Double-stranded RNA Bacteriophage phi6 : Self-assembly and Maturation of the Procapsid

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Title: Double-stranded RNA Bacteriophage phi6 : Self-assembly and Maturation of the Procapsid
Author: Sun, Xiaoyu
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Department of Biosciences
Institute of Biotechnology
Publisher: Helsingin yliopisto
Date: 2014-05-09
Language: en
Belongs to series: Dissertationes Biocentri Viikki Universitatis Helsingiensis - URN:ISSN:1799-7372
Thesis level: Doctoral dissertation (article-based)
Abstract: Many viruses protect their genome in a protein capsid. Viral capsid formation involves the association of multiple copies of viral capsid protein subunits, representing single or multiple protein species, and different assembly strategies are utilized. Pseudomonas phage phi6 uses an assembly pathway in which an empty capsid (procapsid, PC) is first assembled, serving as a compartment for the subsequent encapsidation of the RNA genome. During encapsidation, the compact, empty PC undergoes conformational rearrangement to reach its final expanded form. The phi6 PC is composed of the main structural protein, P1, and three minor protein species: the RNA-dependent RNA polymerase P2, the packaging nucleoside triphosphatase (NTPase) P4, and the assembly cofactor P7. In vitro systems of phi6 assembly, genome encapsidation, and transcription have been established, allowing infectious particles to be constructed from purified protein and RNA components. In this thesis, stoichiometric measurements were established to estimate the relative copy numbers of PC proteins in phi6 virions and PCs. Different concentrations of the phi6 minor proteins were employed in in vitro assembly reactions to probe potential PC binding sites. The results indicate that potential binding sites for proteins P2 and P7 are only partially occupied in phi6 virions and recombinant PCs. High P7 occupancy in self-assembled PCs resulted in reduced P2 incorporation, suggesting some correlation between P2 and P7 during PC assembly. Although high P4 hexamer occupancy was critical for initial particle formation, a large excess of P4 in the self-assembly reaction slowed the rate of PC self-assembly, which may be ascribed to excessive production of P1-P4 nucleation complexes. In addition, electrostatic interactions were demonstrated to be the main driving force in phi6 PC assembly. Furthermore, it was shown that P4 hexamers spontaneously dissociate from the empty capsid shell. P4-deficient particles have slower sedimentation velocity and an expanded appearance compared to the PC that has full-occupancy of P4. These particles are also defective in RNA packaging and transcription. However, purified P4 hexamers can efficiently assemble on P4-deficient particles, guiding the particles to their naive compact conformation and rescuing packaging and transcription activities. The results obtained from this study provide new insight into the principles of viral capsid assembly and demonstrate the reversibility of the PC maturation pathway.Useat virukset suojaavat genomiansa pakkaamalla sen proteiinikapsidin sisään. Kapsidin muodostuessa viruksen tuottamat proteiinit ja nukleiinihappomolekyylit liittyvät yhteen. Eri virukset käyttävät erilaisia strategioita kapsidinsa rakentamiseksi. Bakteriofagi phi6 on mallivirus, jonka avulla tätä prosessia voidaan tutkia. phi6 rakentaa ensin tyhjän kapsidin useasta kapsidiproteiinista ja pakkaa tämän jälkeen RNA genominsa kapsidin sisään. Tässä työssä tutkittiin phi6 virionien muodostumista ja viruskapsidin ominaisuuksia käyttäen puhdistettuja komponentteja ja in vitro menetelmiä. Tulokset valoittavat niitä molekyylitason toimintaperiaatteita, joita virukset hyödyntävät kapsidiensa kokoamisessa.
Subject: microbiology
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