Chemical synthesis, calcium-templated in-vitro folding and competitive ELISA assay reveal that only domain 1 of the LRAD3 ectodomain interacts with the severe acute respiratory syndrome-related corona virus 2 (SARS-CoV-2) spike NTD, while domains 2 and 3 do not, providing valuable insight into ACE2-independent viral entry pathways that may contribute to the development of new therapeutic approaches targeting SARS-CoV-2 strains associated with severe neurological complications.

Growing evidence of post-COVID neurological complications, such as encephalopathy, neurodegeneration, and cognitive impairment, suggests severe acute respiratory syndrome-related corona virus 2 (SARS-CoV-2) viral infection into the central nervous system (CNS). Therefore, understanding the mechanisms of viral entry into the CNS, where human angiotensin-converting enzyme 2 (ACE2) is barely expressed, is critical for addressing the neurological consequences of COVID-19. Importantly, the low-density lipoprotein receptor class A domain containing 3 (LRAD3) is overexpressed in brain cells, suggesting a possible ACE2-independent alternate pathway of viral entry into brain cells. Herein, the interaction of the chemically synthesized LRAD3 domains with SARS-CoV-2 spike protein is reported. It is observed that the extracellular domains of LRAD3 depend on calcium for proper folding and maintaining their structural integrity. The results reveal that domain 1 of LRAD3, which is most accessible from the cell surface, engages with the N-terminal domain of the viral spike protein. These findings open up possibilities to develop new therapeutic strategies targeting ACE2 independent viral entry pathways.