Viruses in motion: maturation of an insect virus-like particle – a nanomachine

Castells Graells, Roger (2019) Viruses in motion: maturation of an insect virus-like particle – a nanomachine. Doctoral thesis, University of East Anglia.

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Virus-like particles (VLPs) are particles that lack the infectious genome of the
parent virus but retain the ability to self-assemble. Transient expression of viral coat
proteins in a variety of systems can be used to synthesize VLPs that are morphologically
indistinguishable from the virus from which they have been derived. In this thesis, I have
used VLPs to study virus maturation mechanisms.
The main focus of this thesis has been the study of the maturation of Nudaurelia
capensis omega virus (NωV), an insect virus. NωV has a pH-controlled maturation
mechanism that involves a precise autocatalytic cleavage within the capsid protein that
is accompanied by a dramatic decrease in the diameter of the particles. I used insect cell
and plant-based transient expression of the NωV coat protein to produce procapsids and
showed that reduction in pH results in cleavage of the coat protein and the concomitant
decrease in particle diameter. Mature VLPs purified from plants had a structure
indistinguishable from the authentic virus at 2.7 A resolution, as determined by cryoelectron
microscopy (cryo-EM). However, the plant-produced VLPs showed the
formation of strong interactions between the subunits of NωV, probably representing
covalent crosslinks, that limited their dynamic properties. Given the fact that insect cells
did not present this difficulty, we used them to make the VLPs for cryo-EM analysis of
intermediates in the process of maturation. These analyses revealed the conformational
changes in the structure of the particles that accompany maturation, such as the
formation of the cleavage site and the closure of the pores of the particle.
The culmination of this thesis is represented by a movie of the maturation
process of NωV. This movie provides, for the first time, high-resolution information
about the large conformational changes and protein rearrangements occurring during
the maturation of this virus.
Keywords: Nudaurelia capensis omega virus, virus maturation, autocatalytic cleavage,
virus-like particle, cryo-electron microscopy

Resum (abstract in Catalan)
Les particules similiviriques (VLPs), acronim de l’angles Virus-Like Particles, son
particules que no tenen el material genetic viral pero que conserven la capacitat
d’autoassemblar-se. Per sintetitzar les VLPs es poden utilitzar diversos sistemes
mitjancant els quals es produeixen les proteines de la capsida del virus. Aquestes
particules son morfologicament identiques als virus originals dels quals han estat
derivades. En aquesta tesi he utilitzat les VLPs per estudiar els mecanismes de maduracio
dels virus.
L’objectiu principal d’aquesta tesi ha estat l’estudi del proces de maduracio de
Nudaurelia capensis omega virus (NωV), un virus d’insecte. NωV te un mecanisme de
maduracio, que esta controlat pel pH, que implica una segmentacio autocatalitica de la
proteina de la capsida del virus i va acompanyada d’una reduccio del diametre de les
particules. En aquesta tesi vaig utilitzar cel・lules d’insectes i plantes per tal de produir la
proteina de la capsida de NωV i generar particules, conegudes com a procapsides.
Endemes vaig demostrar que la reduccio en el pH dona lloc a la segmentacio de la
proteina de la capsida i a la reduccio del diametre de les particules. Mitjancant
criomicroscopia electronica (cryo-EM) vaig obtenir, a una resolucio de 2.7 A, l’estructura
de particules que havien estat madurades en plantes i vaig veure que tenien una
estructura identica als virus autentics. Tot i aixo, les VLPs extretes de plantes tenien
interaccions fortes entre les seves subunitats, probablement degut a enllacos covalents,
i aixo va limitar les seves propietats dinamiques. Tenint en compte que les cel・lules
d’insecte no presentaven aquesta dificultat, les vam utilitzar per obtenir les estructures
dels intermedis en el proces de maduracio mitjancant analisis amb cryo-EM. Aquestes
analisis van revelar els grans canvis conformacionals en l’estructura de les particules que
ocorren durant la maduracio, com ara la formacio del punt de segmentacio i el tancament
dels porus de la particula.
La conclusio d’aquesta tesi esta representada per una pel・licula del proces de
maduracio de NωV. Aquesta pel・licula proporciona, per primera vegada, informacio
d’alta resolucio sobre els grans canvis conformacionals i els reordenaments de proteines
que es produeixen durant la maduracio d’aquest virus.
Paraules clau: Nudaurelia capensis omega virus, maduracio viral, segmentacio
autocatalitica, particules similiviriques, criomicroscopia electronica

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Zoe White
Date Deposited: 03 Jan 2020 11:52
Last Modified: 03 Jan 2020 11:52


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