Structural insight into the remarkable multivalent plant virus Helper Component Proteinase

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 and mechanistically characterize the multifunctional protein HCPro. We will prepare recombinant HCPro (various forms, such as fusion proteins, mutants) and determine its three-dimensional structure. Next, to get insight into the mechanism of action, we will study the interactions of HCPro (and variants) with selected binding partners, i.e. various forms of CP and small RNAs, using biochemical and biophysical approaches, and determine three-dimensional structures of selected complexes of HCPro with CP or sRNAs. We will compare interactions between the HCPro/CP cognate pairs of aphid transmissible and non-transmissible viral isolates. HCPro and CP protein sequences will be from the potyviruses PVY and potato virus A (PVA). 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.