Localization-Based Super-Resolution Microscopy Reveals Relationship between SARS-CoV2 Spike and Phosphatidylinositol (4,5)-bisphosphate.

Localization microscopy circumvents the diffraction limit by identifying and measuring the positions of numerous subsets of individual fluorescent molecules, ultimately producing an image whose resolution depends on the uncertainty and density of localization, and whose capabilities are compatible with imaging living specimens. Spectral resolution can be improved by incorporating a dichroic or dispersive element in the detection path of a localization microscope, which can be useful for separation of multiple probes imaged simultaneously and for detection of changes in emission spectra of fluorophores resulting from changes in their environment. These methodological advances enable new biological applications, which in turn motivate new questions and technical innovations. As examples, we present fixed-cell imaging of the spike protein SARS-CoV2 (S) and its interactions with host cell components. Results show a relationship between S and the lipid phosphatidylinositol (4,5)-bisphosphate (PIP2). These findings have ramifications for several existing models of plasma membrane organization.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function.

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.