Influenza A virus hemagglutinin remodels membranes into a vRNP clustering platform

Pandemics caused by influenza A virus are associated with co-infections of zoonotic and human-adapted viruses. To form an infectious entity, the virus must incorporate eight different ribonucleoproteins (vRNPs) into virions budding at the plasma membrane. The process of vRNP clustering is poorly understood and has not been investigated at a molecular resolution. Here we employ in situ cryo-correlative light and electron microscopy at late stages of viral infection to identify hemagglutinin and neuraminidase as drivers of membrane remodelling. We show that hemagglutinin, but not neuraminidase-remodelled compartments serve as vRNP clustering platforms. Our nearest-neighbour analysis demonstrates that vRNPs cluster on hemagglutinin-remodelled membranes in a Rab11a-dependent manner. Additionally, we uncover that matrix protein 1 (M1) forms multi-layered cylindrical assemblies in the nucleus which restructure in the cytoplasm. Further, we show that M1 layer assembly precedes its attachment to the plasma membrane and M1 thereby coordinates the closure of the virion budding neck. In summary, we demonstrate that hemagglutinin is involved in intracellular vRNP clustering by inducing membrane remodelling to provide a large interaction surface. Thus hemagglutinin-membrane remodelling may play a role during vRNP sorting and reassortment that lead to the adaptation of zoonotic influenza A viruses.