Influenza A virus (IAV) is an important human pathogen that causes annual epidemics and occasional pandemics. Like many enveloped viruses, IAV interacts with host membranes and lipids at multiple stages of its replication cycle, frequently altering lipid homeostasis to facilitate viral replication. IAV utilizes Rab11a for the membrane trafficking of its segmented genome, which is divided into eight viral ribonucleoproteins (vRNPs). While vRNP sorting during traf-ficking has been demonstrated, the associated membrane remodeling has not been charac-terized in situ using cryo-electron tomography. Moreover, although IAV assembles at choles-terol-enriched plasma membrane domains, the lipid distribution in infected cells remains in-completely understood. Here, we applied in situ cryo-electron tomography to study membrane remodeling during IAV assembly and budding. Our results reveal that HA- and NA-containing membranes mediate Rab11a-dependent, membrane-assisted vRNP clustering, indicating that genome assembly is guided by specific membrane contexts rather than by HA alone. The characteristic 7 + 1 vRNP bundle forms during budding, coordinated by M1 layer assembly that precedes plasma membrane attachment. Intracellular M1 forms multilayered helical assemblies of antiparallel dimers that likely serve as a reservoir for budding. These findings uncover a membrane-guided mechanism coordinating genome assembly and virion formation in IAV. Together with our collaborators at the Luxembourg Institute of Science and Technology, we have established a novel cryo-secondary ion mass spectrometry (cryo-SIMS) technology. This method is now ready to map lipid distributions and membrane organelles in both uninfect-ed and IAV-infected cells. It will, for the first time, enable visualization of cholesterol distribution within the 3D context of cellular membranes under native conditions. Using this approach, we aim to elucidate the role of cholesterol distribution and nanodomains in membranes remodeled by IAV infection.
Petr Chlanda (Thu,) studied this question.