Award details

How does Musashi 1 enhance Zika virus replication?

Reference	BB/V000292/2
Principal Investigator / Supervisor	Dr Trevor Sweeney
Co-Investigators / Co-Supervisors	Dr Fanni Gergely
Institution	The Pirbright Institute
Department	Viral Gene Expression
Funding type	Research
Value (£)	766,415
Status	Current
Type	Research Grant
Start date	01/12/2021
End date	31/07/2024
Duration	32 months

Abstract

Zika virus is a member of the Flaviviridae family of positive-sense single stranded RNA viruses that is spread by mosquitos. A hallmark of Zika virus infection is the destruction of neural progenitor cells in the foetus of infected mothers, and a causal link between Zika virus infection and microcephaly was recently established. Advances in proteomics techniques has made it possible to identify novel viral RNA-protein interactions in relatively high-throughput. However, assessing the role and importance of these interactions remains an important challenge. We demonstrated that an RNA-binding protein, Musashi-1 (MSI1), is enriched in neural progenitor cells and enhances Zika virus replication while a mutation that reduces MSI1 RNA binding is linked to congenital microcephaly. MSI1 is known to positively or negatively regulate expression of specific proteins by binding directly to their mRNAs. However, the role of MSI1 in promoting Zika virus replication is unknown. Furthermore, we found that ZIKV infection reduced binding between MSI1 and its endogenous transcript targets and deregulated their expression. This observation therefore suggests that ZIKV RNA may sequester MSI1 in the infected cells, hence phenocopying MSI1 deficiency. In this project we will elucidate how MSI1 drives viral replication and whether (and how) Zika infection blocks MSI1 function. We will determine at what stage in the replication cycle MSI1 exerts its pro-Zika viral effect, exploiting a Zika virus reverse genetics system in which MSI1 binding sites will be mutated. Using primary cells and cerebral organoid we will examine if the Zika viral genome acts a sink for sequestering MSI1 away from its normal targets and thereby affect neuronal progenitor cell proteostasis and development. In these 'mini-brains' we will also investigate infection characteristics and replication of mutant Zika viruses lacking MSI1 binding sites to assess their potential as attenuated vaccine candidates.

Summary

Viruses spread by insects cause some of the most important emerging human diseases. As the range of their insect hosts expands, increasing populations are becoming at risk to infection. An outbreak of Zika virus, spread by mosquitos, in Central and South America in 2015/2016 was accompanied by an increase in microcephaly cases in new-born babies. Microcephaly is a condition where the head circumference is smaller than usual and is typically associated with developmental defects. A causal link between Zika virus infection and defects in neural cell and brain development has since been firmly established with neural stem cells being particularly susceptible to infection, and destruction, by the virus. Recent evidence has also demonstrated a link between Zika virus infection and long-term cognitive dysfunction, suggesting that both adults and foetuses are at risk of debilitating disease following exposure. Like other viruses, Zika virus can only replicate once it enters a host cell. The virus genome consists of a single piece of nucleic acid called RNA that is replicated and packaged into new virus particles before being released to spread the infection. Virus replication can be positively and negatively affected by proteins present in the infected cell. Many of these are RNA-binding proteins that interact directly with the viral genome. We recently demonstrated that a host protein called Musashi-1 strongly enhances Zika virus replication. Musashi-1 is known to affect the expression of specific proteins by binding directly to their corresponding RNAs. High levels of Musashi-1 are present in the neural precursor cells that are highly susceptible to Zika virus infection. A mutation in Musashi-1 that affects its ability to interact with RNA is also linked to congenital microcephaly, independent of Zika virus infection. Together, our previous findings support the hypothesis that the presence of Musashi-1 in Zika virus infected cells may contribute to the neural destruction and associated brain development defects observed in affected individuals. In this project we will examine the interplay between Musashi-1 and Zika virus using cutting-edge techniques. We will focus on three main questions: 1) At what stage of the Zika virus replication cycle does Musashi-1 exert its effect and can mutation of Musashi-1 binding sites in the Zika virus RNA affect its ability to reproduce in neuronal cells? 2) Does sequestration of Musashi-1 through binding to the Zika virus RNA disrupt the normal function of Musashi-1 in neuronal cell development? 3) Does Musashi-1 promote the ability of Zika virus to replicate in neural stem cells in mini brains? From this work we expect to advance our knowledge of how the presence of a protein that normally promotes neural cell development can make cells more permissive for Zika virus replication. This research can also reveal novel aspects of basic neurodevelopmental processes. Understanding how Musashi-1 enhances Zika virus replication may ultimately help us to develop safer vaccines with reduced risk of damaging side effects.

Impact Summary

Public sector and Societal Impact Arboviruses are a huge public health burden and as the range of their vectors expands a greater proportion of the world's population will become at risk for infection. The link between Zika virus infection and microcephaly may not be the only long-term public health burden. Recent studies have indicated that developmental defect in the absence of microcephaly may also result from Zika virus infection in utero while evidence is also emerging of long-term cognitive impairment in adults flowing infection. The overall cost of the recent Zika virus outbreaks may, therefore, not be yet fully appreciated. Our research seeks to help explain how Zika virus infects and destroys neural cells with such devastating effect. This information can help with designing and developing new therapeutics with reduced risk of damaging side effects which would have significant impact with decreased public care costs. Industrial and Economic Impact Our preliminary data indicates that the presence of Musashi1 may contribute to the enhanced permissiveness of neural precursor cells to Zika virus replication. Vaccine development for controlling flavivirus infection has been hampered by cross reactivity between different viruses and the antibody dependent enhancement effect. Expanding the possibility of using safer live attenuated vaccines may contribute to the eventual development of broadly targeting therapies. We have submitted a national patent for the use of Musashi1 binding site depleted Zika viruses as attenuated vaccine candidate through Cambridge Enterprise, the University of Cambridge research commercialisation arm showing the potential commercial impact of our research. Improving our knowledge of how Musashi1 promotes Zika virus replication may help build a stronger case for how attenuated viruses could be exploited in the future. Training of skilled researchers Two PDRAs will be recruited and will receive extensive training in modern virological andneural model system culture techniques, both of which are in high demand in the UK and internationally. PDRA1 will be trained in molecular biology techniques to study RNA structure and RNA-protein interactions, and virology reverse genetics systems for flaviviruses. PDRA2 will be trained in the production, culture and modification of iPS cells, derived neural precursor cells and neuronal organoids or 'mini-brains'. Training in the use of these cutting-edge methodologies 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.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology, Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/V000292/1 How does Musashi 1 enhance Zika virus replication?

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