20.500.12556/RUL-190031 Strukturne in funkcijske raziskave večopravilnih proteinov CP in HCPro iz rastlinskih virusov rodu Potyvirus Structural and functional studies of the multitasking proteins CP and HCPro from plant viruses of the genus Potyvirus Potivirusi (rod Potyvirus, družina Potyviridae) so ssRNA-virusi, ki z okužbo kulturnih rastlin povzročajo obsežno gospodarsko škodo. Njihov majhen genom kodira 11 proteinov, med katerimi je plaščni protein (CP) edini strukturni protein, ki sodeluje v več fazah okužbe. Med rastlinami se virioni prenašajo z listnimi ušmi, kjer ima ključno vlogo interakcija CP s potivirusno pomožno komponento proteinazo (HCPro), večopravilnim proteinom s proteazno aktivnostjo in vlogo pri zaviranju gostiteljskega RNA-utišanja. Cilj doktorske naloge je bila karakterizacija molekulskih interakcij HCPro s CP in siRNA na biokemijski, biofizikalni in strukturni ravni. Kot modela smo uporabili virus A krompirja (PVA) in virus Y krompirja (PVY). Ker PVA še ni bil strukturno okarakteriziran, smo najprej s krio-EM smo določili strukturo virusa in njegovih virusu podobnih delcev (VLP-jev). Pokazali smo, da je osnovna arhitektura PVA podobna tisti od PVY, vendar vsebuje majhne razlike, ki omogočajo vrstno specifične interakcije CP–CP in CP–RNA. Te razlike se odražajo tudi v sestavljanju VLP-jev - rekombinantni PVACP v bakterijah tvori dva tipa VLP-jev z levosučno vijačno simetrijo: filamente z RNA (strukturno podobne virionom) in brez RNA, pri čemer se slednji izrazito razlikujejo od VLP-jev brez RNA, ki jih tvori PVYCP. Na osnovi strukturne analize in mutageneze smo identificirali aminokislinske ostanke na interakcijskih površinah CP–CP in CP–RNA, ki so ključni za variabilnost VLP-jev. Njihovo vlogo smo dodatno potrdili s strukturno analizo VLP-jev virusa jedkanja tobaka (TEV), virusa lisavosti paprike (PepMoV) in virusa mozaika divjega sirka (JGMV), s katerimi smo tudi dodatno pokazali vrstno specifičnost arhitektur VLP-jev brez RNA. Z izražanjem v bakterijah smo pripravili PVAHCPro in PVYHCPro z N-končnim fuzijskim partnerjem maltozo vezavnim proteinom (MBP). Oba proteina sta nagnjena k agregaciji ter tvorita več homo-oligomernih zvrsti. Pokazali smo interakcijo PVAHCPro in PVYHCPro s PVACP in PVYCP (v obliki VLP-jev in monomera) znotraj potivirusne vrste in med vrstama. Pokazali smo tudi, da PVAHCPro in PVYHCPro vežeta siRNA neodvisno od nukleotidnega zaporedja. Pri specifičnih pogojih MBP-PVAHCPro (ne pa MBP- PVYHCPro) tvori filamente zaradi polimerizacije PVAHCPro. To omogoča strukturno stabilizacijo PVAHCPro in določanje njegove krio-EM strukture. Določili smo približno 60 % strukture - tri odseke polpeptidne verige, ki tvorijo osrednjo in C-končno domeno. Interakcijske površine med proteini PVAHCPro v filamentih bi lahko bile pomembne tudi pri oligomerizaciji potivirusnih HCPro v naravi. Naše delo pokaže, da majhne, vrstno specifične razlike v potivirusnih CP usmerjajo specifične interakcije v virionih in VLP-jih, ki verjetno uravnavajo sestavljanje stabilnih virionov. Hkrati to delo razširja razumevanje strukturnih značilnosti HCPro in njegove oligomerizacije ter predstavlja osnovo za razumevanje interakcij HCPro s CP in siRNA. Delo predstavlja osnovo za nadaljnje raziskave v rastlinskem kontekstu. Potyviruses (genus Potyvirus, family Potyviridae) are ssRNA viruses that cause extensive economic damage by infecting cultivated plants. Their small genome encodes 11 proteins, among which the coat protein is the only structural protein and participates in multiple stages of infection. A key role in aphid-mediated transmission of virions is played by the interaction of CP with the potyviral helper component-proteinase (HCPro), a multifunctional protein with protease activity and a role in suppressing host RNA silencing. The aim of this doctoral thesis was characterization of HCPro molecular interactions with CP and siRNA at the biochemical, biophysical, and structural levels. As model systems, we used potato virus A (PVA) and potato virus Y (PVY). Since PVA has not yet been structurally characterized, we first determined the structure of the virus and its virus-like particles (VLPs) by cryo-EM. We showed that the overall architecture of PVA is similar to that of PVY, but contains subtle differences that enable species-specific CP–CP and CP–RNA interactions. These differences are also reflected in VLP assembly - recombinant PVACP expressed in bacteria forms two types of left-handed helical VLPs: RNA-containing filaments (structurally similar to virions) and RNA-free filaments - the latter differing significantly from the RNA-free VLPs formed by PVYCP. Based on structural analysis and mutagenesis, we identified amino acid residues at CP–CP and CP–RNA interaction interfaces that are critical for VLP variability. Their role was further confirmed by structural analysis of VLPs of tobacco etch virus (TEV), pepper mottle virus (PepMoV), and Johnsongrass mosaic virus (JGMV), which additionally demonstrated the species specificity of RNA-free VLP architectures. Using bacterial expression, we produced PVAHCPro and PVYHCPro as fusion with N-terminal maltose-binding protein (MBP). Both proteins are prone to aggregation and form multiple homo-oligomeric species. We demonstrated interactions of PVAHCPro and PVYHCPro with PVACP and PVYCP (in the form of VLPs and monomers), both within and between potyvirus species. We also showed that PVAHCPro and PVYHCPro bind siRNA independently of nucleotide sequence. Under specific conditions, MBP-PVAHCPro (but not MBP-PVYHCPro) forms filaments due to PVAHCPro polymerization, enabling its structural stabilization and cryo-EM structure determination. Approximately 60% of PVAHCPro structure was resolved - three segments of the polypeptide chain that form the central and C-terminal domains. The interaction interfaces observed between PVAHCPros in the filaments may also be relevant for HCPro oligomerization in nature. Our work demonstrates that small, species- specific differences in potyviral CPs direct specific interactions in virions and VLPs, likely governing the assembly of stable virions. It also expands the understanding of HCPro structure and oligomerization and provides the basis for understanding of HCPro interactions with CP and siRNA. The work provides a foundation for future studies in the plant context. Potyvirus CP HCPro krio-EM molekulske interakcije Potyvirus CP HCPro cryo-EM molecular interactions true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-10-21 07:15:12 2026-10-21 07:15:32 2026-10-22 04:13:13 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-10-13 43416 11049.pdf 11049.pdf 1 E34A9AA4AA1C409ED20CEB57BB2673FA 080e68493edd2f711549467bd64a5a1a19a655e11a624ab5c9310df6193c9749 4ff6b7a0-cd0e-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=251021 11050.pdf 11050.pdf 1 D64AC3FDCA83D4A3F984AFA550EE530C 0e38d9218f99afb2154376f50a01dcef9a9d6a4a47bf1e3cc01a5976cbba445a 502725a8-cd0e-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=251022 Medicinska fakulteta 0 0 0