The SARS-CoV-2 E protein is an enigmatic viral structural protein with reported viroporin activity associated with the acute respiratory symptoms of COVID-19, as well as the ability to deform cell membranes for viral budding. Like many viroporins, the E protein is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the structure of the protein complex have yielded inconclusive results, suggesting several possible oligomers, ranging from dimers to pentamers. Here, we combined patch-clamp, confocal fluorescence microscopy on giant unilamellar vesicles, and atomic force microscopy to show that E protein can exhibit two modes of membrane activity depending on membrane lipid composition. In the absence or the presence of a low content of cholesterol, the protein forms short-living transient pores, which are seen as semi-transmembrane defects in a membrane by atomic force microscopy. Approximately 30 mol% cholesterol is a threshold for the transition to the second mode of conductance, which could be a stable pentameric channel penetrating the entire lipid bilayer. Therefore, the E-protein has at least two different types of activity on membrane permeabilization, which are regulated by the amount of cholesterol in the membrane lipid composition and could be associated with different types of protein oligomers.

The structural study of plant viruses is of great importance to reduce the damage caused by these agricultural pathogens and to support their biotechnological applications. Nowadays, X-ray crystallography, NMR spectroscopy and cryo-electron microscopy are well accepted methods to obtain the 3D protein structure with the best resolution. However, for large and complex supramolecular structures such as plant viruses, especially flexible filamentous ones, there are a number of technical limitations to resolving their native structure in solution. In addition, they do not allow us to obtain structural information about dynamics and interactions with physiological partners. For these purposes, small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) are well established. In this review, we have outlined the main principles of these two methods and demonstrated their advantages for structural studies of plant viruses of different shapes with relatively high spatial resolution. In addition, we have demonstrated the ability of AFM to obtain information on the mechanical properties of the virus particles that are inaccessible to other experimental techniques. We believe that these under-appreciated approaches, especially when used in combination, are valuable tools for studying a wide variety of helical plant viruses, many of which cannot be resolved by classical structural methods.

We have studied the process of electroporation of bilayer lipid membranes (BLMs) from dioleoylphosphatidylcholine (DOPC). We obtained experimental data on the average lifetime of the membrane as a function of applied voltage in the range of 200–375 mV. The analysis of the data obtained showed that the dependence is nonmonotonic and cannot be described in terms of the classical theory of electroporation. These results are consistent with modern models of the process of through conductive pores formation in a membrane. The above models imply a complex pore energy profile and its dependence on membrane tension and external electric field. Thus, we have shown that the classical theory of electroporation does not satisfy the experimentally observed dependencies of the average membrane lifetime on the applied potential difference and requires further refinement.

We have studied the process of electroporation of bilayer lipid membranes (BLMs) made of dioleoylphosphatidylcholine (DOPC). We obtained experimental data on the average lifetime of the membrane as a function of applied transmembrane voltage in the range of 200–375 mV. The analysis of the obtained data showed that the dependence is nonmonotonic and cannot be described in terms of the classical theory of electroporation. These results are consistent with modern models of the process of formation of through conductive pores in a membrane. The above models imply a complex pore energy profile and its dependence on a membrane tension and an external electric field. Thus, we have shown that the classical theory of electroporation does not satisfy the experimentally observed dependencies of the average membrane lifetime on the applied potential difference and requires further refinement.