Human picornaviruses : uncoating, assembly and interaction with cellular receptors

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Title: Human picornaviruses : uncoating, assembly and interaction with cellular receptors
Author: Shakeel, Shabih
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Department of Biosciences, Genetics
Institute of Biotechnology
Publisher: Helsingin yliopisto
Date: 2014-04-11
Language: en
Thesis level: Doctoral dissertation (article-based)
Abstract: Pathogenic human picornaviruses are known to cause a wide variety of diseases ranging from mild colds to severe paralysis. In addition to their importance in causing disease, they also serve as models for understanding the basic mechanisms of host-pathogen interactions, virus entry, viral genome release, viral synthesis and viral assembly. In picornaviruses, the majority of the structural and host-cell interaction studies have been conducted on polioviruses and human rhinoviruses. Picornaviruses like coxsackievirus A 7, coxsackievirus A 9 and human parechovirus 1 have not been so well studied because of difficulties in culturing them. Recently, the number of cases reported for infection by these viruses has increased dramatically due to better detection methods, thus making structural studies of these viruses and their interactions with their host cells important in order to understand their mode of infection so that better therapeutics can be designed against them. I have studied coxsackievirus A 7, coxsackievirus A 9 and human parechovirus 1, which are all pathogenic picornaviruses, in order to understand the mechanism of pathogenesis, tropism, viral entry and assembly for these viruses in particular and for picornaviruses in general. Two studies dealt with determining the structure of coxsackievirus A 7, a Human Enterovirus A species for which there was no structural information available at the time when this study was conducted. The genome-filled and empty structure of coxsackievirus A 7 were determined using cryo electron microscopy to sub-nanometer resolution which helped in building pseudo-atomic models for them using homology modelling and flexible fitting. With the help of these models, the majority of the strain variations in the capsid proteins were identified on the surface of VP1. Such variations are the likely cause of differences in pathogenesis and tropism between strains. Furthermore, superimposition of these models showed that the capsid underwent a conformational change on RNA release. In the process, generalised methods for optimising and comparing results from flexible fitting were developed. The next structural study elucidated the interaction of coxsackievirus A 9, a Human Enterovirus B species, with a cellular receptor. Integrins were found to bind sub-stoichiometrically to the capsid using electron cryo-tomography (cryo-ET). Asymmetric reconstruction indicated that this was probably due to steric hindrance. The affinity of this interaction was calculated to be 1nM using surface plasmon resonance. Additionally, the conformational changes which occur on its RNA release were quantified. The fourth study explained the importance of viral RNA in picornavirus assembly. Pentameric intermediates of human parechovirus 1 were isolated and used to identify packaging signals in the viral RNA required for capsid assembly using aptamer library screening and next generation sequencing analysis. Poly-U was identified as the common motif for these packaging signals present on the stem or the loop of the RNA secondary structure. Overall, this thesis gives an insight into many important aspects of host-virus interactions especially the events occurring on viral RNA exit and during its encapsidation. The work in this thesis could be utilized to identify potential targets for antiviral synthesis and also to define general virus assembly principles.N/A
Subject: genetics
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