Heterologous expression of human norovirus GII.4 VP1 leads to assembly of T=4 virus-like particles

Abstract

Human noroviruses are a leading cause of acute gastroenteritis, yet there are still no vaccines or antivirals available. Expression of the norovirus capsid protein (VP1) in insect cells typically results in the formation of virus-like particles (VLPs) that are morphologically and antigenically comparable to native virions. Previous structural analysis of norovirus VLPs showed that the capsid has a T=3 icosahedral symmetry and is composed of 180 copies of VP1 that are folded into three quasi-equivalent subunits (A, B, and C). In this study, we determined the cryo-EM VLP structures of two GII.4 variants, termed CHDC-1974 and NSW-2012. Surprisingly, we found that greater than 95% of these GII.4 VLPs were larger than virions and 3D reconstruction showed that these VLPs exhibited T=4 icosahedral symmetry. We found that the T=4 VLPs showed several structural differences to the T=3 VLPs. The T=4 particles assemble from 240 copies of VP1 that adopt four quasi-equivalent conformations (A, B, C, and D) that form two distinct dimers, A/B and C/D. The T=4 protruding domains were elevated ~21-Å off the capsid shell, which was ~7-Å more than the previously studied GII.10 T=3 VLPs. A small cavity and flap-like structure at the icosahedral twofold axis disrupted the contiguous T=4 shell, a consequence of the Dsubunit S-domains having smaller contact interfaces with neighboring dimers.Overall, our findings that old and new GII.4 VP1 sequences assemble T=4 VLPs might have implications for the design of potential future vaccines.

Norovirus Capsid Protein-Derived Nanoparticles and Polymers as Versatile Platforms for Antigen Presentation and Vaccine Development

“…The structures of norovirus VLPs have been thoroughly studied via electron cryo-microscopy (cryo-EM) [8,9] and X-ray crystallography [10]. Noroviral capsids are composed of 180 VP1s that organize in a T = 3 icosahedral symmetry.…”

N-terminal VP1 Truncations Favor T = 1 Norovirus-Like Particles

“…However, in a follow-up study, GI.4 Chiba mutants were shown to form 23 nm or T = 1 particles, putatively due to freezing and thawing of preparations or pH-dependent processes [53]. Previously, T = 4 particles were identified in hNoVLP preparations [20,21]. Interestingly, one study included GII.2 Snow Mountain virus forming T = 1 particles [21].…”

“…Additional hNoVLPs were investigated to pinpoint whether the truncation seen in GII.4 Saga causes T = 1 formation. Norovirus-like particle polymorphism has been described as putatively genotype-dependent [20,21,40]. Therefore, we extended our sampling to GII.10 Vietnam and GII.17 Saitama (Figure 1).…”

“…However, VP1 60-mers of T = 1 symmetry have been described as byproducts of hNoVLP production coexisting with other particle sizes and independent of the expression system [19]. Recently, VP1 240-mers of T = 4 symmetry have also been described so far only for GII.4 variants expressed in insect cells as well as in plants [20,21]. In studies on virions of different norovirus variants T = 3 as well as T = 1, formations were detected [22].…”

N-terminal VP1 truncations favorT=1 norovirus-like particles

“…However, VP1 60-mers of T =1 symmetry have been described as byproducts of hNoVLP production coexisting with other particle sizes and independent of the expression system (19). Recently, also VP1 240-mers of T =4 symmetry have been described so far only for GII.4 variants expressed in insect cells as well as in plants (20, 21). In studies on virions of different norovirus variants T =3 as well as T =1 formations were detected (22).…”