Dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (N) and membrane (M) proteins of SARS coronavirus

When expressed in mammalian cells, the nucleocapsid (N) and membrane (M) proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) are sufficient to form pseudoparticles. To identify region(s) of the N molecule required for pseudoparticle formation, we performed biochemical analysis of the interaction of N mutants and M in HEK293 cells. Using a peptide library derived from N, we found that amino acids 101–115 constituted a novel binding site for M. We examined the ability of N mutants to interact with M and form pseudoparticles, and our observations indicated that M bound to NΔ(101–115), N1–150, N151–300, and N301–422, but not to N1–150Δ(101–115). However, pseudoparticles were formed when NΔ(101–115) or N301–422, but not N1–150 or N151–300, were expressed with M in HEK293 cells. These results indicated that the minimum portion of N required for the interaction with M and pseudoparticle formation consists of amino acids 301–422.