The structure and function of native bunyavirus ribonucleoproteins

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Viruses have the potential to cause global crisis, as illustrated by the recent COVID-19 pandemic. In view of future pandemic preparedness, the World Health Organisation has published a list of just 11 viruses that are considered a high priority for research, and this includes 3 viruses from the order Bunyavirales (also known as bunyaviruses) highlighting their importance to worldwide public health. The genetic material of bunyaviruses is made of RNA, which is covered by the viral nucleoprotein and interacts with the viral polymerase (the viral protein responsible for producing more viral RNA). Together, RNA, nucleoprotein (NP) and polymerase form the ribonucleoprotein complex, or RNP. A primary function of this complex is to allow expression of the viral genes, allowing the virus to hijack the cell and force it into making new viruses; thus, the RNP plays a central role in the infection process and disease. However, many fundamental aspects of bunyavirus RNP structure remain unknown, such as how the nucleoprotein units connect with each other, and how the polymerase is attached to the RNA chain. To understand these important aspects of RNP structure, this proposal will build on our recently published ground-breaking results on the structure of the NP-RNA complex from the model bunyavirus, Bunyamwera virus (BUNV). In this work we purified RNPs from infectious viruses and characterised them using a combination of microscopy approaches. This allowed us to generate an atomic model of the NP-RNA chain, which conclusively showed for the first time that it is a flexible helix, and this flexibility is critical for many viral functions. The model showed how the NP molecules link up to form a helical chain, revealing the molecular basis for RNP flexibility. We confirmed our model by using a non-infectious system known as a replicon, which allowed us to mutate regions of the NP involved in forming this flexible helix, and show that these changes caused a reduction in RNP function.In this proposal we aim to study, for the first time, the structure of RNPs from the most pathogenic bunyaviruses.First, we will improve our protocol for purifying BUNV RNPs, to improve on our published structure, as well as visualizing RNPs that also contain the viral polymerase. Then we will use advanced electron microscopy and atomic force microscopy approaches (both of which allow close to atomic resolution of molecules) to improve our understanding of the NP arrangement within the RNP, and to characterise how and where the viral polymerase is attached within the RNA chain.Secondly, we will use the expertise we have gained with BUNV to generate RNP structures from the highly pathogenic bunyaviruses on the WHO priority list. To do this we will take advantage of less pathogenic viruses within the same virus families, a valid approach since the RNP structures of species within each family are known to be similar. As for BUNV, we will purify large amounts of these viruses, purify their RNPs and image them using microscopy approaches. This will allow us to generate high resolution models of the different RNPs, which we will test using our lab-based replicon systems. To translate our findings to the most pathogenic bunyaviruses, we will computationally adapt our models to the viruses listed by the WHO, and we will test these models using replicon systems specific for these viruses.Finally, for one bunyavirus, we will directly image on-going RNA synthesis by the RNP within the context of an RNP. Of note this information is completely lacking for any bunyavirus. This will allow us to correlate recently atomic models of the viral polymerase, with their corresponding conformations within an authentic RNP.Overall, we will provide an understanding of the arrangement of the RNPs of some of the most dangerous viruses in existence and of their mechanism of RNA synthesis. In turn, this will enhance epidemic preparedness against these viruses.