PhD Discussions Sessions: Maria Arista

Super-resolution microscopy as a powerful tool for understanding the formation and inhibition of influenza virus structures in mammalian cells

Super-resolution microscopy is a mighty tool that has the ability to study fluorescence samples beyond the diffraction limit, achieving a spatial resolution around 20 nm. The study of viruses can greatly benefit from super-resolution imaging, mainly due to their small size, between 50 and 200 nm. Here we show that, thanks to this technique, we are able to visualize and study two relevant viral structures: filaments of influenza virus using stochastic optical construction microscopy (STORM) and virus-like particles formed from influenza using DNAPAINT (Points accumulation for imaging in nanoscale topography) .
Influenza A virus is highly pleomorphic, and virions can have either spherical or filamentous morphology. Influenza A virus strain A/Udorn/72 (H3N2) produces copious amounts of long and thin filaments on the surface of infected cells, led mainly by the matrix protein M1 and the membrane protein M2. These filaments are strongly related to the infectivity of influenza and cell-to-cell communication, however, due to the small size of these filaments (200 nm of width), they are hard to characterize in detail using immunofluorescence microscopy.

Here, we show with super-resolution microscopy that filament formation was inhibited by the treatment of cells with specific IgG2a and IgG1 antibodies but was not inhibited with the isotype control antibodies. Our results demonstrate that M2e-specific IgGs reduces the level influenza A virus replication in vitro and suggest that the inhibition of virus replication lead by M2especific antibodies is due to the fragmentation of filamentous virions and the loss of filament formation from the surfaces of infected cells.
Moreover, we study virus-like particles produced from influenza proteins transfected on mammalian cells. These structures mimic viruses but they lack viral genetic material, for this reason they are great models to study influenza particles without risks. Influenza expresses 3 different proteins on the surface of the particle and the distribution and homogeneity between particles is not well understood. To study this distribution, we are analyzing with DNA-PAINT the differential expression and distribution of these 3 proteins on the surface of the particles. Overall we show how super-resolution is suitable to study nanoscale viral structures and can provide new insights into anti-viral therapies.

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