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Abstract

Influenza A virus (IAV) poses a persistent threat to human and animal health, causing annual epidemics and devastating pandemics responsible for millions of deaths. The emergence of pandemic variants is closely tied to co-infection events involving zoonotic and human IAVs, which can lead to reassortment of their segmented genomes. This genetic exchange can produce new variants with novel antigenic properties and enhanced host adaptation. The increasingly intensive factory farming of poultry and swine amplifies the chances for co-infection events, and each is a dice roll for a new pandemic strain. This threat underscores the need to understand the molecular mechanisms of IAV assembly and reassortment.

A critical aspect of the IAV replication cycle is the assembly process. Each progeny virus must incorporate all eight unique genome segments to be replication competent. To achieve this, it subverts the Rab11-regulated recycling endosome to transport viral ribonucleoproteins (vRNPs) from the nucleus, where they are replicated, to the plasma membrane, where budding occurs. On this route, vRNPs undergo phase-separation and form liquid organelles characterized by high local concentrations of vRNPs. This process has been extensively studied, yet key questions remain unanswered: Where and how are vRNP bundles formed? What is the structural organization of this process? Are other viral or cellular components involved? This thesis aims to address these questions using in situ cryo-electron tomography.

Surprisingly, I discovered that the sites of vRNP clustering were predominantly associated with intracellular membranes, likely originating from endoplasmic reticulum, that were heavily decorated with the viral glycoprotein hemagglutinin (HA). The HA proteins engaged in head-to-head interactions, inducing a reorganization of the membranes into zipper-like double membrane structures that exhibited diverse morphologies and sizes. This membrane remodeling was highly conserved and solely driven by HA. To quantify vRNP clustering, I developed a cluster identification algorithm which revealed that vRNPs form clusters comprising fewer than eight vRNPs on these membranes. This suggests that HA membranes serve as platforms for the formation of vRNP bundles.

Notably, this study also yielded several intriguing serendipitous findings. Matrix protein 1, which determines the virus particle shape by forming a matrix layer in virions, formed multilayered, helical coils in the nuclei of infected cells that disassembled in the cytoplasm. During budding, it initiated the formation of the matrix layer around vRNPs prior to attachment to the plasma membrane, which hints towards a mechanism to ensure that eight vRNPs are incorporated into budding virions.

In sum, this thesis highlights the power of high resolution, in situ imaging using cryo-electron tomography. By directly visualizing cellular processes in their native environment, this work has revealed fundamental mechanisms underlying of IAV assembly, laying the groundwork for future research and opening new avenues for investigation.