A clot to uncover: SARS-CoV-2 nucleocapsid can outcompete the FOXP3 forkhead domain for DNA binding in vitro.

Abstract

During COVID-19, systemic coagulopathy can lead to strokes and embolisms and may also contribute to long COVID. This coagulopathy is the result of overactivated platelets in circulation that lead to inappropriate clot formation. FOXP3 is a transcription factor involved in platelet development. Loss of FOXP3 function leads to abnormal platelets resembling those seen during COVID-19. Thus, FOXP3 may be dysregulated in COVID-19. The SARS-CoV-2 nucleocapsid (NC) is a multifunctional protein typically associated with viral genome packaging and virion assembly. However, it is also capable of binding DNA and may alter host gene expression. Here, potential interactions between the DNA-binding forkhead domain (FHD) of FOXP3 and the SARS-CoV-2 NC were investigated. Identification of a novel interaction between FOXP3 and SARS-CoV-2 NC may provide new clues to the pathophysiology of COVID-19. To address this aim, both proteins were overexpressed in T7 E. coli, purified via immobilised metal affinity chromatography, and monitored for interactions in the absence and presence of DNA using pull-down assays, electrophoretic mobility shift assays, and fluorescence anisotropy. A direct interaction was identified between the two proteins in the absence of DNA in vitro. Additionally, both proteins were found to bind DNA simultaneously under limiting conditions, but competed for binding under saturating conditions, where excess NC led to dissociation of FHD from the FHD-NC-DNA complex. This result implicates NC in FOXP3 dysfunction, potentially contributing to the coagulopathy and other symptoms observed during COVID-19. This work may inform future therapeutic strategies for severe COVID-19.