Odseki

Šifra projekta / Project code: J1-4410

Vodja: izr. prof. dr. Marjetka Podobnik

1. Vsebinski opis projekta:

   Na svetovni ravni rastlinski škodljivci in patogeni povzročijo 40 % izgube pridelka letno. Med rastlinskimi patogeni so virusi odgovorni za skoraj polovico rastlinskih endemičnih infekcij. Potyvirusi (rod Potyvirus, družina Potyviridae) predstavljajo največjo skupino znanih rastlinskih RNA-virusov. Tipska vrsta, krompirjev virus Y (angl. potato virus Y ali PVY), je gospodarsko najpomembnejši virus, ki močno ogroža pridelavo krompirja in drugih razhudnikov po vsem svetu. Potyvirusni virioni so fleksibilni filamentozni delci z 10 kb dolgim genomom, ki je enoverižna RNA. Kot je značilno za RNA-viruse, omejitev velikosti genoma povzroči ekononomično porazdelitev funkcij znotraj majhnega števila večopravilnih proteinov z večdomensko strukturo in intrinzično neurejenimi regijami, da se omogoči promiskuitetnost vezave in funkcionalna raznolikost.
   Med enajstimi potivirusnimi proteini, ki se sproščajo iz virusnega poliproteina po translaciji, po svojih izjemnih večopravilnih sposobnostih izstopata pomožna komponenta-proteinaza (HCPro) in plaščni protein (CP). HCPro je ključen za prenos virionov z listnimi uši, kateri je glavni način prenosa potivirusov z rastline na rastlino. HCPro deluje tudi pri zorenju virusnih poliproteinov, razmnoževanju virusa, kopičenju virusa, gibanju od celice do celico in sistemskem gibanju, intenzivnosti simptomov in povečanju donosa virusnih delcev. HCPro veže tudi nukleinske kisline, kot so majhne eno- in dvoverižne RNA, zaradi česar je bistven dejavnik pri zaviranju utišanja RNA, s čimer kljubuje protivirusnemu imunskemu odzivu rastlin. CP je edini virusni strukturni protein in gradi virusne delce, vključen pa je tudi v prenos virionov preko listnih uši, gibanje od celice do celice in sistemsko gibanje ter pomnoževanje  genoma. Interakcije med HCPro in CP so bile opisane pri potyvirusih, ki se prenašajo preko listnih uši in tudi pri tistih, ki se ne, kar kaže da te interakcije presegajo zgolj prenos virionov z listnimi ušmi.
   Namen naše raziskave je dobiti vpogled v molekularne mehanizme virusnega prenosa z listnimi ušmi in zaviranja protivirusne odpornosti rastlin z uporabo integriranih pristopov molekularne in strukturne biologije. Glavni cilj je strukturno, funkcionalno in mehanistično opisati večnamenski protein HCPro, tako samega, kot njegove interakcije z drugimi virusnimi in gostiteljskimi molekulami. Pri tem projektu bomo sodelovali s strokovnjaki s področja rastlinske virologije. Pričakujemo, da bodo rezultati naše raziskave pomembno prispevali k razumevanju potivirusne biologije in rastlinske obrambe. Poznavanje strukturne in mehanistične osnove vezave kofaktorjev na HCPro bo prispevalo k boljšemu razumevanju procesov, kot so prenos virusov z listnimi ušmi in virusna obramba pred imunskim sistemom rastlin. Rezultati bodo tudi aplikativne narave. 3D struktura HCPro bo namreč omogočila vpogled v to, kako je mogoče v majhnem volumnu posameznega proteina izvajati različne ali celo neodvisne funkcije, s čimer bo zagotovila molekularno platformo za sintezne biologe za pripravo (umetnih) večopravilnih proteinov za uporabo v biotehnologiji. Poleg tega se virusni strukturni proteini, kot je CP in njihove in vitro pripravljene različice v bakterijskem ekspresijskem sistemu, lahko uporabijo za pripravo virusom podobnih delcev s potencialno uporabo v medicini, dostavi zdravil, razvoju cepiv ali sintezi hibridnih (nano)materialov.

