Secretion of the rotavirus VP7 capsid glycoprotein from polarised epithelial cells

Despite the widespread implementation of rotavirus vaccination, rotaviruses remain a major cause of severe diarrhoeal disease in infants and young children worldwide. Rotavirus is a double-stranded RNA (dsRNA) non-enveloped virus that infects polarised epithelial cells lining the upper region of the small intestine villi. Among the structural proteins of rotavirus, the VP7 glycoprotein forms the outer capsid layer of the virion where it is a key target for neutralising antibodies, which act to prevent virion decapsidation. Both polarised and non-polarised cell culture models have been widely used in rotavirus research. However, cell type-dependent differences in rotavirus release and viral protein processing have been described, arising from the reduced viability of non-polarised cells compared to polarised cells during rotavirus infection. This thesis describes the identification of a higher molecular weight form of VP7 found in the medium of polarised Caco-2 cells following rotavirus infection. This secreted VP7 represents the second viral glycoprotein identified as being secreted during rotavirus infection. Investigation into this novel form of VP7 revealed that it is apically secretion through a Golgi-dependent pathway, resulting in modification of its N-linked glycan. A purification protocol was developed to enable isolation of the protein from infected cells, allowing for further biophysical and biological characterisation. The purified form of secreted VP7 (sVP7) enabled the use of size exclusion chromatography, which demonstrated that VP7 is secreted as a soluble trimer. Furthermore, this soluble protein retains its ability to bind neutralising antibodies, suggesting a role as an antigen decoy. The immunostimulatory effect of sVP7 was explored through TLR receptor binding assays and whole blood stimulation assay, revealing a previously uncharacterised immune response profile. Collectively, this work highlights the difference in rotavirus infection of polarised and non-polarised cells through the identification and characterisation of secreted VP7.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biological Science

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2025

Author dc:creator

• Nutsford, Ashley Nicole

Advisors dc:contributor.advisor

• Taylor, John
• Goldstone, David

• Virology
• Rotavirus
• Molecular Biology

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/72870

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/72870