The host interactome of the Rift Valley fever virus RNA-dependent RNA polymerase: targets for inhibition of viral replication?

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Abstract	Rift Valley fever virus (Bunyavirales, Peribunyaviridae) or short RVFV is emerging arbovirus that can cause disease and death in humans and animals. There are no drugs to treat infection, and during outbreaks time frames for vaccination efforts might be too short. It is a WHO priority pathogen, and WHOA listed virus. Replication of the negative strand viral genome involves the RNA-dependent RNA polymerase L, which is a protein of over 100kD. Here we propose to study the host protein interactome of L, to assess what cellular partners and pathways are required for its activity. Tagged L protein has been used in the past, and even introduced into virus by reverse genetics; this approach be used here for producing tagged L to be used in virus- free minigenome systems that mimick virus replication, and wlld type RVFV with a tagged L that be used to infect human cells where the full life cycle can be completed to assess the spectrum of L interactors regardless of context. LC-MS-based proteomics will be used to identify protein interactions of L.Silencing or KO of interactors can then be used to assess their importance, and suitability as targets (or indirectly, targeting cellular pathways they are involved in).
Where does the project lie on the Translational Pathway?	T1 – Basic Research
Expected Outputs	Data on the RVFV L protein interactome. Contribution of host interactors on virus replication and virus life cycle. Information on targets for therapeutic intervention. Publication expected.
Training Opportunities	Conferences & seminars; hands-on proteomics sample preparation, proteomics data analysis and statistics.
Skills Required	Basic understanding of virus replication; some skills in cell culture and molecular biology.
Key Publications associated with this project	Gestuveo, R. J., J. Royle, C. L. Donald, D. J. Lamont, E. C. Hutchinson, A. Merits, A. Kohl* *, M. Varjak. Analysis of Zika capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for establishing infection. Nature Communications 12: 2766. 2021. Co-corresponding authors, *Lead contact
	Alexander A. J. T., M. P. Confort, S. Desloire, J. I. Dunlop, S. Kuchi, V. B. Sreenu, D. Mair, G. S. Wilkie, A. Da Silva Filipe, B. Brennan, M. Ratinier, F. Arnaud, A. Kohl. Development of a reverse genetics system for Toscana virus (lineage A). Viruses 12: 441. 2020.
	Alexander A. J. T., M. Salvemini, V. B. Sreenu, J. Hughes, E. L. Telleria, M. Ratinier, F. Arnaud, P. Volf, B. Brennan, A. Kohl. Characterisation of the antiviral RNA interference response to Toscana virus in sand fly cells. PLOS Pathogens 19(3): e1011283. 2022.
	Hosmillo M., J. lu, M. R. McAllaster, J. B. Eaglesham, X. Wang, E. Emmott, P. Domingues, Y. Chaudry, T. J. Fitzmaurice, M. K. Tung, M. D. Panas, G. McInerney, N. Locker, C. B. Wilen, I. G. Goodfellow. Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation. eLife: e46681. 2019.
	Stewart H., Y. Lu, S. O’Keefe, a. Valpadashi, L. D. Cruz-Zaragoza, H. A. Michel, S. K. Nguyen, G. W. Carnell, N. Lukhovitskaya, R. Milligan, I. Jungreis, V. Lulla, A. D. Davidson, D. A. Matthews, S. High, P. Rehling, E. Emmott, J. L. Heeney, James R. Edgar, G. L. Smith, A. E. Firth. The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity. BioRxiv doi: https://doi.org/10.1101/2022.11.15.516323