   
   At the global level, plant pests and pathogens cause 40 % yield losses annually. Among plant pathogens, viruses account for nearly half of the plant endemics. Potyviruses (genus Potyvirus, family Potyviridae) constitute the largest group of known plant-infecting RNA viruses. The type species, potato virus Y (PVY), is the most economically important virus that severely harms production of potato and other solanaceous crops worldwide. Potyviral virions are flexuous filamentous particles with 10 kb positive-sense single-stranded RNA genome. As typical for RNA viruses, limitation of the genome size results in economic distribution of functions within small number of multitasking proteins of multidomain structure and regions of intrinsic disorder, to enable binding promiscuity and functional diversity.
   Among eleven potyviral proteins that are released from the viral polyprotein posttranslationally, the Helper Component Proteinase (HCPro) and the Coat Protein (CP) stand out for their amazing multitasking abilities. HCPro is essential for transmission by aphids, which is the main way of plant-to- plant transmission of potyviruses. HCPro also acts in the viral polyprotein maturation, virus multiplication, virus accumulation, cell-to-cell and systemic movement, symptom intensity and enhancement of viral particle yield. HCPro also binds nucleic acids, such as small single and double stranded RNAs, which makes it an essential factor in the suppression of RNA silencing, thereby defying plant’s antiviral immune response. CP is the only viral structural protein, essential for formation of viral particles, and also involved in aphid transmission, cell-to-cell and systemic movement and genome replication. Interactions between HCPro and CP have been described in aphid transmissible and non-transmissible viruses, suggesting the importance of such interactions beyond aphid-mediated transmission.
   In our study, we will address molecular mechanisms behind viral transmission and suppression of plant antiviral resistance, using integrated molecular and structural biology approaches. The main objective is to structurally, functionally and mechanistically characterize the multifunctional protein HCPro. We will study the protein itself as well as its interactions with other viral and plant host molecules. Throughout the project we will closely collaborate with expert plant virologists.
   We expect that results of our study will importantly contribute to understanding of the potyviral biology and plant defence. Knowing the structural and mechanistic basis of co-factor binding to HCPro will contribute to better understanding of processes such as potyviral transmissions by aphids and viral defence against the plant immune system. Results will also have an applicative note. Namely, the 3D structure of HCPro will provide insight into how different or even independent functions can be carried out in a small volume of a single protein, thus providing a molecular platform for synthetic biologists to prepare (artificial) multitasking proteins for the use in biotechnology. Furthermore, viral structural proteins such as CP and their variants made in vitro in bacterial expression system (as planned in this study), can be used in preparation of virus-like particles with potential applications in medicine, drug delivery, vaccine development or synthesis of hybrid (nano)materials.
   
   Sodelujoče organizacije:
   
   Nacionalni inštitut za biologijo
   
   
   Osnovni podatki glede financiranja:
   Projekt financira ARRS v okviru cenovne kategorije C za obdobje treh let v obsegu 2187 letnimi urami za obdobje 3 let. Pričetek financiranja je 1. 10. 2022.
   The project is co-financed by ARRS with 2187 annual hours of price class C for a period of 3 years. Funding starts on October 1, 2022.
   
   Sestava projektne skupine s povezavami na SICRIS
   Na Kemijskem inštitutu v projektni skupini sodelujejo / At the National Institute of Chemistry the project group includes:

   Marjetka Podobnik; SICRIS št. 12048
   Neža Koritnik; SICRIS št. 54671
   Luka Kavčič; SICRIS št. 50722
   Andreja Kežar; SICRIS št. 38255
   Matic Kisovec; SICRIS št. 35382
   Tanja Peric; SICRIS št. 55663
   Maja Jamnik; SICRIS št. 39711
    

2. Faze projekta in njihova realizacija
    

   WP1. Priprava rekombinantnih proteinov. Realizacija: 100 %

   WP2. Strukturne študije apo oblike HCPro. Realizacija: 95 %

   WP3. Karakterizacija interakcije med HCPro in CP. Realizacija: 100 %

   WP4. Vodenje projekta. Realizacija: 100 %

   WP5. Razširjanje (objave, konference itd.) Realizacija: 100 %

    

