doktorska disertacija
Luka Kavčič (Author), Marjetka Podobnik (Mentor), Brigita Lenarčič (Thesis defence commission member), Nada Žnidaršič (Thesis defence commission member), Eva Žerovnik (Thesis defence commission member)

Abstract

Rastlinski (+)ssRNA virusi predstavljajo zbirko evolucijsko optimiziranih proteinskih nanodelcev različnih oblik in velikosti, ki pri sesalcih ne morejo razviti virusne okužbe in so zato zelo zanimivi za uporabo. V nasprotju z ikozaedričnimi in paličastimi virusi pa potencial široko zastopanih gibljivih nitastih virusov ni dobro raziskan. Krompirjev virus Y (PVY) je predstavnik največje skupine gibljivih nitastih virusov (rod potivirusov), in predstavlja svetovno najpomembnejši virusni patogen krompirja. Njegov plaščni protein (angl. coat protein, CP) poseduje visoko stopnjo intrinzične neurejenosti ter s tem strukturne plastičnosti, kar mu omogoča sodelovanje v praktično vseh stopnjah virusne okužbe. Kot edini strukturni protein, se v virusnih delcih združuje okoli virusne (+)ssRNA v obliki leve vijačnice in tako tvori gibljivo nitasto kapsido. Čeprav še ni znano, v kakšni strukturni obliki je CP udeležen v različnih virusnih procesih v naravi, lahko informacije o strukturni pokrajini, dostopni potivirusnemu CP, pridobimo s študijo njegovih virusom podobnih delcev (VLP), pridobljenih z rekombinantno proizvodnjo v bakterijah. S pomočjo strukturnih in biofizikalnih pristopov smo pokazali vlogo intrinzično neurejenih regij (IDR) v samosestavljanju rekombinantnega CP PVY, pri čemer smo v vzorcu očiščenih VLP sočasno zaznali prisotnost treh različnih arhitektur nitk (strukturni polimorfizem). Na podlagi njihovih strukturnih modelov visoke ločljivosti, pridobljenih s krio-elektronsko mikroskopijo, smo z dodatnim inženiringom pripravili nove različice proteinskih nanodelcev z izboljšanimi lastnostmi, ki bi pomagale premostili vrzel do njihove uporabe v bionanotehnologiji. S širokim naborom pripravljenih različic CP ter biokemijsko, biofizikalno ter strukturno analizo sestavljenih delcev smo pokazali, da lahko nadzorujemo obliko, velikost, sposobnost pakiranja RNA, simetrijo, stabilnost in površinsko funkcionalizacijo nanodelcev. Pri visoki ločljivosti smo določili tudi tridimenzionalno strukturo teh nanodelcev, ki obsegajo polimorfne ali monomorfne nitke ter manjše obročaste, kubične ali sferične delce s točno definirano arhitekturo. Poleg tega smo pokazali, da lahko preprečimo samosestavljanje CP v bakterijah s fuzijo globularnega proteina na C-koncu CP, kar omogoča nadzorovano tvorbo nanodelcev v pogojih in vitro. Skupno smo pokazali, da je plaščnemu proteinu PVY dostopna zelo obsežna strukturna pokrajina in da lahko lastnosti nastalih nanodelcev dodatno prilagajamo za namene specifičnih uporab. Poleg novih možnosti za proizvodnjo biološko razgradljivih biomaterialov pa naši rezultati podajajo tudi iztočnice za prihodnje raziskave o polimorfizmu CP PVY v biološkem kontekstu.

Keywords

krompirjev virus Y;plaščni protein;proteinski nanodelci;virusom podobni delci;samosestavljanje;polimorfizem;krio-elektronska mikroskopija;inženiring;doktorske disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [L. Kavčič]
UDC: 602.3:578(043.3)
COBISS: 203027203 Link will open in a new window
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Downloads: 16
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Other data

Secondary language: English
Secondary title: Engineering of potato virus Y-based virus-like particles for bionanotechnological applications
Secondary abstract: The capsids of plant ssRNA viruses represent a collection of evolutionarily optimized proteinaceous nanoparticles of different shapes and sizes that are not infectious to mammals and therefore have a high application potential. In contrast to icosahedral and rod-shaped viruses, the potential of the widespread flexible filamentous plant viruses has not yet been fully explored. Potato virus Y (PVY) belongs to the group of potyviruses, the largest group of flexible filamentous viruses, and is the most important viral pathogen of potato worldwide. The capsid/coat protein (CP) exhibits a high degree of intrinsic disorder and structural plasticity, which is strongly related to its numerous functions during the viral infection cycle. As the sole structural protein it is assembled in a left-handed helix around viral (+)ssRNA and forms flexible filamentous virions. While it remains unclear in what structural context the CP fulfils its multiple roles during the viral life cycle, the structural landscape available to the potyviral CP can be ascertained by studying recombinantly produced virus-like particles (VLPs). Using combined structural and biophysical approaches, we have elucidated the role of intrinsically disordered regions (IDRs) in the polymorphic self-assembly of recombinant CP, which results in three distinct VLP architectures. Based on their high-resolution cryo-EM models, we successfully engineered variants with increased application potential for their use in bionanotechnology, produced via deletions at the C- and/or N-terminus, as well as carrying single-site mutations in distinct CP regions. With a wide range of variants combined with their biochemical, biophysical and structural analysis we have shown that we can control the shape, size, ability to encapsidate RNA, symmetry, stability and surface functionalization of PVY CP-derived nanoparticles. We have determined the structure of such nanoparticles, ranging from polymorphic to monomorphic filaments, rings, cubes, spherical particles and more, all with precisely defined architectural parameters. Furthermore, we have shown that we can prevent the self-assembly of CP in bacteria and allowed control of the nanoparticle formation process in space and time. In summary, we have unraveled the vast structural landscape available to PVY CP and that the properties of the assembled nanoparticles can be tailored for specific purposes. Our results not only provide novel possibilities for the production of biodegradable nanoparticles, but could also advance future studies on the polymorphism of CP in a biological context.
Secondary keywords: potato virus Y;coat protein;virus-like particle;self-assembly;polymorphism;cryo-EM;engineering;Virusi RNK;Biotehnologija;Univerzitetna in visokošolska dela;
Type (COBISS): Doctoral dissertation
Study programme: 1000381
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Pages: 163 str.
ID: 24492566