Presence of a surface-exposed loop facilitates trypsinization of particles of Sinsiro virus, a genogroup II.3 norovirus

Noroviruses (NoVs) are the causative agents of nonbacterial acute gastroenteritis in humans. NoVs that belong to genogroup II (GII) are quite prevalent and prone to undergo recombination, and their three-dimensional structure is not yet known. Protein homology modeling of Sinsiro virus (SV), a member of the GII.3 NoVs, revealed the presence of a surface-exposed 20-amino-acid (aa) insertion in the P2 domain of the capsid protein (CP) relative to the Norwalk virus (NV) CP, which is a well known hot spot for mutations to counter the host immunological response. To further characterize the role of the long insertion in SV, the capsid protein gene was expressed using the recombinant baculovirus system. Trypsinization of the resultant virus-like particles yielded two predominant bands (31.7 and 26.1 kDa) in sodium dodeeyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. N-terminal sequencing and analysis of the mass spectroscopic data indicated that these fragments correspond to residues 1 to 292 (26.1 kDa) and 307 to 544 (31.7 kDa). In addition, the above data taken together with the comparative modeling studies indicated that the trypsin cleavage sites of the Sinsiro virus CP, Arg292 and Arg307, are located at the beginning of and within the 20-aa insertion in the P2 domain, respectively. This study demonstrates that the presence of the surface-exposed loop in the GII.3 NoVs facilitates the trypsinization of the capsid protein in the assembled form. The SV particles remain intact even after trypsin digestion and retain the suggested receptor binding linear epitope of residues 325 to 334. The above results are distinct from those obtained from the trypsinization studies performed earlier on the NV (GI) and VA387 (GII) viruses, both of which lack the large surface insertion and associated basic residues. These new observations may have implications for host receptor binding, cell entry, and norovirus infection in general.