Structure and Function of RNA Binding Proteins from Negative Sense-RNA Viruses

Simbu orthobunyavirus (SIMV) is a member of the Peribunyaviridae family and is a segmented negative-sense RNA virus. Transmitted by mosquitos this family of viruses can cause outbreaks across the globe infecting humans and animals causing fever, birth defects and great economic loss. Despite on-going outbreaks there is no antiviral or FDA-approved vaccine for use in humans. The nucleoprotein (NP) functions to encapsidate the viral RNA genome for protection and is a suitable target for structure- based drug design. This thesis presents five high resolution crystal structures of NP in complex with RNA from two viruses of the Peribunyaviridae family; SIMV and Akabane orthobunyavirus (AKAV). We critically analysed the interactions formed between NP and RNA and further assessed their RNA binding contributions in fluorescence anisotropy (FA) assays with comparison to closely related Oropuche Orthobunyavirus (OROV). The structures presented here lay the foundations for structure-based drug design of small molecules that interfere with critical residues for ribonucleoprotein functioning.
Ebolavirus (EBOV) is a non-segmented, negative sense RNA virus within the Filoviridae family of the Mononegavirales order. EBOV is a highly contagious and virulent pathogen, transmitted by mosquitos and bodily fluids. The largest EBOV outbreak in 2013-2016 (40% mortality) highlighted the need for an effective therapeutic. Structure- based drug design has arisen as an investigation method for antiviral drug candidates. A better understanding of structure, multimerization and interactions of viral proteins will aid therapeutic development. Viral protein (VP) -35 functions within the ribonucleoprotein complex, interacting with NP to maintain NP in a monomeric state for newly synthesised RNA encapsidation. VP30 is an activator of viral transcription and is regulated by phosphorylation. The oligomeric state of both proteins is currently debated within the literature. This thesis optimises the expression and purification of both VP35 and VP30 from EBOV and closely related Marburg marburgvirus (MARV) to confirm their oligomeric states and interactions with RNA by FA.
Human orthopneumovirus (hRSV) is a non-segmented, negative sense RNA virus classified within the Mononegavirales order and Pneumovirade family, transmitted by aerosol droplets. hRSV is the leading cause of lower respiratory tract illness in infants and the immunocompromised, causing over 250,000 death annually. Up to 79% of deaths are reported in children under five, with 99% of the mortality occurring in developing countries. The M2-1 protein of hRSV represents a promising potential anti- viral target. M2-1 is a transcription anti-terminator with an essential role in viral gene expression; binding both viral RNA and the polymerase co-factor phosphoprotein (P). Here, we describe the optimised protocol for M2-1 and its known binding partner P90- 160 purification, and the less well characterised binding partner M (matrix protein). Structural studies using X-ray crystallography revealed a 1.99 Å structure of M but no ligand. We confirmed the interaction of M2-1 and M. We implemented structural studies by electron microscopy with M2-1:P90-160 complex to fully characterise this interaction.