Journal: J Virol / Year: 2015 Title: Antigenic and Cryo-Electron Microscopy Structure Analysis of a Chimeric Sapovirus Capsid. Authors: Naoyuki Miyazaki / David W Taylor / Grant S Hansman / Kazuyoshi Murata / 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 ...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. IMPORTANCE: 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 ...IMPORTANCE: 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.
History
Deposition
Dec 14, 2015
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Header (metadata) release
Jan 27, 2016
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Map release
Feb 10, 2016
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Update
Feb 8, 2017
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Current status
Feb 8, 2017
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Energy filter - Name: Omega-type / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 15.0 eV
Sample stage
Specimen holder model: GATAN LIQUID NITROGEN
Temperature
Min: 90 K / Max: 105 K / Average: 100 K
Date
Jul 15, 2012
Image recording
Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 80 / Average electron dose: 20 e/Å2 / Od range: 5 / Bits/pixel: 16
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Image processing
CTF correction
Details: Each particle
Final reconstruction
Applied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 6000
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