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

Ebola virus (EBOV) is one of the most serious human pathogens in existence, responsible for
devastating hemorrhagic fevers with fatality rates of up to 70%. The recent outbreak of EBOV in West
Africa has resulted in over 10,000 human deaths, as well as widespread collapse of essential
infrastructure and economies of affected countries. The extent and duration of this outbreak, as well
as the massive impact on human well-being, have highlighted the urgent need for effective pre- and
post exposure therapies for EBOV infection. Particularly concerning is the long term health
implications of EBOV and its ability to be harboured in immune privileged sites such as the eye, the
testes and the central nervous system. Most of the sustained re-emergence of the virus has been
caused by transmission from asymptomatic carriers. Anti-viral therapy is needed to access and
destroy the virus in these immune privileged sites. EBOV is a negative strand RNA virus, which
expresses 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 the
EBOV 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 a
machine needed to decipher the information stored within EBOV genes and thus cause disease. To
do its job, VP30 must interact with other EBOV proteins, and also RNA that makes up the EBOV
genome. 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. When
we identify these interaction interfaces, we can design inhibitory compounds that will prevent VP30
from making these interactions and thus prevent it from working. To investigate the association of
these components within cells, we will make use of an EBOV replicon system developed by our
collaborator Professor Julian Hiscox at The University of Liverpool. This system allows the
reconstitution of active EBOV replication complexes able to perform authentic replication and
transcription, but does not require specialized biological containment. We will use confocal
microscopy to first locate components of the EBOV replication complex in cells, progressing towards
super resolution imaging to better define their sub-cellular localisation and to investigate the kinetics
of the interactions made by components of the complex. We also have the potential to image these
cellular locations using cryo-EM imaging of intact cells. Furthermore, if components of the polymerase
complex can be expressed recombinantly and purified, high-resolution structures of the complex will
be 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 its
surface. Secondly, that interaction of VP30 with these other components can be disrupted by
specifically designed molecules. Thirdly, that these inhibitory compounds prevent VP30 function in the
context of a working polymerase in cells, providing a potential anti-Ebola therapeutic.