title: Antigenic and Cryo-Electron Microscopy Structure Analysis of a Chimeric Sapovirus Capsid.
abstract: The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S domain forms a scaffold surrounding the nucleic acid, while the P domains form a homodimer that interacts with receptors. The P domain is further subdivided into two subdomains, termed P1 and P2. The P2 subdomain is likely an insertion in the P1 subdomain; consequently, the P domain is divided into the P1-1, P2, and P1-2 subdomains. In order to investigate capsid antigenicity, N-terminal (N-term)/S/P1-1 and P2/P1-2 were switched between two sapovirus genotypes GI.1 and GI.5. The chimeric VP1 constructs were expressed in insect cells and were shown to self-assemble into virus-like particles (VLPs) morphologically similar to the parental VLPs. Interestingly, the chimeric VLPs had higher levels of cross-reactivities to heterogeneous antisera than the parental VLPs. In order to better understand the antigenicity from a structural perspective, we determined an intermediate-resolution (8.5-Å) cryo-electron microscopy (cryo-EM) structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that the P domain dimers were raised slightly (∼5 Å) above the S domain. The VP1 homology model allowed us predict the S domain (67-229) and P1-1 (229-280), P2 (281-447), and P1-2 (448-567) subdomains. Our results suggested that the raised P dimers might expose immunoreactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes.
  
  
  OBJECTIVE
  We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera than the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5-Å resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. The overall structure of Yokote/Mc114 contained features common among other caliciviruses. We showed that the P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had fewer interactions.
authors.affiliation: National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Miyazaki
authors.affiliation: National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi, Japan Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Taylor
authors.affiliation: Schaller Research Group at the University of Heidelberg and the DKFZ, Heidelberg, Germany Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany g.hansman@dkfz.de kazum@nips.ac.jp.
authors.forename: Grant S
authors.initials: GS
authors.lastname: Hansman
authors.affiliation: National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi, Japan g.hansman@dkfz.de kazum@nips.ac.jp.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Murata
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D003429:Cross Reactions; D020285:Cryoelectron Microscopy; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D011995:Recombination, Genetic; D029341:Sapovirus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000648:ultrastructure*; D022701:Virosomes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000648:ultrastructure*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; D022701:Virosomes