Investigating Animal Rotavirus Receptor Binding Domain Interactions with Human Receptors to Understand Zoonotic Potential

Abstract

Understanding binding of animal rotavirus strains offers insight into zoonotic potential
The goal of this project is to determine if bat P[10] binds in an excited state to the human glycans that P[II] strains bind
CEST NMR data and 2D 1H-15N-HQSC spectra revealed that L80, T137, G116, and G103 residues may participate in slow confirmational changes of the VP8* receptor binding domain on the VP4 spike protein

Introduction

•Rotaviruses are a primary cause of severe gastroenteritis in children and responsible for ~200,000 deaths per year

•Zoonosis, the process of disease transmission from animals to humans, may be at play in rotavirus

•The first step in infection is successful binding of rotavirus to cell surface carbohydrates, which is mediated by the VP8* domain on the VP4 spike protein

•P[10] is genetically similar to P[II] genotypes, including P[19]

•P[10] does not show binding activity in the ground state, but binding in the excited state has not been investigated

•Chemical exchange saturation transfer (CEST) nuclear magnetic resonance (NMR) technology can be applied to detect “invisible” protein excited states

Methodology

•VP8* sequences from human P[10] with N-terminal glutathione S-transferase (GST) tag were expressed in E.coli and purified with glutathione column

•GST tag removed using thrombin and protein was further purified with size exclusion chromatography

•CEST experiments done in the absence of ligands, followed by experiments done in presence of ligands

•CEST detects dilute proteins in vitro through the interaction of bulk water proteins and labile solute protons

•Sparsely populated excited states can be detected when they are present for a short amount of time

•Heteronuclear Single Quantum Coherence (HSQC) provides the correlation between nitrogen and amide in peptide bond

•2D 1H-15N-HSQC experiments were completed using Lacto-N-fucopentaose I (LNFPI), Lacto-N-difucohexaose I (LNDFH I), and Lacto-N-tetaose (LNT) as glycan reagents

•The assigned HSCQ spectrum for P[10] was used to identify the amino acid residues that correspond to the peaks that showed activity in CEST experiments

CEST schematic: amplify relatively small excited state signal compared to ground state signal

Results

CEST Activity for Peak 23

Assigned HSQC Spectrum for Peak 23 (highlighted in red)

CEST Activity for Peak 69

Assigned HSQC Spectrum for Peak 69 (highlighted in red)

CEST Activity for Peak 101

Assigned HSQC Spectrum for Peak 101 (highlighted in red)

CEST Activity for Peak 108

Assigned HSQC Spectrum for Peak 108 (highlighted in red)

Human P[19] with bound ligand

Bat P[10] with CEST active residues highlighted

P[10] with CEST active residues highlighted in red

Conclusions

•Bat P[10] exhibits β⍺-binding site, similar to the P[19] binding site

•Four residues involved in slow confirmational changes of P[10] VP8* upon ligand binding are L80, T137, G116, and G103

References

Yang, L., Jiang, X. & Kennedy, M. A. (2016). Glycan Specificity of P[19] Rotavirus and Comparison with Those of Related P Genotypes. Journal of Virology, 90(21), 9983-9996. https://doi.org/10.1128/jvi.01494-16
Shenyun, X., Shuisong, N., Jiang, X. & Kennedy, M.A. (2021). Structural basis of P[II] rotavirus evolution and host ranges under selection of histo-blood group antigens. PNAS, 118(36). https://doi.org/10.1073/pnas.2107963118
Kogan, F., Hariharan, H. & Ravinder, R. (2013). Chemical Exchange Saturation Transfer (CEST) Imaging: Description of Technique and Potential Clinical Applications. Current Radiology Reports, 1, 102-114. https://doi.org/10.1007/s40134-013-0010-3
Liu, Y., Huang, P., Tan, M., Liu, Y., Biesiada, J., Meller, J., Castello, A. A., Jiang, B., & Jiang, X. (2012). Rotavirus VP8*: phylogeny, host range, and interaction with histo-blood group antigens. Journal of virology, 86(18), 9899–9910. https://doi.org/10.1128/JVI.00979-12
Huang, P., Xia, M., Tan, M., Zhong, W., Wei, C., Wang, L., Morrow, A., & Jiang, X. (2012). Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner. Journal of virology, 86(9), 4833–4843. https://doi.org/10.1128/JVI.05507-11
Vallurupalli P, Bouvignies G, Kay LE. Studying "invisible" excited protein states in slow exchange with a major state conformation. J Am Chem Soc. 2012 May 16;134(19):8148-61. doi: 10.1021/ja3001419. Epub 2012 May 3. PMID: 22554188.

NACE Career Readiness Competencies

Teamwork
- Worked with other undergraduate researchers and advisors to make progress in the research process

Technology
- Utilized a novel application of CEST technology to study invisible protein excited states

Critical Thinking
- Solved issues and barriers that presented themselves throughout the research process
- Analyzed findings and made conclusions about the importance of excited proteins in rotavirus binding

Professionalism
- Prepared samples for analysis weekly and communicated with advisors about next steps in the research process

Sofia DePasquale

Department of Chemistry and Biochemistry, Miami University

Class of 2025

B.S. in Mathematics and Statistics

B.A. in Biochemistry

Email: depasqsn@miamioh.edu

Advisor: Dr. Michael Kennedy

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