Structural studies of the influenza genome and its interactions with human innate immune factors

Thesis

Influenza viruses cause an estimated 3-5 million severe cases of contagious respiratory disease and regularly lead to up to 650,000 deaths annually according to the World Health Organisation, posing a significant burden to global public health systems. Despite decades of research, our structural understanding of key viral components remains incomplete. Central to the viral lifecycle is the transcription and replication of the segmented negative sense viral RNA genome, packaged into the viral ribonucleoprotein complex (vRNP). The vRNP comprises one copy of viral RNA corresponding to one genome segment, one copy of the heterotrimeric influenza polymerase complex, and multiple copies of the viral nucleoprotein. The nucleoprotein serves the dual purpose of protecting viral RNA from host immune recognition, while also facilitating genome transcription and replication, making it both essential for viral function and a prime target for the host innate immune response.

The mode of RNA and immune factor binding to the nucleoprotein remains structurally unresolved. Further, despite significant effort both in this laboratory and in others, the native structure of the vRNP remains unknown.

During this DPhil, I have aimed to answer these questions using biochemical, biophysical, and structural techniques. In this thesis, I present the structure of a previously-unsolved avian influenza A virus nucleoprotein using cryo-electron microscopy single particle analysis. I biochemically and biophysically characterise the interaction of two human innate immune factors with susceptible avian influenza A virus nucleoproteins and show that the interaction of these immune factors is selective for avian strains. I describe novel approaches to solving vRNP structure, a method of coating influenza virus vRNPs with binding factors of interest, and present a low-resolution 3D reconstruction of the vRNP.

The findings presented in this thesis advance our understanding of influenza virus architecture, host-pathogen interactions, and demonstrate the successful application of a number of structural techniques to the study of structural virology.

Authors: Whitehead, J

• DOI: 10.5287/ora-dodj6bbdb
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: influenza; nucleoprotein; X-ray Crystallography; MxA; vRNP; Cryo-ET; BTN3A3; immune factor; Cryo-EM
• Subjects: Structural Virology; Cryo-EM