Unveiling viral structures by single-molecule localization microscopy

Abstract

[eng] Influenza A virus is one of the most outstanding human viruses. The major treatments against influenza are small analogues, monoclonal antibodies and vaccines, however, due to the fast mutation of the seasonal influenza strain, these methods are easily outdated, so vaccine production and antiviral development need to be in continuous growth and study to improve immunity and fight against influenza disease. The characterization of the viral structure and the identification of the mechanisms of action of newly synthesized antivirals are crucial to develop fast and powerful treatments, nonetheless, due to the small size of viruses, conventional fluorescence techniques are lacking the resolution power to resolve individual viral structures. In this context, super-resolution microscopy has positioned in the last decade as a powerful technique to characterize viral constructs by achieving resolutions up to 10 nm. Here, we have optimized and established a type of super-resolution microscopy technique entitled single-molecule localization microscopy (SMLM) to study viral structures at single-particle level by characterizing several viral structures, antivirals and vaccines. Firstly, we could characterize the filament formation of influenza virus and described how monoclonal antibodies disturbed the development of those filaments, deforming them. Further, we could relate this malformation with an inhibition of the infectivity, suggesting the crucial role of filament formation in the infectivity of influenza. Moreover, we optimized and implemented a novel SMLM called DNA-PAINT in the study of the target distribution and quantification in the nanoscale, validating this method using commercial nanoparticles for its further implementation in the study of the expression of recombinant proteins of influenza and the corresponding virus-like particle produced. In addition, we studied and identified several other viral structures and antivirals interactions using SMLM such as the distribution of Hepatitis B and Hepatitis Delta on paraffin tissues, the interaction of analogues of sialic acid on four strains of influenza and the uptake of nanovaccines from antigen-presenting cells, obtaining features on how these viruses and antivirals interact in order to produce a smart design of antivirals and vaccines, corroborating how SMLM could increase the knowledge of the mechanism of action of viruses.