Chimeric GII.3/GII.6 norovirus capsid (VP1) proteins: characterization by electron microscopy, trypsin sensitivity and binding to histo-blood group antigens

Abstract

GII.3 and GII.6 noroviruses (NoVs) are similar in several aspects, including the presence of a short sequence insertion in the P2 domain of the major capsid protein (VP1) and trypsin susceptibility of VP1-containing virus-like particles (VLPs). In this study, we generated two constructs with the S or P domains of VP1 from GII.3 and GII.6 NoV strains exchanged (GII.3S/GII.6P and GII.6S/GII.3P), and the resultant chimeric capsid proteins were expressed from recombinant baculoviruses. The assembly of VLPs was confirmed by electron microscopy, and the susceptibility of assembled VLPs to trypsin digestion was analyzed by SDS-PAGE. Salivary histo-blood group antigen (HBGA) binding and binding blockade assays were performed to determine the binding characteristics of chimeric VP1-containing VLPs with and without trypsin digestion. Our results indicated that both expressed GII.3S/GII.6P and GII.6S/GII.3P chimeric proteins successfully assembled into VLPs. Trypsin digestion of VLPs assembled from both chimeric proteins led to the generation of two fragments with molecular sizes similar to those of wild-type VP1-containing VLPs. An in vitro salivary HBGA binding assay demonstrated that VLPs assembled from both chimeric proteins exhibited enhanced binding after trypsin cleavage. An HBGA binding blockade assay indicated that the binding of GII.3S/GII.6P and GII.6S/GII.3P VLPs against salivary HBGAs could only be blocked by GII.3 and GII.6 NoV VLP-specific hyperimmune sera, respectively. For GII.6 and GII.3S/GII.6P VLPs, a difference in binding enhancement after trypsin cleavage was observed. Our results demonstrate that the S domains of GII.3 and GII.6 NoV VP1 are interchangeable and that the S domain affects the binding of the P domain to HBGAs.

Mouse intestinal villi as a model system for studies of norovirus infection

“…Determination of trypsin concentration. Some authors suggest that VP1 capsid proteins need to be digested for proper virus infection (Ma et al, 2018;Tan and Jiang, 2006). Intestinal villi-enriched preparation was plated in 96-well cell culture plates with 100 µl of MEM without fetal bovine serum (FBS).…”

A signal-flexible gene diagnostic strategy coupling loop-mediated isothermal amplification with hybridization chain reaction

“…As a response to the urgent demand for more accurate and reliable detection, here the gene segments of NoV GII was specifically employed as the model target [39]. In spite of their high sensitivity, earlier reports like electron microscopy [40,41], immunology-based detection (radioimmunoassay (RIA) [42,43], enzyme-linked immunosorbent assays (ELISA) [44,45]) and nucleic acids biomarkers detection based on gene amplification techniques (PCR [46,47], nucleic acid sequence-based amplification (NASBA) [48,49], LAMP [50,51]) always lack of specificity and false positive results can easily arise. Herein, the powerful exponential amplification of LAMP results in ultrasensitive NoV gene detection with a detection limit of 30 copies, while the HCR gives rise to very good specificity and reliable signal changes in all kinds of readouts.…”