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.
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