Correlative Light and Electron Microscopy of Influenza Virus Entry and Budding

Abstract

Influenza A virus (IAV) entry is a stepwise process regulated by viral and cellular cues, facilitating cellular functions. Virus entry begins by attachment of hemagglutinin to cell surface sialic acids, followed by endocytic uptake, vesicular transport along microtubules, low-pH-mediated viral membrane fusion with the late endosomal membrane, capsid uncoating, viral ribonucleoprotein (vRNP) release, and nuclear import of vRNPs. Here we show a basic methodology to visualize incoming and egressing IAV particles by correlative light and electron microscopy (CLEM). We combine fluorescence microscopy of virus-infected human lung carcinoma A549 cells with high-pressure freezing (HPF) and in-resin fluorescence CLEM and the Tokuyasu CLEM method. This approach forms a basis to study the virus life cycle and virus-host interactions at the ultrastructural level.