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

Our aim is to understand a new mechanism of protein production by viruses of clinical and economical importance and examine how it contributes to escaping detection from the host.

Cells within the body respond to external stimuli in many ways, the most common of which is via the regulation of gene expression. In response to external stresses such as infection, cells can pause protein synthesis, or translation, and thus the decoding of genetic information. This defense mechanism allows cells to survive by limiting the use of energy and nutrients that protein synthesis requires until the stress is resolved. It also blocks the spread of viruses as viruses are dependent on host cell resources to produce viral proteins and replicate. Because of this, viruses have developed strategies to produce their proteins using alternative mechanisms.

Using different viruses as models, we have previously made significant advances in identifying new mechanisms that viruses use to manipulate the host cell by regulating translation. Viruses transmitted by mosquitoes represent a major burden on human and animal health. Among these, dengue virus is a prominent human health threat causing millions of infections every year worldwide, with no broadly effective or specific treatment. We previously showed that dengue virus infection results in a block in host protein synthesis, yet it remains a mystery how viral proteins are translated to support viral propagation. This is important because understanding how viruses hijack host resources can reveal a new Achilles' heel in the viral armour.

Based on our results, we propose that dengue virus proteins are translated via a novel mechanism that uses a known cellular translation factor, eIF3, in a non-conventional manner to mediate translation. We also propose that this mechanism could help dengue virus escape detection by the host. Therefore, our objectives are to 1- characterize how eIF3 interacts with the viral RNA; 2- elucidate how this contributes to a new mechanism of translation and 3- establish how this mechanism competes with non-self detection of the viral RNA by the host.

From this work we expect to advance our knowledge of how viruses take control of host resources to ensure viral proteins are made. This mechanism is novel, and we think it represents a new line of counterdefence evolved by viruses. Therefore, it can help identify new ways to inhibit virus replication and develop novel antiviral therapies for an important group of viruses. Understanding the fundamental mechanisms of gene regulation is important for virologists, but also for broader academic communities. It may also help us to appreciate better a basis of several pathologies, such as cancer or neurodegenerative diseases, that are linked to alternative translation mechanisms.

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 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 (in high UK demand). PDRA2 will be equipped the unique skillset of reconstituting translation in vitro from native factors (few laboratories worldwide can do this, including NL/TS). 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.