doktorska disertacija
Andreja Kežar (Author), Marjetka Podobnik (Mentor)

Abstract

Krompirjev virus Y (PVY) je eden najnevarnejših rastlinskih virusov in obenem najbolj pomemben virusni patogen krompirja. Virus povzroči ogromno škode na kmetijskih pridelkih, najbolj nevaren pa je različek PVYNTN, ki povzroča bolezen obročkaste nekroze gomoljev. PVY virioni so okrog 740 nm dolgi fleksibilni delci, sestavljeni iz okoli 2000 kopij plaščnega proteina (CP) ter virusne RNA. Heterologno izražanje CP-ja v bakterijskih celicah vodi v produkcijo virusu podobnih delcev (VLP), ki so sestavljeni le iz CP-jev v odsotnosti virusne RNA. Natančna zgradba fleksibilnih filamentoznih virusov do pred kratkim ni bila poznana, sedaj pa je razvoj krio-elektronske mikroskopije (krio-EM) omogočil določevanje zgradbe tudi tem virusom. Tekom doktorske naloge smo s krio-EM določili prostorsko zgradbo viriona PVYNTN z ločljivostjo 3,4 A ter virusu podobnega delca z ločljivostjo 4,1 A. CPPVY je sestavljen iz osrednje globularne regije ter iztegnjenih N- in C-končnih regij, ki mu omogočata prilagajanje zgradbe različnim vezavnim partnerjem ter sestavljanje v filament z edinstveno arhitekturo. CPPVY se namreč ob prisotnosti virusne RNA sestavijo okoli RNA v obliki vijačnice, medtem ko se v odsotnosti virusne RNA CP-ji med seboj povežejo v oktamerne obroče, le-ti pa se naložijo v dolge VLP-filamente. Vlogo iztegnjenih N- in C-končnih regij pri sestavljanju VLP-jev ter infektivnosti virusa smo preučevali z delecijskimi mutacijami plaščnega proteina v VLP-ju in virusu. Z biokemijsko in biofizikalno karakterizacijo smo pokazali, da je N-končna regija nujno potrebna za sestavljanje CP-jev v VLP-je, medtem ko delecija C-končne regije nima negativnega vpliva na sestavljanje. Po drugi strani pa smo z in planta poizkusi pokazali, da sta obe regiji ključni za infektivnost virusa. Virus z delecijo N-končnih 50 aminokislinskih ostankov na CP-ju (?N50-CP) je zmožen šibkega pomnoževanja RNA, vendar je njegovo širjenje po rastlini onemogočeno, medtem ko sta C-končni deleciji 40 oz. 60 aminokislinskih ostankov na CP-ju (?C40-CP in ?C60-CP) virusu preprečili že pomnoževanje virusne RNA. V nasprotju z ?N mutantom, smo pri ?C mutantih v homogenatu listov okužene rastline opazili VLP-jem podobne delce. Poznavanje tridimenzionalne zgradbe PVY-a ter pripadajočih VLP-jev predstavlja ključno osnovo za nadaljnji razvoj preventivnih strategij za zaščito rastlin in nanobiotehnoloških aplikacij na podlagi VLPPVY.

Keywords

molekularna biologija;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL MF - Faculty of Medicine
Publisher: [A. Kežar]
UDC: 577.2
COBISS: 22015491 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Structural studies of Potato virus Y capsid and corresponding virus-like particles
Secondary abstract: Potato virus Y (PVY) is economically one of the most important plant viruses and the most important viral pathogen of potato. Virus can cause enormous damage to growing crops, with the PVYNTN strain being the most harmful as it causes potato tuber necrotic ringspot disease. PVY virions are around 740 nm long flexible filaments, assembled from around 2000 copies of the coat protein (CP) and viral RNA. The heterologous expression of CP in bacterial cells produces virus-like particles (VLP), which are assembled from the CPs in the absence of viral RNA. The detailed structure of flexible filamentous viruses was not known until recently, when the revolutionary advances in cryo-electron microscopy (cryo-EM) enabled determination of such viruses. During this research, we used cryo-EM to determine spatial structure of the PVY at 3,4 Å resolution and of the corresponding VLP at 4,1 Å resolution. CPPVY is comprised of three structural regions: the globular core domain and extended N- and C-terminal regions, which allow structural adaptations to different binding partners and assembly into the filaments with the unique architecture. Namely, CPPVY assemble in helical form around the viral RNA, while in the absence of the viral RNA, the CPs assemble into octameric rings that stack together into long VLP filaments. We analysed the role of N- and C-terminal regions in VLP assembly and viral infectivity by deletion mutations of CP in VLP and virus. The biochemical and biophysical characterization showed that the N-terminal region is crucial for VLP assembly, while deletion of the C-terminal region had no negative impact on filament assembly. On the other hand, the in planta experiments showed the importance of both regions for the infectivity of the virus. The virus with the deletion of 50 N-terminal amino acid residues on the CP (ΔN50-CP) is capable of limited RNA replication but its movement is abolished. However, we showed that C-terminal deletions of 40 or 60 amino acid residues on the CP (ΔC40-CP and ΔC60-CP) basically completely hinder the viral RNA replication. In contrast to the ΔN mutant, we could observe some VLP-like particles in the homogenate of plant leaves infected with the cDNA of ΔC mutants. Determination of the 3D structures of PVY and corresponding VLPs is a crucial basis for the development of prevention strategies for plant protection and for the development of nanobiotechnological applications based on VLPPVY.
Secondary keywords: Plants;Potyvirus;Cryoelectron microscopy;Nanotechnology;Biosynthesis;Amino acid sequence;Polymerase chain reaction;Viruses;Isolation and purification;Cromatography;Rastline;Potivirus;Krioelektronska mikroskopija;Nanotehnologija;Biositeza;Zaporedje aminokislin;Polimerazna verižna reakcija;Virusi;Izolacija in čiščenje;Kromatografija;
Type (COBISS): Dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. Ljubljana, Medicinska fak.
Pages: XVI, 109, [13] str.
ID: 11519223