High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization

Abstract

Many viruses form highly pleomorphic particles; in influenza, these particles range from spheres of ~ 100 nm in diameter to filaments of several microns in length. Virion structure is of interest, not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. Detailed images of virus morphology often rely on electron microscopy, which is generally low throughput and limited in molecular identification. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline to image many thousands of individual influenza virions, gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. This large-scale analysis revealed that influenza particles can be reliably characterised by length, that no spatial frequency patterning of the surface glycoproteins occurs, and that RNPs are preferentially located towards filament ends within Archetti bodies. Our analysis pipeline is versatile and can be adapted for use on multiple other pathogens, as demonstrated by its application for the size analysis of SARS-CoV-2. The ability to gain nanoscale structural information from many thousands of viruses in just a single experiment is valuable for the study of virus assembly mechanisms, host cell interactions and viral immunology, and should be able to contribute to the development of viral vaccines, anti-viral strategies and diagnostics.

Single influenza A viruses induce nanoscale cellular reprogramming at the virus-cell interface

“…We then investigated the intensity distribution of the immobilized IAV particles, which shows a single peak that overlaps with the intensity distribution of 100 nm fluorescent beads, recorded in a parallel experiment (Figure 1C). This indicates that virus particles did not aggregate during the labelling and immobilization procedure in spite of the pleomorphism of IAVs 13,14 . Together, this one-step silanization protocol provides a homogenously distributed viral attachment pattern on the microscopy glass slides.…”

The nanoscale organization of the Nipah virus fusion protein informs new membrane fusion mechanisms

“…Nonetheless, a collaborative effort of multiple copies of viral envelope proteins is favorable for overcoming the energy barrier required for membrane fusion. Nanoclusters formed by viral glycoproteins have been observed for HIV-1 27,28 , Herpes virus 29 , and Influenza virus 30 . The viral restriction factor, serine incorporator protein 5 (SERINC5), inhibits HIV-1 fusion by disrupting the HIV env nanoclusters, highlighting the importance of the nanoclusters in the function of the viral glycoproteins 31 .…”

Optimized production and fluorescent labeling of SARS-CoV-2 virus-like particles

“…The incorporation of other viral or cellular proteins could also be considered for tuning particle assembly. In another study, McMahon et al 32 used high-throughput super-resolution microscopy to measure the size of the SARS-CoV-2 particles, showing that the virus’s diameter distribution is mono-dispersed and centered at 143 nm. This size is in accordance with the one we found using FCS measurements of M(GFP)NES VLPs size in solution (Fig.…”