N-terminal insertion peptides produce varied effects over the cleavage and assembly of norovirus major capsid protein VP1.

The N-terminal cleavage of the norovirus major capsid protein VP1 during in vitro expression is a widely observed phenomenon, yet the underlying mechanisms remain poorly understood. This study aimed to determine how N-terminal insertion sequences affect the cleavage and assembly of virus-like particles (VLPs). To this end, a series of recombinant GII.6 VP1 proteins with varied N-terminal insertion peptides were constructed and expressed using a baculovirus expression system. The expression, integrity, and assembly status of these proteins were analyzed using Western blot (WB), SDS-PAGE, transmission electron microscopy (TEM), and peptide fingerprinting analysis. Furthermore, a recombinant protein with a N-terminal FLAG tag was also constructed and expressed to investigate the characteristics of N-terminal cleavage. Our findings indicate that varied N-terminal insertion peptides produced different cleavage patterns with some peptide sequences showing inhibition of N-terminal cleavage. N-terminal FLAG-tagged fragment was not detected in cell lysate, further suggesting the complexity of the N-terminal cleavage. These results provide new insights into the molecular mechanisms of VP1 processing and its implications for virus-like particle (VLP) assembly.