title: The interactome of Rift Valley fever phlebovirus- towards the identification of new intervention strategies
Creators: name: Mottram, Timothy James
Publisher: name: University of Glasgow
Year: 2018
Subjects: subject: Rift Valley fever phlebovirus, emerging, Zika, interactome, proteomics, WNT signalling, nucleocapsid, mutagenesis.

description: Rift Valley fever phlebovirus (RVFV) is an ongoing threat to both humans and animals across the continent of Africa. RVFV is a member of the Phlebovirus genus and Phenuviridae family, within the Bunyavirales order. Members of the Phlebovirus genus are characterised by a negative sense tripartite RNA genome. The large (L) segment encodes the RNA-dependent RNA polymerase (L), the medium (M) segment encodes the two glycoproteins Gn and Gc, and the small segment (S) encodes the nucleocapsid (N) protein and the non-structural protein NSs. The N protein performs a number of important functions, including encapsidation of the viral genome allowing viral RNA replication and transcription. Research into N protein-protein interactions has been limited. The work presented in this thesis characterises previously unidentified functional residues of RVFV N protein and describes new insights into virus-host protein-protein interactions. Two previously uncharacterized N protein residues, F11 and F149, when substituted for alanine, performed all its known functions; Encapsidation of the viral genome, N-N multimerisation and L protein interaction. However, utilising a minigenome assay still showed these mutants lack replication capacity. This indicates that currently unknown interactions with these residues are disrupted. Furthermore, a proteomics study on N protein immunoprecipitated from lung epithelial A549 cell infections was performed to identify RVFV N interaction partners, revealing 23 potential candidates. A subsequent siRNA knockdown of candidates identified β-catenin, Polyadenylate binding protein 1 and 4, Annexin 1 and 2, and Scaffold attachment factor B as important for functional viral replication. Previous research indicated β-catenin, the effector molecule of the WNT pathway, was involved with RVFV replication. Utilising a TOPFlash reporter assay, it was determined that the WNT pathway, of which β-catenin is the effector molecule, was inhibited by RVFV infection. The generation of a CRISPR-Cas9 β-catenin knockout cell line provided a useful tool for further study into N protein-protein and RVFV-β-catenin interactions. The knockout of β-catenin significantly reduced RVFV replication, similarly to siRNA-mediated knock down. Additionally, it was observed through the use of confocal microscopy that upon infection with RVFV, β-catenin relocalised from the plasma membrane to a diffuse pattern across the cytoplasm. Furthermore, during the course of this study, it was investigated whether RVFV N protein can affect mosquito antiviral pathway(s), similarly to yellow fever virus (genus flavivirus) capsid protein. Using alphavirus Semliki Forest virus (SFV) as a model, allowing work to be carried out in a CL-2 lab setting, it was found that N protein does not possess such properties. However, Zika virus (genus flavivirus) capsid protein (ZIKV C) showed significant proviral properties, however, this effect did not occur via disruption of the siRNA pathway, the most efficient mosquito antiviral mechanism, as evidenced by ZIKV C having no effect within our siRNA assay. To summarise, the data in this thesis reveals new interactions between RVFV nucleocapsid protein and mammalian host proteins that are important for RVFV replication. It provides a basis for future research on RVFV (or phleboviruses, in general) nucleocapsid research. The disruption of RVFV N-host protein interactions or direct disruption of N function could lead to new therapeutic strategies against this important emerging virus.