A Small Region of Porcine Hemagglutinating Encephalomyelitis Virus Spike Protein Interacts with the Neural Cell Adhesion Molecule

Objective: The spike (S) protein of porcine hemagglutinating encephalomyelitis virus (PHEV) may mediate infection by binding to a cellular neural cell adhesion molecule (NCAM). This study aimed to identify the crucial domain of the S1 subunit of the S protein that interacts with NCAM. Methods: Three truncated segments (S₁₋₂₉₁, S₂₇₇₋₇₉₄ and S₅₄₈₋₈₆₈) of the S gene of PHEV and the NCAM gene were cloned individually into the Escherichia coli expression vectors and yeast two-hybrid expression vectors. The interaction between S₁₋₂₉₁, S₂₇₇₋₇₉₄, S₅₄₈₋₈₆₈ and NCAM were detected by a GST pull-down experiment and yeast two-hybrid assay. Results: Three fusion proteins (S₁₋₂₉₁, S₂₇₇₋₇₉₄ and S₅₄₈₋₈₆₈) were screened for their interactions with NCAM by protein-protein interaction assays. The results of these assays clarified that S₂₇₇₋₇₉₄ interacted with NCAM, while S₁₋₂₉₁ and S₅₄₈₋₈₆₈ did not. Conclusions: A small fragment (258-amino-acid fragment, residues 291-548) on the PHEV S protein was posited to be the minimum number of amino acids necessary to interact with NCAM. This fragment may be the receptor-binding domain that mediates PHEV binding to NCAM.