Analyzing Influenza A Virus Entry Through Lattice Light Sheet Microscopy

Influenza A virus (IAV) is an RNA virus that causes the flu in birds and some mammals, including humans.  Human influenza virus usually refers to those subtypes that spread widely among humans: H1N1, H1N2, and H3N2 are the only known influenza A virus subtypes currently circulating among humans.

A research group led by Prof. Allen Liu at the University of Michigan, USA, used  ZEISS Lattice Lightsheet 7  to study  cellular entry mechanisms of this virus. They focused on the membrane-bending protein epsin.  Their findings support the hypothesis that epsin plays a biomechanical role in IAV entry.

Part of the lab focuses on investigating intracellular trafficking using various state of the art live cell imaging and super resolution fluorescence imaging techniques. They are particularly interested in clathrin-mediated endocytosis (CME), a process by which cells uptake proteins and other macromolecules by forming budded structures coated with clathrin on the plasma membrane. Various viruses, specifically viruses like Influenza A virus, SARS CoV-2, and HIV, can hijack CME and similar processes to gain access to the cell.

In their recent work, Prof. Liu's team investigated the role of epsin, a membrane bending protein, in enabling IAV entry via CME. It has been shown that upon IAV binding to the plasma membrane, epsin can specifically bind to cell surface ubiquitinated receptors. They showed that epsin’s interaction with proteins at the site of IAV binding initiated its membrane bending mechanism. Their findings show the ability of IAVs to hijack activity of membrane bending proteins to initiate membrane bending and receptor-mediated endocytosis for cellular entry.

The team utilized various high resolution live cell imaging techniques to study IAV entry in engineered cell lines with epsin and clathrin proteins tagged with fluorescent proteins. Using lattice light sheet imaging, they were able to visualize viral entry and how IAVs interact with epsin and clathrin at the plasma membrane.

Since the technique  utilizes  lattice light sheets which excite a thin region of the cell, they could reduce the background noise created by out-of-focus excited fluorescence proteins. This allowed the generation of high-quality viral particle tracks from which they could determine the amount of epsin and clathrin recruitment associated with the viruses. Light sheet microscopy also reduces the amount of photobleaching which enables the continuous  imaging of cells for a longer period without compromising  the signal quality.