All negative sense RNA viruses (NSRVs) encapsidate their RNA genomes into ribonucleoprotein complexes (RNPs). RNPs are formed as viral Nucleocapsid (N) protein wraps around the genomic (and anti-genomic) RNA. RNPs must associate with their cognate polymerase to form active templates for viral RNA synthesis, resulting in generation of encapsidated replication products and unencapsidated mRNAs. RNP formation is also required for genome packaging into progeny virus particles, and for arenaviruses and bunyaviruses, virus assembly is mediated through direct association between the RNP and viral glycoproteins. RNP formation is thus essential for virus multiplication and therefore represents a potential therapeutic target.
In addition to RNP formation and virus assembly, the N proteins of arenaviruses and bunyaviruses are implicated in other critical functions, many of which relate to interactions with components of the host cell. RNP formation is therefore a process that it is essential to understand, and may provide a target for anti-viral therapy.
Objectives:
Objective 1. Express (in E. coli) and purify recombinant N protein from example bunya and arena viruses such as Oropouche (an Orthobunyanvirus), Seoul virus (a Hantavirus), tomato spotted wilt virus (a Tospovirus), and Lujo virus (an Arenavirus). These will be used for in vitro testing using RNA binding assays e.g. by fluorescence anisotropy, and we will attempt to crystallise them both with and without RNA.
Objective 2. Grow viruses in cell culture from each of the above families (choosing representatives of the appropriate containment level to allow them to be cultured at Leeds), and purify RNPs from live virus for EM work. Initially these will be characterized by 2D negative stain before taking the best examples into 3D cryo-EM.
Objective 3. Use the structural data derived from the first two objectives to generate mutants to test function in cells such as mini-genome assays. Reverse genetics systems are already in use for Bunyamwera virus (in Leeds), Crimean Congo Hemorrhagic Fever virus and Oropouche virus.
Novelty:
Until very recently, nothing was known about the high-resolution structures of N proteins from NSRVs. We, and others, have been using a combination of classical cell and molecular biology with modern structural biology techniques to investigate N protein structure-function relationships to better understand virus biology. While progress in being made towards understanding RNPs, several questions have emerged relating to how RNPs are assembled, and how the encapsidated RNA is replicated by the viral polymerase. We aim to use an interdisciplinary approach to answer these pressing questions.
Timeliness:
The world is under constant threat from emergence of new virus strains including influenza, MERS and topically, Ebola virus. We aim to study the structural biology of RNP assembly and its architecture in two families of segmented NSRVs – Bunyaviruses and Arenaviruses. These are highly pathogenic in their own right and also are closely related to others such as influenza, and more distantly to Ebola, Mumps, Measles and Rabies viruses. Information of RNP structure and function will inform us on basic mechanisms such as control of viral gene expression and evading host immunity, and may therefore suggest routes to new antivirals.
