Structural and functional characterisation of feline calicivirus entry

Conley, Michaela Jayne (2018) Structural and functional characterisation of feline calicivirus entry. PhD thesis, University of Glasgow.

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Abstract

The Caliciviridae are a group of small, non-enveloped viruses with a positive sense, single stranded RNA genome. Caliciviruses include the noroviruses, responsible for winter vomiting disease, as well as several important veterinary pathogens. Feline calicivirus (FCV) is an excellent model for studying calicivirus entry, having a known protein receptor and being readily propagated in cell culture. Here we explore calicivirus entry, using FCV. Virus entry is the critical first step of infection and is therefore an important area of study.

Both alpha 2-6 linked sialic acid and feline junctional adhesion molecule A (fJAM-A) have been identified as receptors for FCV. The attachment of FCV to fJAM-A, is followed by uptake via clathrin mediated endocytosis. Little is known, however, on the viral escape mechanism leading to delivery of the viral RNA into the cytoplasm. We set out to explore the nature of FCV attachment and uncoating using structural, biochemical and biophysical analyses. By cryogenic electron microscopy we have characterized the virus-receptor interaction at high-resolution. Using electron microscopy and an RNA release assay, we have investigated virion uncoating. Finally, we have explored the importance of receptor glycosylation, and oligomerisation.

Our analysis has allowed us to construct an atomic model of the major capsid protein VP1. Upon binding to fJAM-A, FCV undergoes a conformational change (rotation and tilting of the capsomeres). Flexibility in the receptor decorated virion has prevented high-resolution structure analysis of the conformational change or the virus-receptor interaction. We have, however, seen that the structural changes are limited to the capsid spikes. We hypothesised that the conformational change may be a priming step that would prepare the virus for uncoating upon internalisation. We found that upon lowering the pH below 5, receptor decorated virions disassembled, supporting this hypothesis. Disassembly of the virus-receptor complex at low pH presented a tool for estimating the quantity of receptor needed to prime the capsid for uncoating.

Cryo-EM studies reveal that FCV bound fJAM-A is monomeric although the receptor was found to be dimeric in solution as previously described for the human and murine homologues. Furthermore, it is hypothesised that this is the form found at tight junctions between cells. We propose that disruption of fJAM-A homodimers may be the mechanism by which induction of viral uptake by endocytosis is triggered. Finally, we have confirmed the presence of an N-linked glycosylation on fJAM-A and show that the removal of this carbohydrate moiety does not affect viral binding in vitro.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Feline calicivirus, FCV, cryo-EM, electron microscopy, virus entry, RNA virus, fJAM-A, feline junctional adhesion molecule A, RNA release assay, VP1, conformational change.
Subjects: Q Science > QR Microbiology
Q Science > QR Microbiology > QR355 Virology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Centre for Virus Research
Funder's Name: Biotechnology and Biological Sciences Research Council (BBSRC)
Supervisor's Name: Bhella, Dr. David and Willett, Professor Brian
Date of Award: 2018
Depositing User: Miss Michaela J Conley
Unique ID: glathesis:2018-8920
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 05 Apr 2018 10:27
Last Modified: 23 May 2018 07:27
URI: http://theses.gla.ac.uk/id/eprint/8920
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