Journal: Vet Res / Year: 2016 Title: Structural analysis and insertion study reveal the ideal sites for surface displaying foreign peptides on a betanodavirus-like particle. Authors: Junfeng Xie / Kunpeng Li / Yuanzhu Gao / Runqing Huang / Yuxiong Lai / Yan Shi / Shaowei Yang / Guohua Zhu / Qinfen Zhang / Jianguo He / Abstract: Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against ...Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against betanodavirus. But vaccine and viral vector designs of betanodavirus VLP based on their structures remain lacking. Here, the three-dimensional structure of orange-spotted grouper nervous necrosis virus (OGNNV) VLP (RBS) at 3.9 Å reveals the organization of capsid proteins (CP). Based on the structural results, seven putative important sites were selected to genetically insert a 6× histidine (His)-tag for VLP formation screen, resulting in four His-tagged VLP (HV) at positions N-terminus, Ala220, Pro292 and C-terminus. The His-tags of N-terminal HV (NHV) were concealed inside virions while those of 220HV and C-terminal HV (CHV) were displayed at the outer surface. NHV, 220HV and CHV maintained the same cell entry ability as RBS in the Asian sea bass (SB) cell line, indicating that their similar surface structures can be recognized by the cellular entry receptor(s). For application of vaccine design, chromatography-purified CHV could provoke NNV-specific antibody responses as strong as those of RBS in a sea bass immunization assay. Furthermore, in carrying capacity assays, N-terminus and Ala220 can only carry short peptides and C-terminus can even accommodate large protein such as GFP to generate fluorescent VLP (CGV). For application of a viral vector, CGV could be real-time visualized to enter SB cells in invasion study. All the results confirmed that the C-terminus of CP is a suitable site to accommodate foreign peptides for vaccine design and viral vector development.
History
Deposition
Sep 3, 2015
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Header (metadata) release
Nov 18, 2015
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Map release
Oct 19, 2016
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Update
Oct 19, 2016
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Current status
Oct 19, 2016
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Shell ID: 1 / Diameter: 380 Å / T number (triangulation number): 3
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
1.5 mg/mL
Buffer
pH: 8 / Details: PBS buffer
Grid
Details: 300 mesh Quantifoil copper grid with thin carbon support, glow discharged in air atmosphere
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK IV / Method: blot for 5 min before plunging
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Electron microscopy
Microscope
FEI TITAN KRIOS
Temperature
Min: 95 K / Max: 105 K / Average: 100 K
Alignment procedure
Legacy - Astigmatism: Objective lens astigmatism was corrected at 200000 times magnification
Details
Weak beam illumination
Date
Feb 26, 2011
Image recording
Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 2.5 µm / Number real images: 1359 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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