   WP1. Preparation of recombinant proteins. Realization: 100 %

   WP2. Structural studies of the apo form of HCPro. Realization: 95 %

   WP3. Characterization of interactions between HCPro and CP. Realization: 100 %

   WP4. Project management. Realization: 100 %

   WP5. Dissemination and outreach (publications, conferences etc.). Realization: 100 %

    

3. Bibliografske reference, ki izhajajo neposredno iz izvajanja projekta

   
   Članki:
   1. KAVČIČ, Luka, KEŽAR, Andreja, KORITNIK, Neža, TUŠEK-ŽNIDARIČ, Magda, KLOBUČAR, Tajda, VIČIČ, Žiga, MERZEL, Franci, HOLDEN, Ellie, BENESCH, Justin L. P., PODOBNIK, Marjetka. From structural polymorphism to structural metamorphosis of the coat protein of flexuous filamentous potato virus Y. Communications chemistry. 17. Jan. 2024, vol. 7, article number 14, str. 1-19. DOI: 10.1038/s42004-024-01100-x. [COBISS.SI-ID 183852291]

   2. KORITNIK, Neža, KEŽAR, Andreja, KAVČIČ, Luka, TUŠEK-ŽNIDARIČ, Magda, LEONARDI, Adrijana, DE, Swarnalok, POLLARI, Maija, MÄKINEN, Kristiina, PODOBNIK, Marjetka. Species-specific structural adaptation of the potyviral coat protein in virions and virus-like particles. Communications biology. 2026, vol. 9, 1-19, DOI: 10.1038/s42003-025-09502-w. [COBISS.SI-ID 270994435]

    

   Postri na konferencah:
   3. KORITNIK, Neža, KEŽAR, Andreja, KAVČIČ, Luka, TUŠEK-ŽNIDARIČ, Magda, DE, Swarnalok, POLLARI, Maija, MÄKINEN, Kristiina, PODOBNIK, Marjetka. A comprehensive structural study of filamentous potyviruses and their virus-like particles. V: SEPČIĆ, Kristina (ur.), PAVŠIČ, Miha (ur.). 15th Meeting of the Slovenian Biochemical Society with International Participation; book of abstracts: Portorož, 20-23 September 2023. Str. 154. ISBN 978-961-95941-1-7. [COBISS.SI-ID 169475843] https://portoroz2023.sbd.si/upload/docs/SBD2023_Portoroz-Book_of_Abstracts.pdf

   4. KAVČIČ, Luka, KEŽAR, Andreja, KORITNIK, Neža, TUŠEK-ŽNIDARIČ, Magda, MERZEL, Franci, HOLDEN, Elena, BENESCH, Justin L. P., PODOBNIK, Marjetka. Diverse structural landscape of potyviral coat protein assemblies. V: SEPČIĆ, Kristina (ur.), PAVŠIČ, Miha (ur.). 15th Meeting of the Slovenian Biochemical Society with International Participation: book of abstracts; Portorož, 20-23 September 2023. Str. 100. ISBN 978-961-95941-1-7. [COBISS.SI-ID 169451267] https://portoroz2023.sbd.si/upload/docs/SBD2023_Portoroz-Book_of_Abstracts.pdf

   5. KEŽAR, Andreja, KAVČIČ, Luka, KORITNIK, Neža, GUTIÉRREZ-AGUIRRE, Ion, TUŠEK-ŽNIDARIČ, Magda, MAHKOVEC POVALEJ, Tjaša, COLL RIUS, Anna, STARE, Katja, GRUDEN, Kristina, RAVNIKAR, Maja, PODOBNIK, Marjetka. Structural biology of plant (poty)viruses. V: EMBL workshop: In situ structural biology: from cryo-EM to multi-scale modelling : 8–11 February 2023: Heidelberg: EMBL, 2023. Str. 92. [COBISS.SI-ID 143232771]

