution imaging to better define their sub-cellular localisation and to investigate the kineticsof the interactions made by components of the complex. We also have the potential to image thesecellular locations using cryo-EM imaging of intact cells. Furthermore, if components of the polymerasecomplex can be expressed recombinantly and purified, high-resolution structures of the complex willbe pursued by cryo-electron microscopy. The experimental plan will test three hypotheses; Firstly,that VP30 makes critical contacts with other virus components though specific binding sites on itssurface. Secondly, that interaction of VP30 with these other components can be disrupted byspecifically designed molecules. Thirdly, that these inhibitory compounds prevent VP30 function in thecontext of a working polymerase in cells, providing a potential anti-Ebola therapeutic.