TY  - GEN
CY  - Heidelberg
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/27540/
Y1  - 2020///
TI  - Structural studies of influenza A virus by cryo-electron tomography
AV  - public
ID  - heidok27540
A1  - Peukes, Julia
N2  - Influenza A virus (IAV) is a pleomorphic, enveloped virus known for its yearly epidemics and occasional, but fatal pandemics. The outer surface glycoprotein hemagglutinin (HA) together with the matrix protein 1 (M1) are the most abundant protein components of assembled virions. HA, located at the outside of virions, is involved in cell receptor recognition, membrane fusion and is the most relevant protein for antibody binding. Therefore the structure of isolated HA has been extensively characterised by X-ray crystallography. However, it remains unclear to which extent the structure of isolated HA corresponds to the in situ HA structure on the surface of IAV. M1 determines the morphology of the virus by forming a matrix layer underneath the viral membrane. A high resolution structure of full length M1 is missing and the lack of information about the in situ arrangement of the M1 matrix layer currently limits our understanding of how M1 functions.
Here, I set out to determine the structures of HA and M1 directly from IAV particles using high resolution cryo-electron tomography (cryoET) and subtomogram averaging. I found that virus purification can affect the integrity of the virus HA glycoprotein layer and the morphology of virus particles. I therefore adapted a workflow which allows studying the structure of viral proteins directly from viruses in the vicinity of virus-producing cells. Biosafety regulations required inactivation of IAV samples by chemical fixation prior to cryoEM imaging. To assess effects of fixation, I complemented structural studies of HA from pathogenic, fixed IAV particles with studies of HA from non-infectious, unfixed virus-like particles (VLPs). These studies revealed that fixation captures HA in an open conformation while HA structures determined from unfixed samples perfectly match the closed conformation observed in the trimeric crystal structure. In concordance with recent work by others, this observation suggests that fixation captures HA in a an open, otherwise transient conformation, which is part of a constant opening and closing motion known as breathing motion.
To characterise the in situ structure and arrangement of M1, I established a subtomogram averaging workflow to cope with the challenges presented by the small size of M1. I successfully obtained two independent structures of M1 directly from viruses and VLPs. Comparisons of my structures to existing high resolution models of the N-terminal domain (NTD) of M1 revealed that M1 monomers arrange as parallel strands, with a helical propensity and directly underneath the membrane. For the first time, my data allow to describe the M1-membrane interface as well as relevant M1-M1 interfaces within the matrix layer. Finally, I have gained first structural insights into the M1 C-terminal domain (CTD). I further combined the obtained structural information for M1 with a theoretical model of the mechanics of M1 polymerization and membrane deformation during virus assembly. The obtained results suggest that linear polymerization of M1 into multiple parallel strands efficiently provides energy to drive assembly of new virus particles.
The results presented in this thesis improve our understanding of the arrangement and structure of the two influenza proteins HA and M1 in situ which has implications for current models of HA-mediated membrane fusion, virus architecture and virus assembly.
ER  -