   6. KAVČIČ, Luka, KEŽAR, Andreja, KORITNIK, Neža, TUŠEK-ŽNIDARIČ, Magda, MERZEL, Franci, HOLDEN, Elena, BENESCH, Justin L. P., PODOBNIK, Marjetka. Diverse structural landscape of the engineered potato virus Y coat protein assemblies. European biophysics journal, 14th EBSA congress, July 31 – August 4, 2023, Stockholm, Sweden. 2023, vol. 52, suppl. 1, str. s88. ISSN 1432-1017. https://link.springer.com/article/10.1007/s00249-023-01668-7 [COBISS.SI-ID 162502147].

   7. KORITNIK, Neža, KEŽAR, Andreja, KAVČIČ, Luka, TUŠEK-ŽNIDARIČ, Magda, DE, Swarnalok, POLLARI, Maija, MÄKINEN, Kristiina, PODOBNIK, Marjetka. Investigation of the interactions between the potyviral coat protein P22 and the helper component proteinase. V: ANDERLUH, Gregor (ur.), CRNKOVIĆ, Ana (ur.). 3rd MOSBRI Scientific Conference : 10th - 13th June 2024, Ljubljana, Slovenia : programme & abstracts. Ljubljana: National Institute of Chemistry, 2024. Str. 131. ISBN 978-961-6104-94-4. https://www.mosbri.eu/events/conferences/ljubljana-2024/programme-ljubljana/ . [COBISS.SI-ID 199723523].

   Predavanja:

   8. KORITNIK, Neža, KEŽAR, Andreja, KAVČIČ, Luka, TUŠEK-ŽNIDARIČ, Magda, DE, Swarnalok, POLLARI, Maija, MÄKINEN, Kristiina, PODOBNIK, Marjetka. Cryo-EM studies of potyviral coat protein structural plasticity. V: PODOBNIK, Marjetka (ur.), KISOVEC, Matic (ur.), JAMNIK, Maja (ur.). Structural Biology in Slovenia and Beyond : minisymposium 2024 : book of abstracts : May 14th, 2024, Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia. Electronic version. 2024. Str. 12. ISBN 978-961-6104-95-1. https://www.ki.si/fileadmin/user_upload/datoteke-D11/Dogodki_in_mini_simpozij/MS2024/Minisymposium_14May2024-Abstracts.pdf . [COBISS.SI-ID 199610627]

   9. PODOBNIK, Marjetka. Use of cryo-EM to study filamentous plant viruses and their virus-like particles. V: ŽAGAR, Kristina (ur.), et al. 17th Multinational Congress on Microscopy : 17MCM : Portorož, Slovenia, 7-12 September, 2025 : book of abstracts. Ljubljana: Slovene Society for Microscopy, 2025. Str. 70. ISBN 978-961-94264-4-9. https://17mcm.si/wp-content/uploads/2025/09/Book-of-Abstracts_17MCM_final.pdf . [COBISS.SI-ID 266030339]; (vabljeno predavanje)

   10. PODOBNIK, Marjetka. Remarkable structural plasticity and tunability of the potyviral coat protein. V: 4th MOSBRI Scientific Conference, June 10-13, 2025, Paris : programme book. Paris: Horizon, 2025. Str. 43. https://www.mosbri.eu/events/conferences/paris-2025/ . [COBISS.SI-ID 243177731] (vabljeno predavanje)

    

   Poleg tega smo rezultate naših študij, povezanih na ta projekt, predstavili tudi še nekaj vabljenih predavanjih (M. Podobnik: na inštitutu Human Technopole, Milano, Italija (17. november 2022); na sinhrotronu Elettra, delavnica: Elettra 2.0: New Structural Biology Opportunities, July 3-4, 2023) ter Na letnem srečanju FINStruct v Helsinkih, Finska (14.10.2025). Članek iz strukture HCPro in vezave na CP in dsRNA je v pripravi. Neža Koritnik bo v 2026 doktorirala iz tematike tega projekta.