The battle for the 5'end: dissecting a novel virus-specific translation mechanism driven by eIF3

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This work will provide a novel understanding of strategies evolved by viruses to commandeer the translation machinery and how these compete with other regulatory processes. It may enable new antiviral therapies for the control of viral infection.

Dengue virus (DENV), and related mosquito-borne flaviviruses, are important pathogens of humans and animals. They induce global shut-off of host translation during infection, yet they maintain translation of their own proteins via a previously uncharacterized translation pathway that we recently began investigating. We now present robust preliminary evidence that DENV translation may be driven by novel interactions between the viral RNA 5' end and the cellular protein eIF3, suggesting a new specialized role for this translation factor. Importantly, the virus 5' end is a prime regulatory site targeted by the host for non-self detection and also acts in the switch from translation to replication.

Our aim is to understand the molecular basis for this novel eIF3-driven mechanism of viral translation and how it competes with other regulatory processes. First, we will characterize how DENV motifs in the 5' UTR coordinate the recruitment of eIF3, positioning it in direct contact with the 5' end using biochemical methods. We will also establish the importance of this interaction for DENV replication and its conservation across flaviviruses. Next, we will elucidate how this contributes to the DENV translation pathway by investigating translation initiation complex composition at different time points during infection, reconstituting initiation in vitro from purified components, and by revealing the structure of initiation complexes using cryoEM. Finally, we will dissect the competition for the 5' end between eIF3 and other host proteins that regulate DENV replication and non-self sensing.

The preliminary data presented in this application, and the experiments planned to build on our findings, will lead to a step change in our understanding of the regulation of translation by viruses and the host response to infection. This research will have a direct scientific impact in the fields of virology, translational control and virus-host interactions. As mosquito-borne flaviviruses are important human and animal pathogens, our work may identify new targets for treatment of these economically important infections and therefore has the potential to impact on UK health, society and economy.

Industrial and Economic Impact
Understanding the mechanistic details by which microbial pathogens interact with the translation machinery has long been a source of antimicrobial drugs. It has led to the development of highly successful broad-spectrum antibiotics targeting the bacterial ribosome (i.e neomycin, chloramphenicol, tetracycline). Likewise, we will emulate new avenues to develop antivirals that specifically block viral translation for a group of viruses lacking efficient control strategies. Importantly, and beyond viral systems, specialized translation mechanisms relying on canonical factors, carrying out novel regulatory function are increasingly associated with diseases such as cancer. Therefore our work may have broad significance for human health.
In addition, understanding how viruses commandeer the translation machinery has previously enabled for repurposing of viral elements for the design of expression vectors. Gene therapy or protein expression vectors rely on virus-derived IRES to drive gene expression, while polycistronic constructs often include self-cleaving viral protein elements (i.e FMDV 2A). Therefore, identifying a novel mechanism that confers viruses a translational advantage over the host cell will expand the tool set of regulatory elements used in the design of expression or gene therapy vectors.

Public sector and Societal Impact
The flavivirus DENV is the most significant mosquito-borne virus with nearly half the world's population at risk. It is responsible for 390 million infections annually in humans, including 96 million cases of dengue fever and up to 500,000 cases of the potentially fatal haemorrhagic fever. Related flaviviruses are also important pathogens of humans and animals, such as the re-emerged Zika virus associated with developmental disorders or West Nile virus responsible for encephalitis in horses. Our research has the potential to deliver impact by better understanding of these important pathogens of both humans and animals. The findings from our work will be publicised via the University press office and outreach activities to raise awareness in the general public. The co-PI already has good relationships with TV and radio channels through multiple appearances locally, nationally and internationally during coverage of the Zika and Ebola outbreaks.

Training of skilled researchers
Two PDRAs will be recruited and will receive extensive training in modern biochemical and structural techniques to dissect translation. PDRA1 will be trained in molecular biology techniques to study RNA-protein interactions, and virology reverse genetics systems for flaviviruses, including manipulating Schedule 5 pathogens in BSL3 containment facilities. PDRA2 will be equipped the unique skillset of reconstituting translation in vitro from native factors (few laboratories worldwide can do this, including NL/TS in the UK). S/he will also master challenging structural methodologies to dissect biological complexes through collaborating with a leading expert in cryoEM. This holistic set of skills will prepare the PDRAs for challenges relevant to a wide range of careers both in academia or industry, increasing their career prospects. In addition, our laboratories regularly host both undergraduate and post-graduate students, who will also benefit from exposure to the BBSRC funded research.